Novel lipids and their nanoparticle compositions

Abstract

Provided herein are lipids having the formula (I) and pharmaceutically acceptable salts thereof, wherein R 1 , R 1 ', R 2 , R 2 ', R 3 , R 3 ', R 4 , R 4 ', R 5 , and R 5 " is as defined herein. Also provided herein are lipid nanoparticle (LNP) compositions comprising a lipid having Formula (I) and a capsid-free, non-viral vector (e.g., ceDNA). In one aspect, these LNPs can be used to deliver capsid-free, non-viral DNA vectors to a desired target site (e.g., a cell, tissue, organ, etc.). JPEG2023527747000017.jpg2942

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 026,479, filed 18 May 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Gene therapy aims to improve clinical outcomes for patients suffering from either hereditary disorders or acquired diseases caused by abnormal gene expression profiles. Various types of gene therapies have been developed to date, delivering therapeutic nucleic acids to the patient's cells as drugs to treat the disease.

[0003] The introduction and expression of repair genes into a patient's target cells can be carried out via a number of methods, including the use of engineered viral gene delivery vectors and potentially plasmids, minigenes, oligonucleotides, minicircles, or various closed-end DNAs. Among the many available viral vectors (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated virus (rAAV) is accepted as a versatile and relatively safe vector in gene therapy. However, viral vectors such as adeno-associated vectors are highly immunogenic and can induce humoral and cellular immunity, which may impair efficacy, particularly with regard to re-administration.

[0004] Nonviral gene delivery avoids certain disadvantages associated with viral transduction, particularly humoral and cellular immune responses to viral structural proteins that form vector particles, as well as disadvantages arising from the expression of any novel viral gene. Some nonviral gene delivery technologies utilize cationic lipids as carriers.

[0005] Ionizable lipids are generally composed of an amine moiety and a lipid moiety. The cationic amine moiety and the polyanionic nucleic acid interact electrostatically to form positively charged liposomes or lipid membrane structures. Therefore, cellular uptake is promoted and the nucleic acid is delivered to the cell.

[0006] Widely used ionizable lipids include cationic lipids such as CLinDMA, DLinDMA (also called DODAP), and DOTAP. Notably, these lipids have been used for siRNA delivery to the liver, but have been plagued by suboptimal delivery efficiency along with liver toxicity at high doses. Considering the drawbacks of current cationic lipids, there is a need to provide a lipid scaffold that not only shows reduced toxicity and enhanced potency, but also improves pharmacokinetics and intracellular dynamics such as cellular uptake and nucleic acid release from lipid carriers.

Summary of the Invention

Means for Solving the Problems

[0007] In one aspect, a compound of formula (I):

Chemical formula

[0008] Also provided is a pharmaceutical composition comprising the disclosed ionizable lipid, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0009] Another aspect of this disclosure relates to compositions comprising ionizable lipids or pharmaceutically acceptable salts thereof as described herein, and lipid nanoparticles (LNPs) comprising nucleic acids. In one embodiment, the nucleic acid is encapsulated in the ionizable lipid. In a particular embodiment, the nucleic acid is closed-end DNA (ceDNA).

[0010] According to some embodiments of any aspect or embodiment of this specification, the LNP further comprises a sterol. According to some embodiments, the sterol may be cholesterol or beta-sitosterol.

[0011] According to some embodiments, cholesterol is present in molar percentages of about 20% to about 40%, for example, 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 ionizable lipids are present in molar percentages of about 80% to about 60%, for example, 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, cholesterol is present in a molar percentage of approximately 20% to approximately 40%, for example, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, or approximately 40%. The ionizable lipids present in molar percentages of about 80% to about 60%, for example, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, or about 60%. According to some embodiments, cholesterol is present in molar percentages of about 40%, and the ionizable lipids are present in molar percentages of about 50%. According to some embodiments of any aspect or embodiment of this specification, the composition further comprises cholesterol, PEGylated lipids, and noncationic lipids. According to some embodiments, PEGylated lipids are present in molar percentages of about 1.5% to about 4% or about 1.5% to about 3%, for example, 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, PEGylated lipids are present in molar percentages of approximately 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3%.According to some embodiments, cholesterol is present in molar percentages of approximately 30% to approximately 50%, for example, approximately 30% to approximately 45%, approximately 30% to approximately 40%, approximately 30% to approximately 35%, approximately 35% to approximately 50%, approximately 35% to approximately 45%, approximately 35% to approximately 40%, approximately 20% to approximately 40%, approximately 40% to approximately 50%, or approximately 45% to approximately 50%. According to some embodiments, cholesterol is present in molar percentages of approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, or approximately 50%.

[0012] According to some embodiments, the PEGylated lipid in the composition described herein is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG). According to some embodiments of any aspect or embodiment of this specification, the LNP further comprises a noncationic lipid. According to some embodiments, noncationic lipids include distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and distearoylphosphatidylethanolamine. (DSPE), monomethyl-phosphatidylethanolamine (16-O-monomethylPE, etc.), dimethyl-phosphatidylethanolamine (16-O-dimethylPE, etc.), 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM ), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diylcoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), diylidoyl phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,The noncationic lipid is selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. According to some embodiments, the noncationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

[0013] According to some embodiments, ionizable lipids are present in a molar percentage of approximately 42.5% to approximately 62.5%. According to some embodiments, ionizable lipids are present in approximately 42.5%, approximately 43%, approximately 43.5%, approximately 44%, approximately 44.5%, approximately 45%, approximately 45.5%, approximately 46%, approximately 46.5%, 47.5%, approximately 48%, approximately 48.5%, approximately 49%, approximately 49.5%, approximately 50%, approximately 50.5%, approximately 51%, approximately 51.5%, approximately 47%, approximately 52%. They are present in molar percentages of approximately 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, 60%, 60.5%, 61%, 61.5%, 62%, or 62.5%. According to some embodiments of any aspect or embodiment of this specification, noncationic lipids are present in molar percentages of approximately 2.5% to approximately 12.5%. According to some embodiments of any aspect or embodiment of this specification, cholesterol is present at about 40% molar percentage, ionizable lipids at about 52.5% molar percentage, noncationic lipids at about 7.5% molar percentage, and PEGylated lipids at about 3% molar percentage.

[0014] According to some embodiments of any aspect or embodiment of this specification, the LNP further comprises a tissue-specific targeting ligand. The tissue-targeting portion may be a peptide, oligosaccharide, etc., 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, the tissue-specific targeting ligand is a ligand for a liver-specific receptor. According to one embodiment, the liver-specific receptor ligand used for liver targeting is an oligosaccharide such as N-acetylgalactosamine (GalNAc) or a GalNAc derivative such as one, two, three, or four-antennary GalNAc. According to some embodiments of any aspect or embodiment of this specification, the tissue-specific targeting ligand is conjugated to a pegylated lipid. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in lipid nanoparticles at molar percentages 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, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.2%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.3%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.4%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.5%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.6%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.7%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.8%.According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 0.9%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 1.0%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of approximately 1.5%. According to some embodiments, PEGylated lipids conjugated with tissue-specific targeting ligands are present in LNPs at a molar percentage of 2.0%.

[0015] According to some embodiments of any aspect or embodiment of this specification, the LNP composition further comprises dexamethasone palmitate.

[0016] According to some embodiments of any aspect or embodiment of this specification, the LNP has an average diameter of about 50 nm to about 110 nm, for example, 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 The sizes are in the range of approximately 85nm, approximately 60nm to 80nm, approximately 60nm to 75nm, approximately 60nm to 70nm, approximately 60nm to 65nm, approximately 70nm to 110nm, approximately 70nm to 100nm, approximately 70nm to 95nm, approximately 70nm to 90nm, approximately 70nm to 85nm, approximately 70nm to 80nm, approximately 70nm to 75nm, approximately 80nm to 110nm, approximately 80nm to 100nm, approximately 80nm to 95nm, approximately 80nm to 90nm, approximately 80nm to 85nm, approximately 90nm to 110nm, or approximately 90nm to 100nm. According to some embodiments of any aspect or embodiment of this specification, the LNP has an average size of less than about 100 nm, for example, a size of 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. According to some embodiments, the LNP has an average size of less than about 70 nm, for example, a size of 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. According to some embodiments, the LNP has an average size of less than about 60 nm, for example, a size of 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.According to some embodiments of any aspect or embodiment of this specification, the LNP composition has a total lipid-to-nucleic acid ratio of about 10:1. According to some embodiments of any aspect or embodiment of this specification, the LNP composition has a total lipid-to-nucleic acid ratio of about 20:1. According to some embodiments of any aspect or embodiment of this specification, the composition has a total lipid-to-nucleic acid ratio of about 30:1. According to some embodiments of any aspect or embodiment of this specification, the composition has a total lipid-to-nucleic acid ratio of about 40:1. According to some embodiments of any aspect or embodiment of this specification, the composition has a total lipid-to-nucleic acid ratio of about 50:1.

[0017] According to some embodiments of any aspect or embodiment of this specification, the LNP composition is prepared in a buffer such as malic acid. In some embodiments, the composition is prepared with about 10 mM to about 30 mM malic acid, for example, 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, and about 20 mM to about 25 mM. According to some embodiments of any of the aspects or embodiments described 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, the composition contains about 20 mM malic acid.

[0018] According to some embodiments of any aspect or embodiment of this specification, the LNP composition is prepared in a solution having about 30 mM to about 50 mM NaCl, for example, 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 aspect or embodiment of this specification, 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, the LNP composition is prepared in a solution having about 40 mM NaCl.

[0019] According to some embodiments, LNP compositions contain about 20 mM to about 100 mM MgCl2, for example, about 20 mM to about 90 mM MgCl2, about 20 mM to about 80 mM MgCl2, about 20 mM to about 70 mM MgCl2, about 20 mM to about 60 mM MgCl2, about 20 mM to about 50 mM MgCl2, about 20 mM to about 40 mM MgCl2, approximately 20 mM to approximately 30 mM MgCl2, approximately 320 mM to approximately 90 mM MgCl2, approximately 30 mM to approximately 80 mM MgCl2, approximately 30 mM to approximately 70 mM MgCl2, approximately 30 mM to approximately 60 mM MgCl2, approximately 30 mM to approximately 50 mM MgCl2, approximately 30 mM to approximately 40 mM MgCl2, approximately 40 MgCl2 of approximately 1 / 5 mM, approximately 40 mM to approximately 80 mM, approximately 40 mM to approximately 70 mM, approximately 40 mM to approximately 60 mM, approximately 40 mM to approximately 50 mM, approximately 50 mM to approximately 90 mM, approximately 50 mM to approximately 80 mM, approximately 50 mM to approximately 70 mM It is prepared in a solution containing approximately 50 mM to 60 mM MgCl2, approximately 60 mM to 90 mM MgCl2, approximately 60 mM to 80 mM MgCl2, approximately 60 mM to 70 mM MgCl2, approximately 70 mM to 90 mM MgCl2, approximately 70 mM to 80 mM MgCl2, or approximately 80 mM to 90 mM MgCl2.

[0020] According to some embodiments of any aspect or embodiment of this specification, ceDNA is a closed-end linear double-stranded DNA. According to some embodiments of any aspect or embodiment of this specification, ceDNA includes an expression cassette comprising a promoter sequence and a transgene.

[0021] According to some embodiments, the ceDNA includes an expression cassette containing a polyadenylated sequence.

[0022] According to some embodiments of any aspect or embodiment of this specification, the ceDNA includes at least one inverted end repeat (ITR) adjacent to either the 5' or 3' end of the expression cassette. According to some embodiments, the expression cassette is adjacent to two ITRs, the two ITRs comprising one 5'ITR and one 3'ITR. According to some embodiments, the expression cassette is ligated to an ITR (3'ITR) at its 3' end. According to some embodiments, the expression cassette is ligated to an ITR (5'ITR) at its 5' end. According to some embodiments, at least one of the 5'ITR and 3'ITR is a wild-type AAV ITR. According to some embodiments, at least one of the 5'ITR and 3'ITR is a modified ITR. According to some embodiments, the ceDNA further includes a spacer sequence between the 5'ITR and the expression cassette.

[0023] According to some embodiments, the ceDNA further includes a spacer sequence between the 3'ITR and the expression cassette. According to some embodiments, the spacer sequence is at least 5 base pairs long. According to some embodiments, the spacer sequence is at least 5 to 100 base pairs long. According to some embodiments, 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 long. According to some embodiments, the spacer sequence is at least 5 to 500 base pairs long. According to some embodiments, the spacer array has lengths of 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, The lengths are 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.

[0024] According to some embodiments of any aspect or embodiment of this specification, the ceDNA has nicks or gaps.

[0025] According to some embodiments, the ITR is an ITR derived from an AAV serotype, an ITR derived from a goose virus, an ITR derived from a B19 virus, and a wild-type ITR from a parvovirus. According to some embodiments, the AAV serotype is selected from the group including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0026] According to some embodiments, the ITR is a mutant ITR, and the ceDNA optionally includes an additional ITR different from the first ITR. According to some embodiments, the ceDNA includes 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 aspect or embodiment of this specification, the ceDNA is CELiD, DNA-based minicircle, MIDGE, ministering DNA, 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. According to some embodiments of any aspect or embodiment of this specification, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0027] According to some embodiments, the Disclosure provides a method for treating a genetic disorder in a subject, the method comprising administering an effective amount of a pharmaceutical composition according to any aspect or embodiment of this Spec. According to some embodiments, the subject is a human. According to some embodiments, hereditary disorders include sickle cell anemia, melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency) and hemophilia B (coagulation factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary hepatic metabolic disorders, Lesch-Neyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Schayet syndrome (MPS type IS), Hurler-Scheyet syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS III) (Types A, B, C, and D), Morquio A and B (MPS IVA and MPS IVB), Maloto-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick disease A / B, C1 and C2, Fabry disease, Schindler's disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis I, II / III and IV, sialidosis I and II, glycogen storage disorder I and II (Pompe disease), Gaucher disease I, II and III, cystinosis, Batten disease, aspartylglucosamiuria, 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, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophy of epidermolysis bullosa (DEB),The genetic disorder is selected from the group consisting of ectonucleotide pyrophosphatase 1 deficiency, systemic arterial calcification in infancy (GACI), Leber congenital amaurosis (LCA), 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, the genetic disorder is Leber congenital amaurosis (LCA). According to some embodiments, LCA is LCA10. According to some embodiments, the genetic disorder is Niemann-Pick disease. According to some embodiments, the genetic disorder is Stargardt macular dystrophy. According to some embodiments, the hereditary disorder is glucose-6-phosphatase (G6Pase) deficiency (glycogen storage disorder type I) or Pompe disease (glycogen storage disorder type II). According to some embodiments, the hereditary disorder is hemophilia A (factor VIII deficiency). According to some embodiments, the hereditary disorder is hemophilia B (factor IX deficiency). According to some embodiments, the hereditary disorder is Hunter syndrome (mucopolysaccharidosis type II). According to some embodiments, the hereditary disorder is cystic fibrosis. According to some embodiments, the hereditary disorder is Usher syndrome. According to some embodiments, the hereditary disorder is epidermolysis bullosa dystrophy (DEB). According to some embodiments, the hereditary disorder is phenylketonuria (PKU). According to some embodiments, the hereditary disorder is progressive familial intrahepatic cholestasis (PFIC). According to some embodiments, the hereditary disorder is Wilson's disease. According to some embodiments, the hereditary disorder is Gaucher disease type I, II, or III. ,

[0028] The embodiments of this disclosure, briefly summarized above and discussed in more detail below, can be understood by referring to exemplary embodiments of this disclosure depicted in the accompanying drawings. However, the accompanying drawings only illustrate typical embodiments of this disclosure and should not be considered limiting in scope, as this disclosure may allow for other equally valid embodiments. [Brief explanation of the drawing]

[0029] [Figure 1] As observed in preclinical studies (dose = 0.25 mg / kg), ceDNA-luciferase expression on day 4 is shown, achieved by using lipid nanoparticles LNP2 and LNP3 formulated with lipid 1 and lipid 3, respectively, as the delivery vehicle, compared to LNP1 formulated with reference lipid A (positive control) and DPBS (negative control). [Modes for carrying out the invention]

[0030] This disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs), such as viral or nonviral 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 therapies has limited the number of patients that can be treated due to existing background immunities and has prevented patient re-administration to titrate to effective levels for each patient or to maintain efficacy over the long term. Furthermore, other nucleic acid modalities are greatly plagued by immunogenicity due to innate DNA or RNA sensing mechanisms that trigger a cascade of immune responses. Due to the lack of existing immunities, 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, including pediatric populations that may require subsequent administrations as tissues grow. Furthermore, it is a finding of this disclosure that lipid compositions containing one or more tertiary amino groups, and TNA lipid particles (e.g., lipid nanoparticles) containing disulfide bonds, provide more efficient delivery of TNA (e.g., ceDNA), better tolerability, and an improved safety profile. Since the TNA lipid particles (e.g., lipid nanoparticles) described herein are not subject to the packaging constraints imposed by space within the viral capsid, theoretically, the only size limitation of TNA lipid particles (e.g., lipid nanoparticles) lies in the efficiency of host cell expression (e.g., DNA replication or RNA translation).

[0031] One of the biggest hurdles in developing therapies, particularly for rare diseases, is the sheer number of individual conditions. Approximately 350 million people worldwide live with rare disorders, and the National Institutes of Health defines rare disorders as those diagnosed in fewer than 200,000 people. About 80% of these rare disorders are of genetic origin, and about 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 the ability to rapidly adapt to multiple diseases that can be treated with specific modalities of TNA, particularly rare monogenic disorders, which could significantly alter the current state of treatment for many genetic disorders or diseases.

[0032] I. Definition The term "alkyl" refers to a monovalent saturated linear or branched hydrocarbon group. 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 others.

[0033] The term "alkylene" refers to a divalent saturated linear or branched hydrocarbon group, and examples include, but are not limited to, those having the same core structure as the alkyl groups exemplified above.

[0034] The term "alkenyl" refers to a linear or branched aliphatic hydrocarbon group having one or more (e.g., one or two) carbon-carbon double bonds, and alkenyl groups include groups having "cis" and "trans" orientations, or, according to other nomenclature, "E" and "Z" orientations.

[0035] As used herein, the term “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt of the ionizable lipids of the present invention. Examples of salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, acidic tartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, sugarates, formates, benzoates, glutamates, methanesulfonates "mesylates", ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), 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 counterions. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. If multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0036] As used herein and in the appended claims, the term “about” means, when referring to a measurable value such as quantity or temporal duration, to include a variation 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, in order to be appropriate for carrying out the disclosed method.

[0037] As used herein, “comprise,” “comprising,” “comprises,” and “comprised of” are synonymous with “include,” “including,” “includes,” or “contain,” “containing,” and “contains,” and are, for example, comprehensive or free-form terms that specify the presence of a component followed by another component, and do not exclude or preclude the presence of additional, unreferenced components, features, elements, members, or steps that are known or disclosed in the art.

[0038] The term "consisting of" refers to the compositions, methods, processes, and their respective components described herein, excluding any elements not enumerated in the description of the embodiments.

[0039] As used herein, the term “essentially derived from” refers to elements necessary for a given embodiment. This term allows for the presence of additional elements that do not materially affect the basic, novel, or functional features of the embodiments of the present invention.

[0040] As used herein, the terms “administer,” “to administer,” and variations thereof mean the introduction of a composition or drug (e.g., nucleic acids, particularly ceDNA) into a subject, including the simultaneous and sequential introduction of one or more compositions or drugs. “Administer” may mean, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administer” also encompasses in vitro and ex vivo treatments. Introduction of a composition or drug into a subject may be by any preferred route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another person. Administration may be carried out by any preferred route. A preferred route of administration allows the composition or drug to perform its intended function. For example, if the preferred route is intravenous, the composition is administered by introducing the composition or drug into a vein of the subject. In one embodiment, “administer” means therapeutic administration.

[0041] As used herein, phrases such as “anti-therapeutic nucleic acid immune response,” “anti-transfer vector immune response,” “immune response to therapeutic nucleic acid,” and “immune response to transfer vector” refer to any undesirable immune response to therapeutic nucleic acid, whether viral or nonviral in origin. In some embodiments, the undesirable immune response is an antigen-specific immune response to the viral transfer vector itself. In some embodiments, the immune response is specific to the transfer vector, which may be double-stranded DNA, single-stranded 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.

[0042] As used herein, the terms “carrier” and “excipient” mean all optional solvents, dispersions, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Complementary active ingredients may also be incorporated into the composition. The phrase “pharmaceutically acceptable” means molecular entities and compositions that, when administered to a host, do not produce a toxic, allergic, or similarly undesirable reaction.

[0043] As used herein, the term “ceDNA” means capsid-free closed-end linear double-stranded (ds) double-stranded DNA for synthesis or other nonviral gene transfer. A detailed description of ceDNA is provided in International Patent Application US2017 / 020828, filed March 3, 2017, which is expressly incorporated herein by reference in its entirety. Certain methods for producing ceDNA containing various inverted-end repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application 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 Patent Application PCT / US2019 / 14122, filed on January 18, 2019, the entire contents of which are incorporated herein by reference. Where used herein, the terms “ceDNA vector” and “ceDNA” are interchangeable. According to some embodiments, ceDNA is a closed-end linear double-stranded (CELiD) CELiD DNA. According to some embodiments, ceDNA is a DNA-based minicircle. According to some embodiments, ceDNA is a minimal immunologically defined gene expression (MIDGE) vector. According to some embodiments, ceDNA is ministering DNA. According to some embodiments, ceDNA is a dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITR at the 5' and 3' ends of the expression cassette. According to some embodiments, ceDNA is doggybone® DNA.

[0044] As used herein, the term “ceDNA-bacmid” means an infectious baculovirus genome that contains a ceDNA genome as an intermolecular double helix that can be propagated as a plasmid in E. coli, thereby enabling it to function as a baculovirus shuttle vector.

[0045] As used herein, the term "ceDNA-baculovirus" means a baculovirus that contains the ceDNA genome as an intermolecular double helix within the baculovirus genome.

[0046] As used herein, the terms “ceDNA-baculovirus-infected insect cells” and “ceDNA-BIIC” are interchangeable and mean invertebrate host cells (including, but not limited to, insect cells (e.g., Sf9 cells)) infected with ceDNA-baculovirus.

[0047] As used herein, the term “ceDNA genome” means an expression cassette further incorporating at least one inverted terminal repeat region. The ceDNA genome may further include one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0048] As used herein, the terms “DNA regulatory sequence,” “regulatory element,” and “regulatory element” are interchangeable herein and mean transcriptional and translational regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., which provide and / or regulate the transcription of non-coding sequences (e.g., DNA-targeted RNA) or coding sequences (e.g., site-directed modified polypeptides or Cas9 / Csn1 polypeptides) and / or regulate the translation of encoded polypeptides.

[0049] As used herein, the term “exogenous” means a substance present in a cell other than its natural source. As used herein, “exogenous” may mean a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide introduced into a biological system such as a cell or organism by a human-involved process, where it is not normally found and it is desirable to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” may mean a nucleic acid or polypeptide introduced into a biological system such as a cell or organism by a human-involved process, where it is found in relatively small amounts and it is desirable to increase the amount of nucleic acid or polypeptide in the cell or organism, for example, to result in ectopic expression or levels. In contrast, as used herein, the term “endogenous” means a substance that is natural to a biological system or cell.

[0050] As used herein, the term “expression” means cellular processes involved in the production of RNA and proteins, and, as appropriate, secreted proteins, including but not limited to, transcription, transcriptional processing, translation, and protein folding, modification, and processing. As used herein, the term “expression product” includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0051] As used herein, the term “expression vector” means a vector that directs the expression of RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often heterologous to the host cell, but not necessarily. An expression vector may contain additional elements; for example, an expression vector may have two replication systems so that it can be maintained in two organisms, such as human cells in the case of expression and a prokaryotic host in the case of cloning and amplification. An expression vector may also be a recombinant vector.

[0052] As used herein, the terms “expression cassette” and “expression unit” mean a heterologous DNA sequence that is interchangeable and manipulably ligated to a promoter or other DNA regulatory sequence sufficient to direct the transcription of a transgene in a DNA vector, such as a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. Promoters may also be of AAV origin.

[0053] As used herein, the term “adjacent” refers to the relative position of one nucleic acid sequence to another nucleic acid sequence. Generally, in sequence ABC, A and C are adjacent to B on both sides. The same is true for arrangement AxBxC. Thus, an adjacent sequence may precede or follow the adjacent sequence, but it does not need to be continuous with or immediately adjacent to the adjacent sequence. In one embodiment, the term adjacent refers to terminal repeats at each end of a linear single-stranded synthetic AAV vector.

[0054] As used herein, the term “gene” is used broadly to refer to any segment of nucleic acid associated with the expression of a given RNA or protein in vitro or in vivo. Thus, a gene includes a region encoding the expressed RNA (usually containing a polypeptide coding sequence) and, often, regulatory sequences necessary 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.

[0055] As used herein, the terms “hereditary disorder” or “hereditary disorder” mean a disorder caused, directly or indirectly, partially or completely, by one or more abnormalities in the genome, particularly a condition present from birth. The abnormality may be a mutation, insertion, or deletion in a gene. The abnormality may affect the coding sequence or its regulatory sequence of a gene.

[0056] As used herein, the term “heterogeneous” means a nucleotide or polypeptide sequence not found in naturally occurring nucleic acids or proteins, respectively. Heterogeneous nucleic acid sequences can be ligated (e.g., by genetic engineering) to naturally occurring nucleic acid sequences (or their variants) to produce chimeric nucleotide sequences encoding chimeric polypeptides. Heterogeneous nucleic acid sequences can be ligated (e.g., by genetic engineering) to variant polypeptides to produce nucleotide sequences encoding fusion variant polypeptides.

[0057] As used herein, the term “host cell” refers to any cell type that is susceptible to transformation, transfection, transduction, etc., by the nucleic acid therapeutics of this disclosure. Non-limiting examples include isolated primary cells, pluripotent stem cells, CD34 + The host cell may be a cell, an induced pluripotent stem cell, or one of several immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be an in situ or in vivo cell in a tissue, organ, or organism. Furthermore, the host cell may be a target cell of a mammalian subject (e.g., a human patient requiring gene therapy).

[0058] Where used herein, “inducible promoter” means one characterized by initiating or enhancing transcriptional activity in the presence of, being influenced by, or being contacted by an inducer or inducer. Where used herein, “inducer” or “inducer” may be endogenous or typically exogenous compounds or proteins administered in a manner that is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducer, i.e., a chemical, compound, or protein, may itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer may be an inducer protein expressed by another component or module) and may itself be under the control of an inducible promoter. In some embodiments, the inducible promoter is induced in the absence of certain agents, such as repressors. Examples of inducible promoters include, but are not limited to, tetracycline, metallothione, ecdysone, mammalian viruses (e.g., the late promoter of adenovirus and the long terminal repeat (MMTV-LTR) of mouse mammary tumor virus), and other steroid-responsive promoters, rapamycin-responsive promoters, etc.

[0059] As used herein, the term "in vitro" means assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and may mean introducing a programmable synthetic biological circuit into a non-cellular system, such as a medium that does not contain cells or cell systems, such as cell extracts.

[0060] As used herein, the term “in vivo” means an assay or process that occurs in or within an organism, such as a multicellular animal. In some embodiments described herein, a method or use may be said to occur “in vivo” when a single-celled organism, such as a bacterium, is used. The term “ex vivo” means a method or use carried out using a multicellular animal or plant, such as, in particular, an explant, cultured cells (including primary cells and cell lines), transformed cell lines, and living cells with intact membranes outside of an extracted tissue or cell (including blood cells).

[0061] As used herein, the term “lipid” refers to a group of organic compounds including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three classes: (1) “simple lipids” including fats, oils, and waxes; (2) “complex lipids” including phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0062] Typical examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other phosphorus-deficient compounds such as sphingolipids, the sphingoglycolipid family, diacylglycerols, and β-acyloxy acids are also included in the group called amphiphilic lipids. In addition, the above amphiphilic lipids can be mixed with other lipids, including triglycerides and sterols.

[0063] In one embodiment, the lipid composition comprises one or more tertiary amino groups, one or more phenyl ester bonds, and a disulfide bond.

[0064] As used herein, the term “lipid conjugate” means a conjugated lipid that inhibits the aggregation of lipid particles (e.g., lipid nanoparticles). Such lipid conjugates include, but are not limited to, PEGylated lipids such as PEG coupled to dialkyloxypropyl (e.g., PEG-DAA conjugate), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugate), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, for example, U.S. Patent No. 5,885,613), ionizable PEGylated lipids, polyoxazoline (POZ)-lipid conjugates, and mixtures thereof.

[0065] As used herein, the term “lipid-encapsulated” means lipid particles that provide an activator or therapeutic agent, such as nucleic acids (e.g., ASO, mRNA, siRNA, ceDNA, viral vectors), in a fully encapsulated, partially encapsulated, or both manner. In preferred embodiments, the nucleic acid is fully encapsulated within the lipid particle (e.g., to form a lipid particle containing the nucleic acid).

[0066] As used herein, the terms “lipid particles” or “lipid nanoparticles” refer to lipid formulations that can be used to deliver therapeutic agents, such as nucleic acid therapeutics (TNAs), to target sites of interest (e.g., cells, tissues, organs, etc.) (referred to as “TNA lipid particles,” “TNA lipid nanoparticles,” or “TNA LNPs”). In one embodiment, the lipid particles of the present invention are therapeutic nucleic acid-containing lipid particles, which are typically formed from ionizable lipids, noncationic lipids, and conjugated lipids that optionally prevent particle aggregation. In other preferred embodiments, therapeutic agents, such as therapeutic nucleic acids, can be encapsulated in the lipid portion of the particles, thereby protecting them from enzymatic degradation. In one embodiment, the lipid particles comprise nucleic acids (e.g., ceDNA) and lipids containing one or more tertiary amino groups, one or more phenyl ester bonds, and disulfide bonds.

[0067] The lipid particles of the present invention are typically approximately 20 nm to approximately 120 nm, approximately 30 nm to approximately 150 nm, approximately 40 nm to approximately 150 nm, approximately 50 nm to approximately 150 nm, approximately 60 nm to approximately 130 nm, approximately 70 nm to approximately 110 nm, approximately 70 nm to approximately 100 nm, approximately 80 nm to approximately 100 nm, approximately 90 nm to approximately 100 nm, approximately 70 nm to approximately 90 nm, approximately 80 nm to approximately 90 nm, approximately 70 nm to approximately 80 nm, or approximately It has an average diameter of 30nm, approximately 35nm, approximately 40nm, approximately 45nm, approximately 50nm, approximately 55nm, approximately 60nm, approximately 65nm, approximately 70nm, approximately 75nm, approximately 80nm, approximately 85nm, approximately 90nm, approximately 95nm, approximately 100nm, approximately 105nm, approximately 110nm, approximately 115nm, approximately 120nm, approximately 125nm, approximately 130nm, approximately 135nm, approximately 140nm, approximately 145nm, or approximately 150nm.

[0068] As used herein, the term "hydrophobic lipid" refers to compounds having nonpolar groups, 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. Preferred examples include, but are not limited to, diacylglycerol, dialkylglycerol, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0069] As used herein, the term “ionizable lipid” means a lipid having at least one protonable or deprotonable group such that the lipid is positively charged below the physiological pH (e.g., pH 7.4) and neutral above a second pH, preferably above the physiological pH, such as a cationic lipid. 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 that references to charged or neutral lipids refer to the properties of the dominant species, and that not all lipids need to exist in charged or neutral forms. Generally, ionizable lipids have a pKa of protonable groups in the range of about 4 to about 7. In some embodiments, ionizable lipids may include “cleavable lipids” or “SS-cleavable lipids.”

[0070] As used herein, the term “neutral lipid” means any of the many lipid species that exist 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, cerebroside, and diacylglycerol.

[0071] 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 bound to neutral lipids.

[0072] As used herein, the term “noncationic lipid” means any amphiphilic lipid and any other neutral or anionic lipid.

[0073] As used herein, the terms “cleavable lipid” or “SS cleavable lipid” refer to lipids containing cleavable units of disulfide bonds. In one embodiment, the cleavable lipid comprises a tertiary amine that responds to disulfide bonds that can be cleaved in reducing environments such as acidic compartments, e.g., endosomes or lysosomes for membrane destabilization, and the cytoplasm. In one embodiment, the cleavable lipid is an ionizable lipid. In one embodiment, the cleavable lipid is a cationic lipid. In one embodiment, the cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

[0074] As used herein, the term "organic lipid solution" means a composition containing an organic solvent that is entirely or partially lipid-containing.

[0075] As used herein, the term “liposome” refers to a lipid molecule assembled in a spherical structure that encapsulates an internal aqueous volume separated from an aqueous external environment. A liposome is a vesicle having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of formulation development. They act by fusing with the cell membrane and repositioning its lipid structure to deliver the drug or active formulation component. Liposome compositions for such delivery are typically composed of phospholipids, particularly compounds having a phosphatidylcholine group, although these compositions may also contain other lipids.

[0076] As used herein, the term “local delivery” means the direct delivery of an activator, such as interfering RNA (e.g., siRNA), to a target site within a living organism. For example, a drug can be delivered locally by direct injection to a disease site such as a tumor, or to another target site such as an inflammatory site, or to a target organ such as the liver, heart, pancreas, or kidney.

[0077] As used herein, the terms “neDNA” or “nicked ceDNA” refer to closed-end DNA having a nick or gap of 2 to 100 base pairs in the stem or spacer region 5' upstream of an open reading frame (e.g., the promoter and transgene being expressed).

[0078] As used herein, the term “nucleic acid” means a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be, for example, in the form of antisense molecules, plasmid DNA, DNA-DNA double helix, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-end linear double-strand DNA (CELiD or ceDNA), doggybone® DNA, dumbbell DNA, minimal immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include those containing known nucleotide analogs or modified backbone residues or ligatures, which are synthetic, naturally occurring, and non-natural, and possess similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholino), phosphoramides, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, locked nucleic acids (LNA®), and peptide nucleic acids (PNA). Unless otherwise limited, this term encompasses nucleic acids containing known analogs of naturally occurring nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise specified, any given nucleic acid sequence implicitly includes its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as any explicitly indicated sequences.

[0079] As used herein, the terms “nucleic acid therapeutic agent,” “therapeutic nucleic acid,” and “TNA” are interchangeable and refer to any modality of therapy that uses nucleic acids as the active ingredient of a therapeutic agent for treating a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-exclusive examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-exclusive examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) 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), and dumbbell-shaped minimal DNA vectors ("dumbbell DNA"). As used herein, the term "TNA LNP" refers to lipid particles containing at least one TNA, as described above.

[0080] As used herein, “nucleotide” comprises a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups.

[0081] As used herein, “operably linked” means a parallel relationship in which components described in this way are allowed to function in the manner intended. For example, if a promoter affects its transcription or expression, the promoter is operably linked to the coding sequence. A promoter can be said to drive the expression or transcription of the nucleic acid sequence it regulates. The terms “operably linked,” “operably positioned,” “operably linked,” “controlled,” and “transcriptionally controlled” indicate that the promoter is in the correct functional position and / or orientation with respect to the nucleic acid sequence and is regulated to control the transcription initiation and / or expression of that sequence. As used herein, “inverted promoter” refers to a promoter in which the nucleic acid sequence is reversed in orientation, so 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 the switch. In addition, in various embodiments, the promoter can be used in conjunction with an enhancer.

[0082] As used herein, the term “promoter” means any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inductive, repressive, tissue-specific, or any combination thereof. A promoter is a regulatory region of a nucleic acid sequence that controls the initiation and rate of the rest of the transcription of the nucleic acid sequence. Promoters may also contain gene elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Within a promoter sequence, a transcription start site and a protein-binding domain involved in RNA polymerase binding will be found. Eukaryotic promoters often, though not necessarily, contain “TATA” and “CAT” boxes. Various promoters, including inductive promoters, can be used to drive the expression of a transgene in the synthetic AAV vectors disclosed herein. A promoter sequence is bound at its 3' end by a transcription start site and extends upstream (5' orientation) to contain the minimum number of bases or elements necessary to initiate transcription at a detectable level above the background.

[0083] A promoter may be naturally associated with a gene or sequence, and can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon of a given gene or sequence. Such a promoter may be called “endogenous.” Similarly, in some embodiments, an enhancer may be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments, the coding nucleic acid segment is positioned under the control of a “recombinant promoter” or “heterogeneous promoter,” both of which refer to promoters not typically associated with an encoded nucleic acid sequence that is manipulably linked in its natural environment. Similarly, “recombinant or heterogeneous enhancer” refers to an enhancer not typically associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers may 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 present,” i.e., 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 the synthetic production of promoter and enhancer nucleic acid sequences, promoter sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, with respect to the synthetic biological circuits and modules disclosed herein (see, for example, U.S. Patents 4,683,202 and 5,928,906, each incorporated herein by reference in its entirety). Furthermore, regulatory sequences that orient the transcription and / or expression of sequences in non-nuclear organelles such as mitochondria and chloroplasts may be used in a similar manner.

[0084] As used herein, “Rep-binding site” (“RBS”) and “Rep-binding element” (“RBE”) are interchangeable and refer to a binding site for a Rep protein (e.g., AAV Rep78 or AAV Rep68), which, upon binding by the Rep protein, enables the Rep protein to perform its site-specific endonuclease activity on the sequence incorporating the RBS. The 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'.

[0085] As used herein, the term “recombinant vector” means 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. In some embodiments, the vector is an episome. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in a subject in high copy number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal incorporation.

[0086] As used herein, the term “reporter” means 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 readily observable.

[0087] As used herein, the terms “sense” and “antisense” refer to the orientation of structural elements on a polynucleotide. The sense and antisense versions of an element are inverse complements of each other.

[0088] As used herein, the term “sequence identity” refers to the relationship between two nucleotide sequences. For the purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, above), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, above), preferably version 3.0.0 or later. Optional parameters used are a gap-open penalty of 10, a gap-expand penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled “Longest Identity” (obtained using the -nobrief option) is used as an identity percentage and is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment - total number of gaps in alignment). The alignment length is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0089] As used herein, the term “spacer region” means an intervening sequence that separates functional elements within a vector or genome. In some embodiments, an AAV spacer region holds two functional elements in a desired treatment for optimal functionality. In some embodiments, the spacer region provides or increases the genetic stability of the vector or genome. In some embodiments, the spacer region facilitates easy genetic manipulation of the genome by providing a convenient location for cloning sites and gaps in base pair design numbers. For example, in certain embodiments, cis-acting factors can be separated by positioning oligonucleotides “polylinkers” or “polycloning sites” containing several restriction endonuclease sites, or non-open reading frame sequences designed not to have known protein (e.g., transcription factor) binding sites, within the vector or genome, for example, by inserting 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc.

[0090] As used herein, the term “subject” means a human or animal to which a treatment, including prophylactic treatment with therapeutic nucleic acids according to the present invention, is provided. Typically, animals are vertebrates such as primates, rodents, domesticated animals, or game animals, but are not limited to these. Examples of primates include, but are not limited to, chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, e.g., domestic cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, ostriches, and fish such as 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, the subject may be a neonatal or fetal subject, for example, the subject is in the womb. Preferably, the subject is a mammal. Mammals may be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. 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 in domesticated animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, for example, Caucasian (white), Asian, African, Black, African American, Afro-European, Latin American, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already receiving treatment. In some embodiments, the subject is an embryo, fetus, neonatal, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonatal, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.

[0091] As used herein, the term “subjects requiring treatment” means, unless the context and usage of the term indicate otherwise, (i) subjects who are to be administered TNA lipid particles (or pharmaceutical compositions containing TNA lipid particles) in accordance with the invention described herein, (ii) subjects who are receiving TNA lipid particles (or pharmaceutical compositions containing TNA lipid particles) in accordance with the invention described herein, or (iii) subjects who have received TNA lipid particles (or pharmaceutical compositions containing TNA lipid particles) in accordance with the invention described herein.

[0092] As used herein, the terms “suppress,” “reduce,” “interfere,” “inhibit,” and / or “reduce” (and similar terms) generally refer to the act of directly or indirectly reducing concentration, level, function, activity, or behavior to natural, expected, or average, or to control conditions.

[0093] As used herein, the terms “synthetic AAV vector” and “synthetic production of AAV vector” refer to AAV vectors and methods for their synthetic production in a completely cell-free environment.

[0094] As used herein, the term “systemic delivery” refers to the delivery of lipid particles that result in widespread biodistribution of an active agent, such as interfering RNA (e.g., siRNA), within a living organism. Depending on the administration technique, systemic delivery of a particular drug may or may not be achieved. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the drug is exposed to most parts of the body. To achieve widespread biodistribution, the drug generally requires a blood lifetime such that it is not rapidly degraded or excreted (by first-pass organs (liver, lungs, etc.) or by rapid nonspecific cell 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 delivery. In preferred embodiments, systemic delivery of lipid particles (e.g., lipid nanoparticles) is by intravenous delivery.

[0095] As used herein, the terms “end-resolved site” and “TRS” are interchangeable herein and mean that Rep refers to a region that forms a tyrosine-phosphodiester bond with 5'-thymidine, which generates a 3'-OH that serves as a substrate for DNA elongation via cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex may be involved in coordination ligation reactions.

[0096] Where used herein, the terms “therapeutic dose,” “therapeutic effective dose,” “effective dose,” “effective dose,” or “pharmaceutically effective dose” of an activator (e.g., TNA lipid particles as described herein) are interchangeable and refer to an amount sufficient to provide the intended therapeutic benefit or effect, e.g., inhibition of the expression of a target sequence compared to the expression level detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include, for example, protein or RNA level testing using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art. Dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, the patient’s condition, the severity of the condition, the route of administration, and the specific activator used. Therefore, the dosage plan may vary considerably but can be routinely determined by a physician using standard methods. Additionally, the terms “therapeutic dose,” “therapeutic effective dose,” and “pharmaceutically effective dose” include prophylactic or preventative doses of the compositions of the present invention as described herein. In the prophylactic or preventative uses of the described invention, the pharmaceutical composition or agent is administered in an amount sufficient to eliminate or reduce risk, decrease severity, or delay the onset of the disease, disorder, or condition to patients who are susceptible to or otherwise at risk of the disease, disorder, or condition, including the biochemical, histological and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes that appear during the onset of the disease, disorder, or condition. According to some medical judgment, it is generally preferable to use the maximum dose, i.e., the highest safe dose. The terms “dose” and “administered dose” are used interchangeably herein. In one embodiment, “therapeutic dose,” “therapeutic effective dose,” and “pharmaceutical effective dose” refer to non-prophylactic or non-preventative uses.

[0097] As used herein, the term “therapeutic effect” refers to the outcome of treatment, which is deemed desirable and beneficial. A therapeutic effect may include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. A therapeutic effect may also include, directly or indirectly, the prevention, reduction, or elimination of disease progression.

[0098] For any therapeutic agent described herein, the effective therapeutic dose can first be determined from preliminary in vitro studies and / or animal models. The effective therapeutic dose can also be determined from human data. The dose applied can be adjusted based on the relative bioavailability and potency of the compound administered. Adjusting the dose to achieve maximum potency based on the above methods and other well-known methods is within the capabilities of those skilled in the art. General principles for determining therapeutic efficacy, as can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th Edition, McGraw-Hill (New York) (2001), which are incorporated herein by reference, are summarized below.

[0099] Pharmacokinetic principles provide a basis for modifying dosing regimens to achieve the desired level of therapeutic effect while minimizing unacceptable side effects. Plasma drug concentrations can be measured, and in situations related to therapeutic concentration ranges, additional guidance on dosage adjustments can be obtained.

[0100] As used herein, “to treat,” “to treat,” and / or “treatment” include inhibiting, inhibiting, delaying, or reversing the progression of a condition, improving the clinical symptoms of a condition, or preventing the appearance of clinical symptoms, or obtaining a beneficial or desirable clinical outcome. Treatment further means achieving one or more of the following: (a) reducing the severity of the disability; (b) limiting the onset of symptoms characteristic of the disability being treated; (c) limiting the exacerbation of symptoms characteristic of the disability being treated; (d) limiting the recurrence of the disability in patients who previously had the disability; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic with respect to the disability. In one embodiment, “to treat,” “to treat,” and / or “treatment” include inhibiting, inhibiting, delaying, or reversing the progression of a condition, or improving the clinical symptoms of a condition.

[0101] Beneficial or desired clinical outcomes, such as pharmacological and / or physiological effects, include, but are not limited to, preventing the onset of a disease, disorder, or condition (preventive treatment) in subjects who may be predisposed to the disease, disorder, or condition but have not yet experienced or exhibited symptoms of the disease; alleviating symptoms of the disease, disorder, or condition; reducing the severity of the disease, disorder, or condition; stabilizing the disease, disorder, or condition (i.e., preventing exacerbation); preventing the spread of the disease, disorder, or condition; slowing or delaying the progression of the disease, disorder, or condition; improving or reducing the disease, disorder, or condition; and combinations thereof; as well as extending survival compared to the survival expected without treatment.

[0102] The terms “vector” or “expression vector” refer to a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, that can be bound to another DNA segment, i.e., an “insert,” “transgene,” or “expression cassette,” in order to result in the expression or replication of the bound segment (“expression cassette”) in a cell. A vector may be a nucleic acid construct designed for delivery to a host cell or for movement between different host cells. As used herein, a vector may 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 can replicate and transfer a gene sequence to a cell when associated with appropriate regulatory elements. In some embodiments, a vector may be a recombinant vector or an expression vector.

[0103] The grouping of alternative elements or embodiments of the present invention disclosed herein should not be construed as limitation. 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 removed from a group for convenience and / or patentability reasons. In the event of any such inclusion or removal, the specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0104] In some embodiments of the embodiments described herein, the disclosures described herein do not relate to human cloning processes, processes for correcting the genetic identity of human germline cells, 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, or processes for correcting the genetic identity of animals resulting from such processes.

[0105] Other terms are defined within the description of various aspects of the present invention as specified herein.

[0106] II. Lipids In the first chemical embodiment, an ionizable lipid of formula (I): [ka] , Or a pharmaceutically acceptable salt thereof is provided, in the formula, R 1 and R 1 ' are each independent of R a A (C1-C6) alkylene that is optionally substituted with one or more groups selected from the following: R 2 and R 2 ' are each independent of (C 1~ C2) It is an alkylene, R 3 and R 3 ' are each independent of R b A (C1-C6) alkyl group that is optionally substituted with one or more groups selected from the following: Alternatively, R 2 and R 3 and / or R 2 'and R 3 ', together with the intervening N atoms, form 4- to 7-membered heterocyclines. R 4 and R 4 ' are (C2~C6) alkylenes interrupted by -C(O)O-, R 5 and R 5 ' are independent of each other, (C2~C 30 ) Alkyl or (C2~C 30 ) are alkenyls, each of which can be optionally terminated with -C(O)O- or (C3~C6)cycloalkyl groups. R a and R b These are either halo or cyano, respectively.

[0107] In a second chemical embodiment, R in an ionizable lipid of formula (I) 1 and R 1’ Each of these is independently an alkylene (C1-C6), and the remaining variables are as described above for formula (I). Alternatively, as part of a second chemical embodiment, R in the ionizable lipid of formula (I) 1 and R 1 ' are (C1~C3) alkylenes, each independently, and the remaining variables are as described above for equation (I).

[0108] In a third chemical embodiment, the ionizable lipid of formula (I) is Formula (II): [ka] , The substance or a pharmaceutically acceptable salt thereof, where the remaining variables are as described above for formula (I).

[0109] In the fourth chemical embodiment, the ionizable lipid of formula (I) is formula (III) or (IV): [ka] , The substance or a pharmaceutically acceptable salt thereof, where the remaining variables are as described above for formula (I).

[0110] In the fifth embodiment, the ionizable lipid of formula (I) is formula (V) or (VI): [ka] , The substance or a pharmaceutically acceptable salt thereof, where the remaining variables are as described above for formula (I).

[0111] In the sixth embodiment, the ionizable lipid of formula (I) is formula (VII) or (VIII): [ka] The substance or a pharmaceutically acceptable salt thereof, where the remaining variables are as described above for formula (I).

[0112] In the seventh chemical embodiment, R in an ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 (C6~C 26 ) Alkyl or (C6~C 26 ) are alkenyls, each of which can be optionally terminated with -C(O)O- or (C3-C6) cycloalkyl, and the remaining variables are as described above for formula (I). Alternatively, as part of the seventh chemical embodiment, R in ionizable lipids of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 (C6~C 26 ) Alkyl or (C6~C 26 ) are alkenyls, each of which can be optionally terminated with -C(O)O- or (C3-C5) cycloalkyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in ionizable lipids of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 (C7~C 26 ) Alkyl or (C7~C 26 ) are alkenyls, each of which can be optionally terminated with -C(O)O- or (C3-C5) cycloalkyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in ionizable lipids of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 (C8~C 26 ) alkyl or (C8~C 26) are alkenyls, each of which can be optionally terminated with -C(O)O- or (C3-C5) cycloalkyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in ionizable lipids of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 (C6~C 24 ) Alkyl or (C6~C 24 ) are alkenyls, each of which can be optionally terminated with -C(O)O- or cyclopropyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in ionizable lipids of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 (C8~C 24 ) alkyl or (C8~C 24 ) is an alkenyl, and the said (C8~C 24 The alkyl group is optionally interrupted with -C(O)O- or cyclopropyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in ionizable lipids of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 (C8~C 10 ) is alkyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in an ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 It was interrupted with cyclopropyl (C 14 ~C 16 ) is alkyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in an ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5(C 10 ~C 24 ) is alkyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in an ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 is, (C 16 ~C 18 ) is an alkenyl, and the remaining variables are as described above for formula (I). In another alternative example, as part of the seventh chemical embodiment, R in an ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 is -(CH2)3C(O)O(CH2)8CH3, -(CH2)5C(O)O(CH2)8CH3, -(CH2)7C(O)O(CH2)8CH3, -(CH2 )7C(O)OCH[(CH2)7CH3]2, -(CH2)7-C3H6-(CH2)7CH3, -(CH2)7CH3, -(CH2)9CH3, -(CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3, or -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remaining variables are as explained above for equation (I).

[0113] In the eighth chemical embodiment, R in an ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 ' is interrupted by -C(O)O- (C 15 ~C 28 ) is alkyl, and the remaining variables are as described above for formula (I) or the seventh embodiment. Alternatively, as part of the eighth embodiment, R in ionizable lipids of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 ' is interrupted by -C(O)O- (C 17 ~C 28) is alkyl, and the remaining variables are as described above for formula (I) or the seventh embodiment. In another alternative, as part of the eighth embodiment, R in the ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 ’ is (C 19 ~C 28 ) alkyl interrupted by -C(O)O-, and the remaining variables are as described above for formula (I) or the seventh embodiment. In another alternative, as part of the eighth embodiment, R in the ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 ’ is (C 17 ~C 26 ) alkyl interrupted by -C(O)O-, and the remaining variables are as described above for formula (I) or the seventh embodiment. In another alternative, as part of the eighth embodiment, R in the ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 ’ is (C 19 ~C 26 ) alkyl interrupted by -C(O)O-, and the remaining variables are as described above for formula (I) or the seventh embodiment. In another alternative, as part of the eighth embodiment, R in the ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) 5 ’ is (C 20 ~C 26 ) alkyl interrupted by -C(O)O-, and the remaining variables are as described above for formula (I) or the seventh embodiment. In another alternative, as part of the eighth embodiment, R 5 ’ is (C 22 ~C 24 ) alkyl interrupted by -C(O)O-, and the remaining variables are as described above for formula (I) or the seventh embodiment. In another alternative, as part of the eighth embodiment, R 5' is -(CH2)5C(O)OCH[(CH2)7CH3]2, -(CH2)7C(O)OCH[(CH2)7CH3]2, -(CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or -(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remaining variables are as described above for Equation (I) or the seventh embodiment.

[0114] Specific examples are provided in Table 1 and the following illustrative section and are included as part of the ninth chemical embodiment herein of the ionizable lipid of formula (I). Pharmaceutically acceptable salts and neutral forms are also included. Furthermore, in one embodiment, lipids of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XI), or (X) and one or more nitrogen atoms on lipids 1-5 may be quaternized. Lipids can be converted to their corresponding quaternized lipids, for example, by treatment with acetonitrile (CH3CN) and chloromethane (CH3Cl) in chloroform (CHCl3). [Table 1-1] [Table 1-2]

[0115] Lipid-nucleic acid particles (LNPs) or pharmaceutical compositions thereof, comprising ionizable lipids and capsid-free, nonviral vectors (e.g., ceDNA) as described herein, may be used to deliver capsid-free, nonviral DNA vectors to target sites of interest (e.g., cells, tissues, organs, etc.).

[0116] In one embodiment, the lipid particle (lipid nanoparticle) formulation is prepared and loaded with TNA (e.g., ceDNA) obtained by a process disclosed in International Patent Application US2018 / 050042, filed September 7, 2018, which is incorporated herein by reference in whole. This can be achieved by high-energy mixing of ethanol lipids and aqueous TNA such as ceDNA at a low pH, which protonates the lipids and provides a favorable energy for ceDNA / lipid association and nucleation of the particles. The particles can be further stabilized by aqueous dilution and removal of organic solvents. The particles can be concentrated to a desired level.

[0117] Generally, lipid particles (e.g., lipid nanoparticles) are prepared with a total lipid-to-nucleic acid (mass or weight) ratio of about 10:1 to 60:1. In some embodiments, the lipid-to-nucleic acid ratio (mass / mass ratio, w / w ratio) may be in 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, and about 30:1 to about 60:1. According to some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with nucleic acids (mass or weight) relative to a total lipid ratio of about 60:1. According to some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with nucleic acids (by mass or weight) to a total lipid ratio of about 30:1. By adjusting the amounts of lipids and nucleic acids, a desired N / P ratio (i.e., the ratio of positively charged polymer amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups), such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, can be provided. Generally, the total lipid content of lipid particle formulations can range from about 5 mg / ml to about 30 mg / mL.

[0118] In some embodiments, the lipid nanoparticles include agents for condensing and / or encapsulating nucleic acid cargo such as ceDNA. Such agents are also referred to herein as condensants or encapsulants. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acids can be used, as long as it is non-fusionable. In other words, agents can condense and / or encapsulate nucleic acid cargo such as ceDNA, but have little or no fusion activity. While we do not wish to be bound by theory, condensants may have some fusion activity if they do not condense / encapsulate nucleic acids such as ceDNA, but the nucleic acids encapsulating the lipid nanoparticles formed with the condensant may be non-fusionable.

[0119] Generally, ionizable lipids are used to condense nucleic acid cargo, such as ceDNA, at low pH and to drive membrane association and membrane fusion. Generally, cationic lipids are lipids containing at least one amino group that is positively charged or protonated under acidic conditions, for example, pH 6.5 or lower. Cationic lipids can also be ionizable lipids, such as ionizable cationic lipids. "Non-fusionable ionizable lipids" means ionizable lipids that can condense and / or encapsulate nucleic acid cargo such as ceDNA, but have little to no fusion activity.

[0120] In one embodiment, ionizable lipids may constitute 20-90% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). For example, the molar content of ionizable lipids may be 20-70% (mol), 30-60% (mol), 40-60% (mol), 40-55% (mol), or 45-55% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, ionizable lipids constitute about 50 mol% to about 90 mol% of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0121] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further contain noncationic lipids. Noncationic lipids can help enhance fusion and improve the stability of LNPs during formation. Examples of nonionizable lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Therefore, noncationic lipids can be neutral, uncharged, zwitterionic, or anionic lipids. Noncationic lipids are typically used to enhance membrane fusion.

[0122] Examples of noncationic lipids include distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and distearoylphosphatidylethanolamine (DS PE), monomethyl-phosphatidylethanolamine (16-O-monomethylPE, etc.), dimethyl-phosphatidylethanolamine (16-O-dimethylPE, etc.), 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diylcoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,Examples include, but are not limited to, 2-difytanol-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholines and diacylphosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably C, 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0123] Other examples of noncationic lipids suitable for use in lipid particles (e.g., lipid nanoparticles) include, 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, dioctadecyldimethylbromone, ceramides, and nonphosphorus lipids such as sphingomyelin.

[0124] In one embodiment, the noncationic lipid is a phospholipid. In one embodiment, the noncationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the noncationic lipid is DSPC. In other embodiments, the noncationic lipid is DOPC. In other embodiments, the noncationic lipid is DOPE.

[0125] In some embodiments, non-cationic lipids may constitute 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the non-cationic lipid content is 0.5-15% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the non-cationic lipid content is 5-12% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the non-cationic lipid content is 5-10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, 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, 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, the non-cationic lipid content is about 7.5% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the noncationic lipid content is approximately 8.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In another embodiment, the noncationic lipid content is approximately 9.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In several embodiments, the noncationic lipid content is approximately 10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In yet another embodiment, the noncationic lipid content is approximately 11% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0126] Exemplary noncationic lipids are described in International Patent Application Publication No. 2017 / 099823 and U.S. Patent Application Publication No. 2018 / 0028664, both of which are incorporated herein by reference in their entirety.

[0127] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further contain components such as sterols to provide integrity and stability to the lipid particle membrane. In one embodiment, exemplary sterols that can be used in the lipid particles are cholesterol or its derivatives. 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-cholestanol, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholesterol hemysuccinate (CHEMS).

[0128] Exemplary cholesterol derivatives are described in International Patent Application No. 2009 / 127060 and U.S. Patent Application Publication No. 2010 / 0130588, both of which are incorporated herein by reference in their entirety.

[0129] In one embodiment, the membrane-integrating component, such as sterols, may constitute 0-50% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 20-50% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 30-40% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 35-45% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 38-42% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles).

[0130] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further comprise conjugated lipid molecules. Generally, these are used to inhibit aggregation of the lipid particles (e.g., lipid nanoparticles) and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEGylated lipids (i.e., lipids conjugated to polyethylene glycol or PEG), polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (e.g., ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEGylated lipid, e.g., a (methoxypolyethylene glycol)-conjugated lipid. In some other embodiments, the PEGylated lipid is PEG 2000 -DMG (Dimyristoyl Glycerol).

[0131] Examples of PEGylated lipids include PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanediol (PEG-S-DMG)), PEG-dialkoxypropylcarbam, and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-diste This includes, but is not limited to, aroyl-sn-glycero-3-phosphoethanolamine sodium salts or mixtures thereof. Additional exemplary PEGylated lipids are described, for example, in US5,885,613, US6,287,591, and in U.S. Patent Application Publications US2003 / 0077829, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904, all of which are incorporated herein by reference in their entirety.

[0132] In one embodiment, the PEG-DAA PEGylated lipid may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEGylated lipid is PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholesta-5-ene-3[beta]-oxy)carboxyamide-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG- DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether) and 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] may be one or more of these. In one embodiment, the PEGylated lipid can be selected from the group consisting of PEG-DMG and 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0133] In one embodiment, lipids conjugated with molecules other than PEG can be used instead of PEGylated 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, namely PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in International Patent Application Publications 1996 / 010392, 1998 / 051278, 2002 / 087541, 2005 / 026372, and 2008 / 147. Issues 438, 2009 / 086558, 2012 / 000104, 2017 / 117528, 2017 / 099823, 2015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 / 0062 U.S. Patent Application Publications No. 82, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0376224, and 2016 / 0317458 These are described in U.S. Patent Nos. 2013 / 0303587, 2013 / 0303587, and 2011 / 0123453, and U.S. Patents Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, all of which are incorporated herein by reference in their entirety. PEG or POZ can be conjugated directly to lipids or bound to lipids via a linker moiety. For example, any linker moiety suitable for binding PEG or POZ to lipids can be used, including non-ester-containing and ester-containing linker moieties. In certain preferred embodiments, a non-ester-containing linker moiety such as an amide or carbamate is used.

[0134] In some embodiments, PEGylated lipids may constitute 0-20% (mol). In some embodiments, the PEGylated lipid content is 0.5-10% (mol). In some embodiments, the PEGylated lipid content is 1-5% (mol). In some embodiments, the PEGylated lipid content is 1-3% (mol). In one embodiment, the PEGylated lipid content is approximately 1.5% (mol). In some embodiments, the PEGylated lipid content is approximately 3% (mol).

[0135] It is understood that the molar ratios of the disclosed ionizable lipids to noncationic lipids, sterols, and PEGylated lipids can be varied as needed. For example, lipid particles (e.g., lipid nanoparticles) may contain 30-70% lipids by mole or total weight of the composition, 0-60% cholesterol by mole or total weight of the composition, 0-30% noncationic lipids by mole or total weight of the composition, and 1-10% PEGylated lipids by mole or total weight of the composition. In one embodiment, the composition contains 40-60% ionizable lipids by mole or total weight of the composition, 30-50% cholesterol by mole or total weight of the composition, 5-15% noncationic lipids by mole or total weight of the composition, and 1-5% PEGylated lipids by mole or total weight of the composition. In one embodiment, the composition comprises 40-60% ionizable lipids by mole or total weight, 30-40% cholesterol by mole or total weight, 5-10% noncationic lipids by mole or total weight, and 1-5% PEGylated lipids by mole or total weight. The composition may also contain 60-70% ionizable lipids by mole or total weight, 25-35% cholesterol by mole or total weight, 5-10% noncationic lipids by mole or total weight, and 0-5% PEGylated lipids by mole or total weight. The composition may also contain up to 45-55% ionizable lipids by mole or total weight, 35-45% cholesterol by mole or total weight, 2-15% noncationic lipids by mole or total weight, and 1-5% PEGylated lipids by mole or total weight.The formulation may also contain, for example, 8-30% ionizable lipids by mole or total weight of the composition, 5-15% noncationic lipids by mole or total weight of the composition, and 0-40% cholesterol by mole or total weight of the composition; 4-25% ionizable lipids by mole or total weight of the composition, 4-25% noncationic lipids by mole or total weight of the composition, 2-25% cholesterol by mole or total weight of the composition, 10-35% conjugate lipids by mole or total weight of the composition, and 5% cholesterol by mole or total weight of the composition; or by mole or The lipid nanoparticle formulation may further contain 2-30% by total weight of ionizable lipids, 2-30% by mole or total weight of noncationic lipids, 1-15% by mole or total weight of cholesterol, 2-35% by mole or total weight of PEGylated lipids, and 1-20% by mole or total weight of cholesterol; or further, up to 90% by mole or total weight of ionizable lipids and 2-10% by mole or total weight of noncationic lipids; or further, 100% by mole or total weight of ionizable lipids. In some embodiments, the lipid particle formulation contains ionizable lipids, noncationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of 50:10:38.5:1.5. In some embodiments, the lipid particle formulation contains ionizable lipids, noncationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:10:38:2. In some embodiments, the lipid particle formulation contains ionizable lipids, noncationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:10:37:3. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation contains ionizable lipids, noncationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:7:40:3. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation contains ionizable lipids, noncationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:8:40:2.In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation contains ionizable lipids, noncationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:9:39:2. In another embodiment, the lipid particle (e.g., lipid nanoparticle) formulation contains ionizable lipids, noncationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:9:38:3.

[0136] In one embodiment, the lipid particles (e.g., lipid nanoparticles) include ionizable lipids, noncationic lipids (e.g., phospholipids), sterols (e.g., cholesterol), and PEGylated lipids (conjugated lipids), where the molar ratio of lipids is in the range of 20 to 70 mole percent for ionizable lipids (target 30 to 60), the molar percentage of noncationic lipids is in the range of 0 to 30 (target 0 to 15), the molar percentage of sterols is in the range of 20 to 70 (target 30 to 50), and the molar percentage of PEGylated lipids (conjugated lipids) is in the range of 1 to 6 (target 2 to 5).

[0137] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Patent Application No. US2018 / 050042, filed on September 7, 2018, which are incorporated herein in their entirety and are intended for use in the methods and compositions disclosed herein.

[0138] The particle size of lipid particles (e.g., lipid nanoparticles) can be determined by quasi-elastic light scattering using Malvern Zetasizer Nano ZS (Malvern, UK), and is approximately 50–150 nm in diameter, 55–95 nm in length, or 70–90 nm in length.

[0139] The pKa of formulated ionizable lipids may correlate with the efficacy of LNPs for nucleic acid delivery (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 in their entirety by reference). In one embodiment, the pKa of each ionizable lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles consisting of ionizable lipids / DSPC / cholesterol / PEGylated lipids (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipids can be prepared using in-line processes described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. The vesicles can be diluted to 24 mM lipids in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH in the range of 2.5 to 11. Aliquots of the TNS solution can be added to a final concentration of 1 mM, and the eddy-mixed emission intensity is subsequently measured at room temperature using an SLM Aminco Series 2 emission spectrophotometer with an excitation wavelength of 321 nm and an oscillation wavelength of 445 nm. S-curve best fit analysis can be applied to the fluorescence data, and the pKa is measured as the pH that produces the semi-optimal fluorescence intensity.

[0140] In one embodiment, 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 expressed as luciferase ng per g of liver measured 4 hours after administration.

[0141] Without limitation, the lipid particles (e.g., lipid nanoparticles) of this disclosure include lipid formulations that can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., a cell, tissue, organ, etc.). Generally, the lipid particles (e.g., lipid nanoparticles) include a capsid-free, non-viral DNA vector and an ionizable lipid or a salt thereof.

[0142] In one embodiment, the lipid particles (e.g., lipid nanoparticles) contain ionizable lipids / noncationic lipids / sterols / conjugated lipids in a molar ratio of 50:10:38.5:1.5. In one embodiment, the disclosure provides a lipid particle (e.g., lipid nanoparticles) formulation comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0143] III. Therapeutic Nucleic Acids (TNA) This 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, small 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 genome) or non-viral 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 mini vectors ("Dumbbell DNA"). Accordingly, aspects of this disclosure generally provide ionizable lipid particles (e.g., lipid nanoparticles) containing TNAs.

[0144] therapeutic nucleic acids Examples of therapeutic nucleic acids in this disclosure include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), ribozymes, closed-end double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministrings"), doggybone®, protelomere-closed DNA, or dumbbell linear DNA), Dicer substrate dsRNAs, small hairpin RNAs (shRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), mRNAs, tRNAs, rRNAs, and DNA viral vectors, viral RNA vectors, and any combination thereof.

[0145] siRNA or miRNAs that can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi) are also intended to be nucleic acid therapeutics according to 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 is achieved by inducing specific disruption of mRNA, which results in the downregulation of the corresponding protein.

[0146] Antisense oligonucleotides (ASOs) and ribozymes, which inhibit mRNA translation into proteins, can be used as nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxynucleic acids have a sequence complementary to the target protein mRNA sequence and can bind to the mRNA via Watson-Crick base pairing. This binding prevents translation of the target mRNA and / or induces RNaseH degradation of the mRNA transcript. As a result, antisense oligonucleotides have increased specificity of action (i.e., downregulation of specific disease-related proteins).

[0147] In any of the methods and compositions provided herein, therapeutic nucleic acid (TNA) may be therapeutic RNA. Such therapeutic RNA may be an mRNA translation inhibitor, an RNA interference (RNAi) agent, a catalytically active RNA molecule (ribozyme), transfer RNA (tRNA), or RNA that binds to mRNA transcripts (ASOs), proteins, or other molecular ligands (aptamers). In any of the methods provided herein, the RNAi agent may be double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, small hairpin RNA, or triple-helix-forming oligonucleotide.

[0148] In any of the methods and compositions provided herein, the therapeutic nucleic acid (TNA) may be therapeutic DNA such as closed-end double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministring"), doggybone®, protelomere closed-end DNA, dumbbell linear DNA, plasmid, minicircle, etc.). Some embodiments of this disclosure are based on methods and compositions comprising closed-end linear double-stranded DNA (ceDNA) capable of expressing a transgene (e.g., 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 variable eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors.

[0149] ceDNA vectors preferably have a linear and continuous structure rather than a discontinuous structure. A linear and continuous structure is considered to be more stable against attack by cellular endonucleases and less likely to undergo recombination and mutagenesis. Therefore, linear and continuous ceDNA vectors are a preferred embodiment. Continuous linear single-stranded intramolecular double-stranded ceDNA vectors may have a covalently bonded terminal without a sequence encoding the AAV capsid protein. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are bacterial circular double-stranded nucleic acid molecules. While the complementary strand of a plasmid can separate following denaturation to produce two nucleic acid molecules, conversely, a ceDNA vector, although having a complementary strand, is a single DNA molecule and therefore remains a single molecule even upon denaturation. In some embodiments, ceDNA vectors, unlike plasmids, can be produced without prokaryotic cell-type DNA base methylation. Therefore, ceDNA vectors and ceDNA plasmids differ in both their structure (particularly linear-to-cyclic) and the methods used to produce and purify these different objects, as well as in their DNA methylation, with ceDNA plasmids being prokaryotic and ceDNA vectors being eukaryotic.

[0150] Provided herein are non-viral capsid-free ceDNA molecules (ceDNA) having covalent closed ends. These non-viral capsid-free ceDNA molecules can be produced in permissible host cells from expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrated cell lines) containing heterologous genes (e.g., transgenes, particularly therapeutic transgenes) positioned between two different inverted end repeat (ITR) sequences, the ITRs being different with respect to each other. In some embodiments, one of the ITRs is modified by deletion, insertion, and / or substitution compared to a wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs includes a functional end degradation site (TRS) and a Rep-binding site. The ceDNA vector is preferably double-stranded over at least a portion of the molecule, such as an expression cassette, e.g., self-complementary (e.g., ceDNA is not a double-stranded circular molecule). The ceDNA vector has covalent closed ends and is therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37°C for more than 1 hour.

[0151] In one embodiment, the ceDNA vector comprises a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, oriented from 5' to 3'. In one embodiment, 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. In exemplary embodiments, the first ITR may be a wild-type ITR and the second ITR may be a mutant or modified ITR, or vice versa; or the first ITR may be a mutant or modified ITR and the second ITR may be a wild-type ITR. In one embodiment, both the first and second ITRs are modified but have different sequences, different modifications, or are not identical modified ITRs, and have different three-dimensional spatial configurations. In other words, a ceDNA vector using asymmetric ITRs may have ITRs in which any modification of one ITR relative to the WT-ITR is not reflected in the other ITRs, or alternatively, if the asymmetric ITRs are modified asymmetric ITR pairs, they may have different sequences and different three-dimensional shapes relative to each other.

[0152] In one embodiment, the ceDNA vector comprises a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, in the 5' to 3' direction, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are symmetric or substantially symmetric with respect to each other, i.e., the ceDNA vector may contain ITR sequences having a symmetric three-dimensional spatial configuration, so that their structures are the same shape in geometric space or have the same A, C-C', B-B' loops in three-dimensional space. In such embodiments, the symmetric ITR pair, or substantially symmetric ITR pair, may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair may have one or more modifications from the wild-type ITR and have the same sequence that is inversely complementary (inverted) to each other. In one embodiment, the modified ITR pair is substantially symmetric as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetric three-dimensional shape. In some embodiments, the symmetric ITR, or substantially symmetric ITR, may be wild-type (WT-ITR) as described herein, i.e., both ITRs have wild-type sequences but do not necessarily have to be WT-ITRs of the same AAV serotype. In one embodiment, one WT-ITR may originate from one AAV serotype, and the other WT-ITR may originate from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they may have one or more conserved nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration.

[0153] The wild-type, mutant, or otherwise modified ITR sequences provided herein represent the DNA sequences contained in expression constructs (e.g., ceDNA plasmids, ceDNA bacmids, ceDNA baculoviruses) for the production of ceDNA vectors. Therefore, the ITR sequences actually contained in ceDNA vectors produced from ceDNA plasmids or other expression constructs may be identical or different to the ITR sequences provided herein as a result of naturally occurring changes (e.g., replication errors) that occur during the production process.

[0154] In one embodiment, a ceDNA vector described herein, comprising an expression cassette having a transgene which is a therapeutic nucleic acid sequence, can be operably ligated to one or more regulatory sequences that enable or control the expression of the transgene. In one embodiment, a polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is adjacent to the first and second ITR sequences, and the first and second ITR sequences are asymmetrical or symmetrical with respect to each other.

[0155] In one embodiment, the expression cassette includes, in this order, a promoter located between two ITRs and operably linked to the transgene, a post-transcriptional regulatory element, and one or more polyadenylation and termination signals. In one embodiment, the promoter is regulated-inducible or repressible. The promoter may be any sequence that promotes transcription of the transgene. In one embodiment, the promoter is a CAG promoter or a variation thereof. The post-transcriptional regulatory element is a sequence that modulates the expression of the transgene and, as a non-limiting example, is any sequence that creates a tertiary structure that enhances the expression of the transgene, which is a therapeutic nucleic acid sequence.

[0156] In one embodiment, the post-transcriptional regulatory element comprises WPRE. In one embodiment, the polyadenylation and termination signal comprises BGH polyA. Any cis-regulatory element known in the art, or a combination thereof, such as the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements (including, but not limited to, thymidine kinase genes of herpes simplex virus or hepatitis B virus (HBV)) may be used in addition. In one embodiment, the expression cassette length in the 5'-3' direction exceeds the maximum length known to be capsidized in AAV virion. In one embodiment, 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.

[0157] In one embodiment, the expression cassette may include more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range of about 4,000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides.

[0158] In one embodiment, 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, the ITR may act as a promoter for the transgene. In some embodiments, the ceDNA vector may include additional components for regulating transgene expression, such as regulatory switches for controlling and regulating transgene expression, and optionally, a regulatory switch that is a kill switch enabling controlled cell death of the cell containing the ceDNA vector.

[0159] In one embodiment, the ceDNA vector may be capsid-free and obtained from a plasmid encoding a first ITR, an expressible transgene cassette, and a second ITR in that order, wherein at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence.

[0160] In one embodiment, the ceDNA vector disclosed herein is used for therapeutic purposes (e.g., medical, diagnostic, or veterinary use) or for immunogenic polypeptides.

[0161] The expression cassette may contain any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector contains any gene of interest in a subject, comprising one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0162] In one embodiment, the sequences provided in the expression cassette, expression construct, or donor sequence of the ceDNA vector described herein can be codon-optimized for host cells. As used herein, the terms “optimized codon” or “codon optimization” refer to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a significant number of codons in an unmodified sequence (e.g., a prokaryotic sequence) with codons that are more or most frequently used in the genes of a vertebrate of interest, such as a mouse or a human, for enhanced expression in that vertebrate's cells. Different species exhibit a particular bias towards certain codons of certain amino acids.

[0163] Typically, codon optimization does not alter the amino acid sequence of the original translated protein. Optimized codons can be determined using, for example, Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another publicly available database.

[0164] Many organisms exhibit codon bias, using specific codons to encode insertions of particular amino acids in growing peptide chains. Codon preference, or codon bias, which is the difference in codon usage frequency between organisms, is brought about by the degeneracy of the genetic code and is well documented across many organisms. Codon bias is often correlated with messenger RNA (mRNA) translation efficiency and is thought to depend particularly on the characteristics of the translated codon and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs within a cell is generally a reflection of the codons most frequently used in peptide synthesis. Therefore, based on codon optimization, genes can be tuned for optimal gene expression in a given organism.

[0165] Considering the large number of gene sequences available in a wide variety of animal, plant, and microbial species, it is possible to calculate the relative frequency 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)).

[0166] inverted terminal repeat (ITR) As described herein, a ceDNA vector is a capsid-free linear double-stranded DNA molecule formed from a continuous strand of complementary DNA having covalent ends (linear, continuous, non-capsid structures), and includes different or asymmetrical 5' inverted end repeat (ITR) sequences and 3' ITR sequences. At least one of the ITRs includes a functional end degradation 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 end repeat (ITR), i.e., a deletion, insertion, and / or substitution of another ITR, and an expressible transgene.

[0167] In one embodiment, at least one of the ITRs is an AAV ITR, for example, a wild-type AAV ITR. In one embodiment, at least one of the ITRs is a modified ITR relative to the other ITRs, i.e., the ceDNA includes ITRs that are asymmetric with respect to each other. In one embodiment, at least one of the ITRs is a non-functional ITR.

[0168] In one embodiment, the ceDNA vector comprises (1) an expression cassette containing a cis-regulatory element, a promoter, and at least one transgene; (2) a promoter operably linked to at least one transgene; and (3) two self-complementary sequences adjacent to the expression cassette, e.g., ITRs, and the ceDNA vector is not associated with a capsid protein. In some embodiments, 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 a terminal degradation site (TRS) of AAV or a functional variant of the RBE, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments, the ceDNA vector may include additional components for regulating the expression of the transgene, e.g., a regulatory switch for controlling and regulating the expression of the transgene, and a regulatory switch that is a kill switch enabling controlled cell death of the cell containing the ceDNA vector.

[0169] In one embodiment, the two self-complementary sequences may be ITR sequences from any known parvovirus, such as a dependent virus, e.g., AAV (e.g., AAV1-AAV12). In addition to a variable palindromic sequence that enables hairpin secondary structure formation, any modified AAV2 ITR sequence may be used, but is not limited to those containing a Rep-binding site (RBS) and terminal degradation site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3'. In some embodiments, the ITR may be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITR does not contain an AAV base sequence. In yet another embodiment, the synthetic ITR preserves the above ITR structure but has little to no AAV source sequence. In some embodiments, the synthetic ITR may preferentially interact with wild-type Rep or Rep of a specific serotype, or, in some cases, may not be recognized by wild-type Rep and may only be recognized by mutant Rep. In some embodiments, the ITR is a synthetic ITR sequence that retains a functional Rep-binding site (RBS) and terminal degradation site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to a variable palindromic sequence that enables hairpin secondary structure formation. In some examples, the modified ITR sequence retains the sequence of the RBS, TRS, and the structure and position of the Rep-binding element that forms the terminal loop portion of a single 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 partial ITR sequences are shown in Figures 26A-26B of International Patent Application US18 / 49996, filed September 7, 2018. In some embodiments, the ceDNA vector may include an ITR with modifications in the ITR corresponding to any of the modifications in the ITR sequence or ITR subsequence sequence shown in one or more of the modifications in the ITR sequence or ITR subsequence sequence shown in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A and 10B of International Patent Application US18 / 49996 filed on September 7, 2018.

[0170] In one embodiment, a ceDNA vector may be produced from an expression construct further comprising a specific combination of cis-regulatory elements. Examples of 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, an ITR may act as a promoter for the transgene. In some embodiments, the ceDNA vector includes additional components for regulating the expression of the transgene, such as a regulatory switch described in International Patent Application US18 / 49996 filed September 7, 2018, which can regulate the expression of the transgene or a kill switch capable of killing cells containing the ceDNA vector.

[0171] In one embodiment, the expression cassette may also include post-transcriptional elements to increase the expression of the transgene. In one embodiment, the Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase the expression of the transgene. Other post-transcriptional elements, such as post-transcriptional elements from the thymidine kinase genes of herpes simplex virus or hepatitis B virus (HBV), can be used. Secretory sequences can be linked to the transgene, for example, the VH-02 and VK-A26 sequences. The expression cassette may include polyadenylated sequences known in the art or variations thereof, such as native sequences isolated from bovine BGHpA or viral SV40pA, or synthetic sequences. Some expression cassettes may also include SV40 late polyA signal upstream enhancer (USE) sequences. USE can be used in combination with SV40pA or heterologous polyA signals.

[0172] Figures 1A–1C of International Patent Application No. 2018 / 050042, filed on 7 September 2018 and incorporated herein in its entirety by reference, show schematic diagrams of corresponding sequences of non-limiting exemplary ceDNA vectors or ceDNA plasmids. The ceDNA vector is capsid-free and may be obtained from a plasmid encoding a first ITR, an expressible transgene cassette, and a second ITR in that order, wherein at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence. The expressible transgene cassette preferably comprises one or more of the following in this order: an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and polyadenylation and termination signals (e.g., BGH polyA).

[0173] promoter Suitable promoters, including those mentioned above, may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Using suitable promoters, expression can be driven by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include the SV40 early promoter, the mouse mammary tumor virus long-terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV very early promoter region (CMVTE), the Roussarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6, e.g., Miyagishi el al., Nature Biotechnology 20, 497-500 (2002)), and the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003). Examples include, but are not limited to, the human H1 promoter (H1), the CAG promoter, and the human alpha-1-anti-trypsin (HAAT) promoter (e.g., etc.). In one embodiment, these promoters are modified at their downstream intron-containing ends to include one or more nuclease cleavage sites. In one embodiment, the DNA containing the nuclease cleavage sites is foreign to the promoter DNA.

[0174] In one embodiment, a promoter may further enhance expression and / or alter its spatial and / or temporal expression by including one or more specific transcriptional regulatory sequences. A promoter may also include distal enhancer or repressor elements that may be located thousands of base pairs away from the transcription start site. Promoters may originate from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may constitutively or differentially regulate the expression of gene components with respect to the cell, tissue, or organ in which expression occurs, or 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, and the promoters listed below. Such promoters and / or enhancers can be used to express any gene of interest, e.g., a therapeutic protein. For example, a vector may include a promoter operably ligated to a nucleic acid sequence encoding a therapeutic protein. In one embodiment, the promoter operably ligated to the therapeutic protein-coding sequence may be a Simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, e.g., a bovine immunodeficiency virus (BIV) long-terminal repeat (LTR) promoter, a Moloney virus promoter, an avens leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter, e.g., a CMV early-stage promoter, an Epstein-Barr virus (EBV) promoter, or a Roussarcoma virus (RSV) promoter. In one embodiment, 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, such as a liver-specific promoter, such as human alpha-1 anti-trypsin (HAAT). In one embodiment, delivery to the liver may be achieved using an endogenous ApoE-specific target of the composition containing the ceDNA vector to hepatocytes via low-density lipoprotein (LDL) receptors present on the surface of hepatocytes.

[0175] In one embodiment, the promoter used is the unvariable promoter of the gene encoding the therapeutic protein. The promoters 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., unvariable), such as enhancers.

[0176] 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.

[0177] Polyadenylated sequence Sequences encoding polyadenylated sequences may be included in the ceDNA vector to stabilize mRNA expressed from the ceDNA vector and to assist in nuclear transport and translation. In one embodiment, the ceDNA vector does not contain a polyadenylated 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, or at least 50 adenine dinucleotides. In some embodiments, the polyadenylated sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range in between.

[0178] In one embodiment, ceDNA can be obtained from a vector polynucleotide encoding a heterogeneous nucleic acid operably positioned between two different inverted terminal repeat sequences (ITRs) (e.g., AAV ITR), where at least one of the ITRs includes a terminal degradation site and a replication protein-binding site (RPS), e.g., a Rep-binding site (e.g., wt AAV ITR), and one of the ITRs includes a deletion, insertion, and / or substitution with respect to the other ITR, e.g., a functional ITR.

[0179] In one embodiment, the host cell does not express the viral capsid protein, and the polynucleotide vector template lacks any viral capsid coding sequence. In one embodiment, the polynucleotide vector template lacks the AAV capsid gene, but also lacks the capsid genes of other viruses. In one embodiment, the nucleic acid molecule also lacks the AAV Rep protein coding sequence. Therefore, in some embodiments, the nucleic acid molecule of the present invention lacks both the functional AAV cap and the AAV rep gene.

[0180] In one embodiment, the ceDNA vector does not have a modified ITR.

[0181] In one embodiment, the ceDNA vector includes a regulatory switch as disclosed herein (or in International Patent Application US18 / 49996 filed on September 7, 2018).

[0182] IV. Production of ceDNA vectors A method for producing the ceDNA vector described herein, including asymmetric ITR pairs or symmetric ITR pairs as defined herein, is described in Section IV of International Patent Application US18 / 49996, filed on 7 September 2018, which is incorporated herein by reference in its entirety. As described herein, a ceDNA vector may be obtained by a process comprising, for example, a) incubating a population of host cells (e.g., insect cells) containing a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), wherein the host cells lack the viral capsid coding sequence for a sufficient period of time in the presence of the Rep protein, and the host cells are free of the viral capsid coding sequence; and b) harvesting and isolating the ceDNA vector from the host cells. The presence of the Rep protein induces replication of the vector polynucleotide having modified ITRs to produce the ceDNA vector in the host cells.

[0183] However, viral particles (e.g., AAV virions) are not expressed. Therefore, there are no size limitations, such as those naturally imposed in AAV or other virus-based vectors.

[0184] 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 having a single recognition site on the ceDNA vector, and by 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.

[0185] In one embodiment, the present invention provides the use of a host cell line that stably incorporates a DNA vector polynucleotide expression template (ceDNA template) into its own genome in the production of a non-viral DNA vector, as described, for example, Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, the Rep is attached to the host cell at an MOI of about 3. If the host cell line is a mammalian cell line, for example, HEK293 cells, the cell line may have a stably incorporated polynucleotide vector template, and the Rep protein can be introduced into the cell using a second vector, such as a herpesvirus, enabling the excision and amplification of ceDNA in the presence of the Rep and helper virus.

[0186] In one embodiment, the host cell used to construct the ceDNA vector described herein is an insect cell, and a baculovirus is used to deliver both the polynucleotide encoding the Rep protein and the non-viral DNA vector polynucleotide expression construct template of the ceDNA. In some embodiments, the host cell is engineered to express the Rep protein.

[0187] Next, the ceDNA vector is harvested and isolated from host cells. The time for harvesting and collecting the ceDNA vector from cells as described herein may be selected and optimized to achieve high yield production of the ceDNA vector. For example, the harvesting time may be selected considering cell viability, cell morphology, cell proliferation, etc. In one embodiment, cells are harvested after sufficient time has elapsed since baculovirus infection to grow under sufficient conditions and produce the ceDNA vector, but before the majority of the cells begin to die due to the toxicity of the baculovirus. The DNA vector may be isolated using a plasmid purification kit such as the Qiagen Endo-Free plasmid kit. Other methods developed for plasmid isolation may also be adapted for DNA vectors. In general, any nucleic acid purification method may be employed.

[0188] DNA vectors can be purified by any means known to those skilled in the art for the purification of DNA. In one embodiment, the ceDNA vector is purified as a DNA molecule. In one embodiment, the ceDNA vector is purified as an exosome or microparticle. The presence of a ceDNA vector can be confirmed by digesting the vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector, and by using gel electrophoresis to analyze both the digested and undigested DNA material to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0189] V. Preparation of Lipid Particles Lipid particles (e.g., lipid nanoparticles) can be spontaneously formed when TNA (e.g., ceDNA) and lipids are mixed. 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 a 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.

[0190] In general, 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, U.S. Patent Application Publications US2013 / 0037977, 2010 / 0015218, 2013 / 0156845, 2013 / 0164400, 2012 / 0225129 and 2010 / 0130588, the contents of each of these are incorporated herein by reference in their entirety. In some embodiments, 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 U.S. Patent Application Publication 2007 / 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 U.S. Patent Application Publication No. 2004 / 0142025, which is incorporated herein by reference in its entirety.

[0191] In one embodiment, lipid particles (e.g., lipid nanoparticles) can be prepared by an impact jet process. Generally, the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, 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 mixing ratio can be about 45-55% lipid and about 65-45% ceDNA.

[0192] The lipid solution may contain the disclosed ionizable lipids, non-cationic lipids (phospholipids such as DSPC, DOPE, and DOPC), PEG or PEG-conjugated molecules (e.g., PEG-lipids), and sterols (e.g., cholesterol) in an alcohol, such as ethanol, at a total lipid concentration of 5 to 30 mg / mL, more preferably 5 to 15 mg / mL, and most likely 9 to 12 mg / mL. In the lipid solution, the molar ratio of lipids may range from about 25 to 98%, preferably about 35 to 65%, for cationic lipids; about 0 to 15%, preferably about 0 to 12%, for non-ionizable lipids; about 0 to 15%, preferably about 1 to 6%, for PEG or PEG-conjugated lipid molecules; and about 0 to 75%, preferably about 30 to 50%, for sterols.

[0193] The ceDNA solution may contain ceDNA in a buffer solution having a pH in the range of 3.5 to 5 at a concentration range of 0.3 to 1.0 mg / mL, preferably 0.3 to 0.9 mg / mL.

[0194] 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 in, for example, an impact jet mixer to immediately form LNPs. The mixing flow rate may be in the range of 10–600 mL / min. The tube ID range may be 0.25–1.0 mm, and the total flow rate may 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 vol:vol ratio in the range of 1:1–1:3, preferably about 1:2 vol:vol. If necessary, this buffer solution may 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. Before anion exchange, the mixed LNPs can be incubated for a certain period, for example, 30 minutes to 2 hours. The incubation temperature can be in the range of 15–40°C or 30–40°C. After incubation, the solution is filtered through a filter such as a 0.8 μm filter, including an anion exchange separation step. This process can use tube IDs ranging from 1 mm to 5 mm and flow rates of 10–2000 mL / min.

[0195] After formation, the LNPs can be concentrated and filtered through an ultrafiltration process, in which alcohol is removed and the buffer is replaced with a final buffer solution, such as phosphate-buffered saline (PBS) at approximately pH 7, approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4.

[0196] In the ultrafiltration process, a tangential flow filtration format (TFF) with a nominal molecular weight cutoff range of 30–500 kD for the membrane can be used. The membrane format is hollow fiber or flat sheet cassette. In a TFF process with an appropriate molecular weight cutoff, LNP can be retained in the holding solution, and the filtrate or permeate contains waste from alcohol, citrate buffer, and final buffer. The TFF process is a multi-step process to an initial ceDNA concentration of 1–3 mg / mL. After concentration, the LNP solution is ultrafiltered against the final buffer at a volume of 10–20 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.

[0197] VI. Pharmaceutical Compositions and Formulations This specification also provides pharmaceutical compositions comprising TNA lipid particles and pharmaceutically acceptable carriers or excipients.

[0198] In one embodiment, TNA lipid particles (e.g., lipid nanoparticles) are provided with complete and partial encapsulation of therapeutic nucleic acids. In one embodiment, the nucleic acid therapeutic agent is fully encapsulated in lipid particles (e.g., lipid nanoparticles) to form nucleic acids containing lipid particles. In one embodiment, the nucleic acid may be encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0199] In one embodiment, lipid particles are arranged in the following wavelengths to ensure effective delivery: approximately 20nm to approximately 100nm, 30nm to approximately 150nm, approximately 40nm to approximately 150nm, approximately 50nm to approximately 150nm, approximately 60nm to approximately 130nm, approximately 70nm to approximately 110nm, approximately 70nm to approximately 100nm, approximately 80nm to approximately 100nm, approximately 90nm to approximately 100nm, approximately 70nm to approximately 90nm, and approximately 80nm to approximately 90nm. The particles have an average diameter of approximately 70 nm to approximately 80 nm, or approximately 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 preparing them are disclosed, for example, in International Patent Application Publication US18 / 50042, U.S. Patent Publication 2004 / 0142025 and U.S. Patent Publication 2007 / 0042031, which are incorporated herein by reference in their entirety for any purpose. In one embodiment, the size of lipid particles (e.g., lipid nanoparticles) can be determined by quasi-elastic light scattering using, for example, the Malvern Zetasizer Nano ZS (Malvern, UK) system.

[0200] Generally, the lipid particles (e.g., lipid nanoparticles) of the present invention have an average diameter selected to provide the intended therapeutic effect.

[0201] Depending on the intended use of the lipid particles, the proportion of their components may vary, and the delivery efficiency of a particular formulation can be measured, for example, using an endosomal release parameter (ERP) assay.

[0202] In one embodiment, ceDNA can be compounded with the lipid portion of a particle or encapsulated in the lipid position of a lipid particle (e.g., lipid nanoparticles). In one embodiment, ceDNA can be completely encapsulated in the lipid position of a lipid particle, thereby protecting it from degradation by nucleases, for example, in aqueous solutions. In one embodiment, ceDNA in lipid particles is substantially undegraded after exposure of lipid particles to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes. In some embodiments, ceDNA in lipid particles is substantially undegraded after incubation of 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.

[0203] In one embodiment, the lipid particles (e.g., lipid nanoparticles) are substantially nontoxic to the target, such as mammals including humans.

[0204] In one embodiment, a pharmaceutical composition comprising the therapeutic nucleic acid of the present disclosure may be formulated into lipid particles (e.g., lipid nanoparticles). In some embodiments, the lipid particles comprising the therapeutic nucleic acid may be formed from the disclosed ionizable lipids. In some other embodiments, the lipid particles comprising the therapeutic nucleic acid may be formed from noncationic lipids. In preferred embodiments, the lipid particles of the present invention are nucleic acid-containing lipid particles formed from disclosed ionizable lipids comprising therapeutic nucleic acids selected from the group consisting of mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or nonviral synthetic 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 covalent closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA").

[0205] In another preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles, which are formed from noncationic lipids and conjugated lipids that optionally prevent particle aggregation.

[0206] In one embodiment, the lipid particle formulation is an aqueous solution. In another embodiment, the lipid particle (e.g., lipid nanoparticles) formulation is a freeze-dried powder.

[0207] In some embodiments, the present disclosure provides lipid particle formulations further comprising one or more pharmaceutical excipients. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation further comprises sucrose, tris, trehalose, and / or glycine.

[0208] In one embodiment, the lipid particles (e.g., lipid nanoparticles) disclosed herein may be incorporated into a pharmaceutical composition suitable for administration to a target for in vivo delivery to a target cell, tissue, or organ. Typically, the pharmaceutical composition comprises the TNA lipid particles (e.g., lipid nanoparticles) disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the TNA lipid particles (e.g., lipid nanoparticles) of this disclosure may be incorporated into a pharmaceutical composition suitable for a desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via hyperbaric intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also intended. Pharmaceutical compositions for therapeutic purposes may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. Sterile injectable solutions may be prepared by filtration sterilization, incorporating the required amount of ceDNA vector compound in a suitable buffer, along with one or a combination of the components listed above, as needed.

[0209] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for topical, systemic, intraamniotic, subarachnoid, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), subarachnoid, intrabladder, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, interstitial, intraacular, and intravitreous), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via hyperbaric intravenous or intraarterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also intended.

[0210] A pharmaceutically active composition containing TNA lipid particles (e.g., lipid nanoparticles) can be formulated to deliver a transgene in nucleic acid to recipient cells, resulting in the therapeutic expression of the transgene. This composition may also contain a pharmaceutically acceptable carrier.

[0211] Pharmaceutical compositions for therapeutic purposes are typically sterile and stable under the conditions of manufacture and storage. The compositions 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 required amount of the ceDNA vector compound in a suitable buffer, optionally with one or a combination of the ingredients listed above, and filtering to sterilize.

[0212] In one embodiment, the lipid particles (e.g., lipid nanoparticles) are solid core particles having at least one lipid bilayer. In one embodiment, the lipid particles have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) form. Without limitation, examples of non-lamellar forms include, for example, three-dimensional tubes, rods, cubic symmetries, etc. The non-lamellar form (i.e., non-bilayer structure) of lipid particles is known to those skilled in the art and can be determined using analytical techniques used by those skilled in the art. Such techniques include, but are not limited to, cryogenic 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 readily evaluated and characterized, for example, using Cryo-TEM analysis as described in U.S.A. Patent Application Publication No. 2010 / 0130588, the content of which is incorporated herein by reference in its entirety.

[0213] In one embodiment, lipid particles having a non-lamellar form have a high electron density.

[0214] In one embodiment, the present disclosure provides lipid particles that are either a single lamellar structure or a multi-lamellar structure. In some aspects, the present disclosure provides a lipid particle (e.g., lipid nanoparticle) formulation comprising multilamellar particles and / or foamed-based particles. By controlling the composition and concentration of the lipid components, the rate at which lipid conjugates exchange outside the lipid particles and, in turn, the rate at which the lipid particles become membrane fusogenic can be controlled. In addition, other variables, such as, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid particles become membrane fusogenic. Other methods that can be used to control the rate at which lipid particles (e.g., lipid nanoparticles) become membrane fusogenic will be apparent to those skilled in the art based on the present disclosure. It will also be apparent that the lipid particle size can be controlled by controlling the composition and concentration of the lipid conjugates.

[0215] In one embodiment, the pKa of the formulated ionizable lipid can correlate with the efficacy of the LNP for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533, Semple et al, Nature Biotechnology 28, 172-176 (2010). Both of these are incorporated herein by reference in their entirety). In one embodiment, a preferred range of pKa is from about 5 to about 8. In one embodiment, a preferred range of pKa is from about 6 to about 7. In one embodiment, a preferred pKa is about 6.5.In one embodiment, the pKa of the ionizable lipid can be determined in lipid particles (e.g., lipid nanoparticles) using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS).

[0216] In one embodiment, ceDNA encapsulation in lipid particles can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or the PicoGreen® assay, using a dye whose fluorescence is enhanced 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 when a small amount of nonionic surfactant is added. The surfactant-mediated breakdown of the lipid bilayer releases the encapsulated ceDNA, allowing it to interact with the membrane-impermeable dye. CeDNA encapsulation can be calculated as E=(Io-I) / Io, where I and Io refer to the fluorescence intensity before and after the addition of the surfactant.

[0217] Unit dose In one embodiment, the pharmaceutical composition may be presented in unit dosage forms. These unit dosage forms will typically be adapted to one or more routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by inhaler. In some embodiments, the unit dosage form is adapted for administration by sprayer. In some embodiments, the unit dosage form is adapted for administration by aerosolizer. In some embodiments, the unit dosage form is adapted for oral administration, buccal administration, or sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intraventricular administration. In some embodiments, 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 the amount of the compound that produces the therapeutic effect.

[0218] VII. Treatment Methods Nucleic acid sequences (e.g., therapeutic nucleic acid sequences) can be introduced into host cells using the ionizable lipid compositions and methods described herein (e.g., TNA lipid particles (e.g., lipid nanoparticles) described herein). In one embodiment, the introduction of nucleic acid sequences into host cells using TNA LNPs (e.g., ceDNA vector lipid particles described herein) can be monitored with appropriate biomarkers from the treated patient to evaluate gene expression.

[0219] The LNP compositions provided herein can be used to deliver transgenes (nucleic acid sequences) for a variety of purposes. In one embodiment, a ceDNA vector (e.g., ceDNA vector lipid particles described herein) can be used in a variety of ways, including, for example, excitation, in vitro and in vivo applications, methodologies, diagnostic procedures, and / or gene therapy regimens.

[0220] Provided herein is a method for treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of TNA LNP (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) into target cells of a subject requiring treatment (e.g., hepatocytes, muscle cells, kidney cells, nerve cells, or other affected cell types) along with an optionally pharmaceutically acceptable carrier. The implemented TNA LNP (e.g., ceDNA vector lipid particles) contains a target nucleotide sequence useful for treating the disease. In particular, the TNA may contain a desired exogenous DNA sequence operably ligated to a regulatory element that, when introduced into the subject, can direct the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence. The TNA LNP (e.g., ceDNA vector lipid particles) can be administered via any preferred route known in the art as described herein. In one embodiment, the target cells are in a human subject.

[0221] Provided herein is a method for providing a diagnostically or therapeutically effective amount of TNA LNP (e.g., ceDNA vector lipid particles as described herein) to a subject in need thereof, the method comprising providing a certain amount of TNA LNP (e.g., ceDNA vector lipid particles as described herein) to cells, tissues, or organs of a subject in need thereof for a time sufficient to enable the expression of a transgene from the TNA LNP, thereby providing the subject with a diagnostically or therapeutically effective amount of a protein, peptide, nucleic acid expressed by the TNA LNP (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)). In one embodiment, the subject is human.

[0222] Provided herein are methods for diagnosing, preventing, treating, or improving at least one symptom of a disease, disorder, dysfunction, injury, abnormal condition, or trauma in a subject. Generally, these methods include at least the step of administering TNA LNPs (e.g., ceDNA vector lipid particles as described herein) to a subject in need for a period of time and in an amount sufficient to diagnose, prevent, treat, or improve one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the subject. In one embodiment, the subject is a human.

[0223] Provided herein are methods for using TNA LNPs as tools for treating one or more symptoms of a disease or disease state. Several genetic diseases exist in which defective genes are known, and these typically fall into two classes: deficiency states of enzymes that are usually generally inherited recessively, and imbalance states of enzymes that may be involved in regulatory or structural proteins but are not typically always inherited dominantly. In the case of deficiency states, TNA LNPs (e.g., ceDNA vector lipid particles described herein) can be used to deliver transgenes to introduce normal genes into affected tissue for replacement therapy, and in some embodiments, antisense mutations can be used to create animal models of the disease. In the case of imbalance 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 that disease state. Thus, the TNA LNPs (e.g., ceDNA vector lipid particles) and methods disclosed herein enable the treatment of genetic diseases. As used herein, a disease condition is treated by partially or completely repairing the deficiency or imbalance that causes or exacerbates the disease.

[0224] In general, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to deliver any transgene in accordance with the above description to treat, prevent, or improve symptoms associated with any disorder of gene expression. Examples of disease conditions 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 mellitus, muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy), Harler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondriopathy (e.g., Leber optic neuropathy (LHON), Leigh syndrome, and subacute sclerosing encephalitis), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathy), and diseases of solid organs (e.g., brain, liver, kidneys, heart). In some embodiments, ceDNA vectors such as those disclosed herein may be advantageously used in the treatment of individuals with metabolic disorders (e.g., ornithine transcarbamylase deficiency).

[0225] In one embodiment, TNA LNPs described herein can be used to treat, improve, 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 described herein) include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disorders); 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)); hematological diseases or disorders (e.g., hemophilia (A and B), thalassemia, and anemia); cancers and tumors; and genetic diseases or disorders (e.g., cystic fibrosis).

[0226] In one embodiment, a heterologous nucleotide sequence can be delivered using TNA LNP (e.g., ceDNA vector lipid particles as described herein) in situations where it is desirable to regulate the expression level of a transgene (e.g., a transgene encoding a hormone or growth factor).

[0227] In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to correct abnormal levels and / or functions (e.g., absence or deletion in a protein) of gene products that cause disease or impairment. TNA LNPs (e.g., ceDNA vector lipid particles) can produce functional proteins and / or repair protein levels to alleviate or reduce symptoms resulting from specific diseases or impairments caused by absence or deletion in proteins, or to provide benefits. For example, treatment of OTC deficiency can be achieved by producing functional OTC enzymes. Treatment of hemophilia A and B can be achieved by correcting the levels of factor VIII, factor IX, and factor X. Treatment of PKU can be achieved by correcting the levels of phenylalanine hydroxylase enzymes. 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 isuronate-2-sulfatase, respectively. Treatment of cystic fibrosis may be achieved by producing functional transmembrane conductance regulators of cystic fibrosis. Treatment of glycogen storage disease may be achieved by restoring functional G6Pase enzyme function. Treatment of PFIC may be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

[0228] In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles) 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, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). For example, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to deliver antisense nucleic acids to cells in vitro or in vivo. For example, if the transgene is an RNAi molecule, the expression of antisense nucleic acid or RNAi in target cells reduces the expression of a particular protein by the cell. Therefore, a transgene that is an RNAi molecule or antisense nucleic acid can be administered to reduce the expression of a particular protein in a target that requires it. Antisense nucleic acids can also be administered to cells in vitro to modulate cell physiology, for example, to optimize a cell or tissue culture system.

[0229] In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles) 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 genome) 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 minimal DNA vectors ("dumbbell DNA"). For example, in one embodiment, a ceDNA vector (e.g., ceDNA vector lipid particles) can be used to deliver minicircles to cells in vitro or in vivo. For example, if the transgene is minicircle DNA, the expression of minicircle DNA in target cells reduces the expression of a particular protein by the cell. Therefore, a transgene that is minicircle DNA can be administered to reduce the expression of a particular protein in a target that requires it. Minicircle DNA can also be administered to cells in vitro to modulate cell physiology, for example, to optimize cell or tissue culture systems.

[0230] In one embodiment, exemplary transgenes encoded by a TNA vector containing an expression cassette include X, a lysosomal enzyme (e.g., hexosaminidase A associated with Tay-Sachs disease, or Hunter syndrome / MPS) Examples include, but are not limited to, isuronate sulfatases related to II, erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., interferon, β-interferon, interferon-γ, 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 factor 1 and 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factors 3 and 4, brain-derived neurotrophic factor (BDNF), glial-derived growth factor (GDNF), transforming growth factors 1 and In some exemplary embodiments, the transgene encodes a monoclonal antibody specific to 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, the transgene encodes an antibody comprising a full-length antibody or an antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an 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.

[0231] Administration In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles as described herein) can be administered to an organism for in vivo cell transduction. In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles) can be administered to an organism for ex vivo cell transduction.

[0232] Generally, administration is by one of the routes typically used to bring the molecule into final contact with blood or tissue cells. Preferred methods for administering such nucleic acids are available and well known to those skilled in the art, and two or more routes may be used to administer a particular composition, although a particular route may often be more immediate and provide a more effective response than another. Exemplary forms of administration 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, perdermal, intraendothelial, 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 skin and mucous membrane surfaces including the airway surface, and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., to the liver, eye, skeletal muscle, myocardium, diaphragm, muscle, or brain).

[0233] Administration of the ceDNA vector (e.g., ceDNA vector lipid particles) can be performed on any site of a subject, including but not limited to sites 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, administration of the ceDNA vector (e.g., ceDNA vector lipid particles) 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 being treated, ameliorated, and / or prevented, as well as the nature of the particular ceDNA vector (e.g., ceDNA vector lipid particles) being used. Additionally, the ceDNA enables administration of two or more transgenes in a single vector or multiple ceDNA vectors (e.g., a ceDNA cocktail).

[0234] In one embodiment, administration of the ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) to skeletal muscle includes, but is not limited to, administration to the skeletal muscle of the limbs (e.g., upper arm, forearm, upper extremity, and / or lower extremity), waist, neck, head (e.g., tongue), pharynx, abdomen, pelvis / perineum, and / or fingers. The ceDNA vector (e.g., ceDNA vector lipid particles) can be delivered to skeletal muscle by intravenous administration, intraarterial administration, intraperitoneal, limb perfusion (optionally, isolated limb perfusion of the leg and / or arm, see, e.g., Arruda et al., (2005) Blood 105:3458 - 3464), and / or direct intramuscular injection. In certain embodiments, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) is administered to the limbs (arms and / or legs) of a subject (e.g., a subject having a muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intraarterial administration). In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be administered without using "hydrodynamic" techniques.

[0235] Administration of TNA LNPs (e.g., ceDNA vector lipid particles) to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. TNA LNPs (e.g., ceDNA vector lipid particles) can be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., to the left atrium, right atrium, left ventricle, right ventricle), and / or coronary artery perfusion. Administration to the diaphragmatic muscle can be carried out by any preferred method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to smooth muscle can be carried out by any preferred method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment, administration may be carried out to endothelial cells present in, near, and / or on the smooth muscle.

[0236] In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles) are administered to skeletal muscle, diaphragmatic muscle and / or myocardium (e.g., to treat, improve, and / or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure)).

[0237] TNA LNPs (e.g., ceDNA vector lipid particles) can be administered to the CNS (e.g., brain or eye). TNA LNPs (e.g., ceDNA vector lipid particles) 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. TNA LNPs (e.g., ceDNA vector lipid particles) can also be administered to different areas of the eye, such as the retina, cornea, and / or optic nerve. TNA LNPs (e.g., ceDNA vector lipid particles) can be delivered into 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).

[0238] In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles) may be administered to a desired region of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of sugars such as mannitol), intranasal, intraocular, intraocular (e.g., intravitreous, subretinal, anterior chamber), and periocular (e.g., subtenon region) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[0239] According to some embodiments, TNA LNPs (e.g., ceDNA vector lipid particles) are administered in a liquid formulation by direct injection (e.g., stereotactic injection) into a desired region or compartment in the CNS. According to other embodiments, TNA LNPs (e.g., ceDNA vector lipid particles) may be provided by topical application to a desired region or by intranasal administration of an aerosol formulation. Administration to the eye may be performed by topical application of droplets. As a further alternative, the ceDNA vector may be administered as a solid sustained-release formulation (see, for example, U.S. Patent No. 7,201,898, which is incorporated herein in whole by reference). In one embodiment, TNA LNPs (e.g., ceDNA vector lipid particles) can be used for retrograde transport to treat, improve, and / or prevent motor neuron-related diseases and disorders (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, TNA LNPs (e.g., ceDNA vector lipid particles) can be delivered to muscle tissue and from there migrate into neurons.

[0240] In one embodiment, the therapeutic product can be repeatedly administered until an appropriate level of expression is achieved. Therefore, in one embodiment, the therapeutic nucleic acid can be administered and re-administered multiple times. For example, the therapeutic nucleic acid can be administered on day 0. Following the initial treatment on day 0, the therapeutic nucleic acid can be administered for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or approximately 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or approximately 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years. A second dose of medication (re-administration) can be given approximately 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 years later.

[0241] In one embodiment, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be contained within 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. Without limitation, the 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, peptide mimes, nucleic acids, nucleic acid analogs and derivatives, extracts made from biomolecules, or any combination thereof.

[0242] In one embodiment, one or more additional compounds can be therapeutic agents. The therapeutic agent can be selected from any class suitable for therapeutic purposes. Thus, the therapeutic agent can be selected from any class suitable for therapeutic purposes. The therapeutic agent can be selected according to the desired therapeutic purpose and biological effect. For example, in one embodiment, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates)). In one embodiment, the additional compound can be an antimicrobial agent (e.g., an antibiotic or an antiviral compound). In one embodiment, the additional compound can be a compound that modulates the immune response (e.g., an immunosuppressive agent, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In one embodiment, different cocktails of different lipid particles containing different compounds, such as TNAs encoding different proteins or different compounds (such as therapeutic drugs), can be used in the compositions and methods of the present invention. In one embodiment, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressive agent. In some embodiments, the additional compound is immunostimulatory.

Example

[0243] The following examples are provided by way of illustration and not limitation. It will be understood by those skilled in the art that the ionizable lipids can be designed and synthesized using the general synthetic methods described below.

[0244] Example 1: General Synthesis The ionizable lipid of formula (I) was synthesized using a similar synthetic method as described in the general procedure of Scheme 1 below. The variables R 1 , R 1 ’, R 2 , R 2 ’, R 3 , R 3 ’, R 4 , R 4 ’, R 5 , and R 5 ’ are as defined in formula (I). R x is R 4 as defined in formula (I).However, it has two fewer carbon atoms in the carbon chain, and similarly, R x ' is defined by R in equation (I). 4 However, it has two fewer carbon atoms in its carbon chain. [ka]

[0245] In Step 1, 4-dimethylaminopyridine (DMAP), followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), was added to a stirred solution of disulfide 1 and acid 2 in dichloromethane (DCM). The resulting mixture was stirred at room temperature for 2 days, and then a saturated sodium bicarbonate solution was added. The reaction mixture was extracted with DCM. The combined organic phase was washed with brine, dried over sodium sulfate (Na2SO4), and concentrated. The residue was purified by silica gel column chromatography using 0-5% methanol (MeOH) in DCM as the eluent to obtain 3. The reagents and conditions in Step 2 were almost identical to those in Step 1, which yielded the lipid of formula (I) as the final product.

[0246] Example 2: Synthesis of Lipids 1-5 The specific synthesis procedures for lipids 1-5 are shown in Scheme 2 and explained below. Variable R 5 and R 5 ' is defined as shown in equation (I). R x R is defined by equation (I). 4 However, it has two fewer carbon atoms in the carbon chain, and similarly, R x ' is defined by R in equation (I). 4 However, it has two fewer carbon atoms in its carbon chain. [ka]

[0247] Synthesis of O'1,O1-(((disulfandiylbis(ethane-2,1-diyl))bis(piperidine-1,4-diyl))bis(ethane-2,1-diyl))9,9'-di(heptadecan-9-yl)di(nonanedioate) (lipid 5) and 1-(heptadecan-9-yl)9-(2-(1-(2-((2-(4-(2-(oleoyloxy))ethyl))piperidine-1-yl)ethyl)disulfanyl)ethyl)piperidine-4-yl)ethyl)nonanedioate (lipid 1) Referring to Scheme 2, a stirred solution of disulfide 1a (1.17 g, 3.1 mmol) and 9-(heptadecane-9-yloxy)-9-oxononanoic acid (2.0 g, 4.6 mmol) in DCM (50 ml) was mixed with DMAP (565 mg, 4.6 mmol), followed by EDCI (878 mg, 4.6 mmol). The resulting mixture was stirred at room temperature for 2 days, then washed with saturated sodium bicarbonate solution (60 ml) and brine (20 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified twice by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. The fraction containing the desired compound is evaporated to obtain lipid 5 (620 mg, 23%) and 1-(heptadecan-9-yl)9-(2-(1-(2-((2-(4-(2-hydroxyethyl)piperidine-1-yl)ethyl)disulfanyl)ethyl)piperidine-4-yl)ethyl)nonanedioate or compound 3a-D (i.e., R y Compound 3a) (389 mg, 22%) of scheme 2, which is equal to D, was obtained. Lipid 5 1 H-NMR (300MHz, d-chloroform): δ4.85(m, 2H), 4.09(t, 4H), 2.91-2.74(m, 8H), 2.63-2.67(m, 4 H), 2.27-2.22(m, 8H), 1.97(t, 4H), 1.75-1.43(m, 24H), 1.45-1.16(m, 66H), 0.86(t, 12H). MS[M+H] + 1194. 3a-D 1¹H-NMR (300MHz, d-chloroform): δ 4.83 (m, 1H), 4.06 (t, 2H), 3.63 (t, 2H), 2.97-2.69 (m, 9H), 2.66 (m, 4H), 2.25 (t, 4H), 1.93 (t, 4H), 1.76-1.43 (m, 16H), 1.39-1.22 (m, 36H), 0.86 (t, 6H).

[0248] Next, to a stirred solution of disulfide 3a-D (185 mg, 0.23 mmol) and oleic acid (131 mg, 0.46 mmol) in DCM (10 ml), DMAP (55 mg, 0.46 mmol), followed by EDCI (87 mg, 0.46 mmol), was added. The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml) and 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 fraction containing the desired compound was evaporated to obtain lipid 1 (165 mg, 68%). Lipid 1 1 H-NMR (300MHz, d-chloroform): δ5.32(m, 2H), 4.85(m, 1H), 4.09(t, 4H), 2.96-2.77(m, 8H), 2.67-2.53( m, 4H), 2.28-2.20(m, 6H), 2.16-1.92(t, 8H), 1.75-1.47(m, 14H), 1.41-1.13(m, 60H), 0.86(t, 9H). MS[M+H] + 1049.

[0249] Note: Disulfide 1a was synthesized using the procedure described in International Patent Application US2021 / 024413, filed on March 26, 2021.

[0250] Synthesis of O'1,O1-(((disulfanediylbis(ethane-2,1-diyl))bis(piperidine-1,4-diyl))bis(ethane-2,1-diyl))9,9'-dinonyldi(nonanedioate)(lipid 4) Referring to Scheme 2, a stirred solution of disulfide 1a (376 mg, 1 mmol) and 9-(octyloxy)-9-oxononanoic acid (629 mg, 2 mmol) in DCM (25 ml) was mixed with DMAP (244 mg, 2 mmol), followed by EDCI (310 mg, 2 mmol). The resulting mixture was stirred overnight at room temperature, and then saturated sodium bicarbonate solution (20 ml) was added. The reaction mixture was extracted with DCM (2 × 50 ml). The combined organic phase was washed with brine (30 ml), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent to obtain lipid 4 (240 mg, 25%) as a pale yellow solid. 1 ¹H-NMR (300MHz, d-chloroform): δ 4.04-4.09 (m, 8H), 2.5-3.0 (m, 10H), 2.25-2.30 (t, 8H), 2.0 (t, 4H), 1.58-1.90 (m, 24H), 1.20-1.40 (m, 42H), 0.87 (t, 6H).

[0251] Synthesis of 1-(heptadecan-9-yl)9-(2-(1-(2-((2-(4-(2-((9-(nonyloxy)-9-oxononanoyl)oxy)ethyl)piperidine-1-yl)ethyl)disulfanyl)ethyl)piperidine-4-yl)ethyl)nonanediolate (lipid 3) Referring to Scheme 2, a stirred solution of disulfide 1a (376 mg, 1 mmol) and 9-(octyloxy)-9-oxononanoic acid (629 mg, 2 mmol) in DCM (25 ml) was to which DMAP (244 mg, 2 mmol) was added, followed by EDCI (310 mg, 2 mmol). The resulting mixture was stirred overnight at room temperature, and then saturated sodium bicarbonate solution (20 ml) was added. The reaction mixture was extracted with DCM (2 × 50 ml). The combined organic phase was washed with brine (30 ml), dried over Na2SO4, and concentrated. The residue is purified by silica gel column chromatography using 0-5% MeOH in dichloromethane as the eluent to obtain 1-(2-(1-(2-((2-(4-(2-hydroxyethyl))piperidine-1-yl)ethyl)disulfanyl)ethyl)piperidine-4-yl)ethyl)9-nonylnonanediate or compound 3a-C (i.e., R y Compound 3a) (250 mg, 26%) of scheme 2, where =C, was obtained and used directly in the next transformation without characterization.

[0252] Next, to a stirred solution of disulfide 3a-C (650 mg, 0.97 mmol) and 9-(heptadecane-9-yloxy)-9-oxononanoic acid (411 mg, 0.96 mmol) in DCM (50 ml), DMAP (117 mg, 0.96 mmol), followed by EDCI (149 mg, 0.96 mmol), was added. The resulting mixture was stirred at room temperature for 2 days, then washed with water 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 fraction containing the desired compound was evaporated to obtain lipid 3 (420 mg, 40%). 1 ¹H-NMR (300MHz, d-chloroform): δ 4.9 (m, 1H), 4.05-4.09 (m, 6H), 2.80-3.0 (m, 8H), 2.60-2.70 (m, 4H), 2.25-2.27 (m, 8H), 1.92-2.01 (t, 4H), 1.48-1.62 (m, 25H), 1.24-1.40 (m, 52H), 0.87 (t, 9H).

[0253] Synthesis of 1-(heptadecan-9-yl)9-(2-(1-(2-((2-(4-(2-((5-(nonyloxy)-5-oxopentanoyl)oxy)ethyl)piperidine-1-yl)ethyl)disulfanyl)ethyl)piperidine-4-yl)ethyl)nonanedioate (lipid 2) To a stirred solution of disulfide 4 (3.76 g, 10 mmol) and 9-(heptadecane-9-yloxy)-9-oxononanoic acid (2.13 g, 5 mmol) in DCM (100 ml), DMAP (776 mg, 5 mmol), followed by EDCI (610 mg, 5 mmol), was added. The resulting mixture was stirred at room temperature for 2 days, and then saturated sodium bicarbonate solution (40 ml) was added. The reaction mixture was extracted with DCM (2 × 100 ml). The combined organic phase was washed with brine (60 ml), dried over Na2SO4, and concentrated. The residue is purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent to obtain 1-(heptadecan-9-yl)9-(2-(1-(2-((2-(4-(2))-hydroxyethyl)piperidine-1-yl)ethyl)disulfanyl)ethyl)piperidine-4-yl)ethyl)nonanediolate or compound 3a-D (i.e., R y Compound 3a) (1.4g, 36%) of scheme 2, which is equal to D, was obtained. 1 ¹H-NMR (300MHz, d-chloroform): δ 4.90 (m, 1H), 4.09-4.10 (m, 3H), 3.68 (t, 2H), 2.79-2.99 (m, 8H), 2.66 (m, 4H), 2.30 (m, 4H), 2.03 (t, 4H), 1.22-1.78 (m, 55H), 0.86 (s, 6H).

[0254] Next, to a stirred solution of disulfide 3a-D (300 mg, 0.38 mmol) and 5-(nonyloxy)-5-oxopentanoic acid (115 mg, 0.45 mmol) in DCM (20 ml), DMAP (49 mg, 0.4 mmol), followed by EDCI (62 mg, 0.4 mmol), was added. The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml) and 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 fraction containing the desired compound was evaporated to obtain lipid 2 (165 mg, 42%). 1 ¹H-NMR (300MHz, d-chloroform): δ 5.85 (m, 1H), 4.05-4.10 (m, 6H), 2.79-2.88 (m, 8H), 2.63-2.66 (m, 4H), 2.33-2.36 (t, 4H), 2.26-2.33 (t, 4H), 1.94-1.98 (m, 6H), 1.55-1.59 (m, 22H), 1.24-1.40 (m, 48H), 0.84-0.89 (t, 9H).

[0255] Example 2: Preparation of lipid nanoparticles Lipid nanoparticles (LNPs) were prepared in a total lipid to ceDNA weight ratio of approximately 10:1 to 30:1. Briefly, the cationic lipids, non-cationic lipids (e.g., distearoylphosphatidylcholine (DSPC)), components for membrane fusion (sterols, e.g., cholesterol), and complex lipid molecules (PEGylated lipid conjugates, e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimiristoylglycerol (having an average PEG molecular weight of 2000 ("PEG-DMG")) of the present disclosure were solubilized in alcohol (e.g., ethanol) in molar ratios of, for example, 47.5:10.0:40.7:1.8, 47.5:10.0:39.5:3.0, or 47.5:10.0:40.2:2.3. The ceDNA was diluted to the desired concentration with buffer. For example, ceDNA was diluted to a concentration of 0.1 mg / ml to 0.25 mg / ml in a buffer 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 to 1:3 (vol / vol) with a total flow rate exceeding 10 ml / min, for example, using a syringe pump or impingement jet mixer. In one example, the alcoholic lipid solution was mixed with aqueous ceDNA at a flow rate of 12 ml / min at a ratio of approximately 1:3 (vol / vol). The alcohol was removed and the buffer was replaced with PBS by dialysis. Alternatively, the buffer was replaced with PBS using a centrifuge. Alcohol removal and simultaneous buffer exchange were achieved, for example, by dialysis or tangential flow filtration. The obtained lipid nanoparticles are filtered through a sterile filter with 0.2 μm pores.

[0256] In one study, lipid nanoparticles containing exemplary ceDNA were prepared using a lipid solution containing reference lipid A (Coatsome®; ss-OP), DSPC, cholesterol, and DMG-PEG2000 (molar ratio 47.5:10.0:40.7:1.8) as a control. In several studies, tissue-specific targeting ligands, such as N-acetylgalactosamine (GalNAc) or its derivatives, were included in formulations containing reference lipid A and the ionizable lipids of this disclosure. GalNAc derivative ligands, such as triantennae GalNAc (GalNAc3) or tetraantennae GalNAc (GalNAc4), can be synthesized as known in the art (see WO2017 / 084987 and WO2013 / 166121) and can be chemically conjugated to lipids or PEG as well 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). Aqueous solutions of ceDNA in buffer solution were prepared. The lipid solution and the ceDNA solution were mixed on a NanoAssembler using an in-house procedure at a lipid-to-ceDNA ratio of 1:3 (v / v) and a total flow rate of 12 mL / min. [Table 2]

[0257] As a general rule, a polydispersity index (PDI) of 0.15 or less indicates good size uniformity of the formed LNPs. All of LNP2, LNP3, LNP4, LNP5, and LNP6, each containing the lipids of this disclosure, were successfully formulated with PDI values ​​of less than 0.15 and good encapsulation efficiency.

[0258] Example 3: Preclinical in vivo study of lipid nanoparticles Preclinical studies were conducted to evaluate the in vivo expression of ceDNA-luciferase containing LNPs in mice. These LNPs included either reference lipid A as a control or one of the lipids disclosed herein. The study designs and procedures included in these preclinical studies are as follows:

[0259] Materials and methods Species (number, sex, age): CD-1 mice (N=65 plus 5 reserves, male, approximately 4 weeks old upon arrival).

[0260] Cage observation: Cage observations were conducted daily.

[0261] Clinical observations: Clinical observations were conducted approximately 1, 5–6, and 24 hours after administration of the test material on day 0. Additional observations were made in exceptional cases. The body weight of all animals was recorded on days 0, 1, 2, 3, 4, and 7 (before euthanasia), where applicable. Additional body weights were recorded as needed.

[0262] Dosage administration: The test product (LNP: ceDNA-Luc) was administered intravenously into the lateral tail vein to groups 1-38 at a rate of 5 mL / kg on day 0.

[0263] In-life imaging: On day 4, all animals were administered 150 mg / kg (60 mg / mL) of luciferin via intraperitoneal (IP) injection at a rate of 2.5 mL / kg. Within 15 minutes of each luciferin administration, all animals underwent an IVIS imaging session according to the in vivo imaging protocol described below.

[0264] Recovery from anesthesia: Animals were continuously monitored while under anesthesia, during recovery, and until they were moved.

[0265] Intermediate blood sampling: Intermediate blood sampling was performed on all animals on day 0, 5-6 hours after the start of the experiment (between 5.0 and 6.5 hours).

[0266] After collection, the animals received 0.5–1.0 mL of Ringer's lactate solution subcutaneously.

[0267] Whole blood for serum was collected by tail vein nicking, saphenous vein puncture, or orbital sinus puncture (under inhaled isoflurane). The whole blood was collected in a serum separator equipped with a coagulation activator tube and processed into one(1) serum aliquots.

[0268] In vivo imaging protocol ● Luciferin stock powder was stored at -20°C as indicated on the label. ●Formulated luciferin was stored in 1 mL aliquots at 2-8°C and protected from light. ●Formulated luciferin was stable for up to 3 weeks at 2-8°C, protected from light, and stable for approximately 12 hours at room temperature (RT). ● Luciferin was dissolved in PBS in sufficient volume to reach a target concentration of 60 mg / mL, and the pH was adjusted to 7.4 as needed with 5M NaOH (approximately 0.5 μl / mg luciferin) and HCl (approximately 0.5 μL / mg luciferin). ●The appropriate amount was prepared according to the protocol, including at least 50% of the excess.

[0269] Injection and imaging ● Shave the animal's fur (if necessary). ●According to the protocol, 150 mg / kg of luciferin in 60 mg / mL PBS was injected intraperitoneally. ● 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 2.5%). ● Isoflurane anesthesia for imaging sessions: The animals were placed in an isoflurane chamber, and we waited for about 2-3 minutes for the isoflurane to become effective. ○Confirm that the anesthesia level indicator on the side of the IVIS device is in the "ON" position. ○The animal was placed in the IVIS device. The desired acquisition protocol was performed with the highest sensitivity setting.

[0270] Preclinical studies were conducted to evaluate the ability of the exemplary lipids described herein, i.e., lipids 1 to 5, to be used in LNP formulations to encapsulate ceDNA molecules, and to assess in vivo expression and tolerance when mice were administered an LNP-ceDNA-luciferase composition at a dose of 0.25 mg / kg (see, for example, Example 2). As shown in Figure 1, groups of mice treated with ceDNA-luciferase containing LNP2 or LNP3 (i.e., LNPs containing lipids 1 or 3 as described in Example 2 and Table 4, respectively) showed equivalent or greater luciferase expression on day 4 compared to those treated with LNP1 containing reference lipid A (commercially available ss-OP). Mice treated with LNP 4, 5, or 6 showed ceDNA expression far above detection, but the expression levels were approximately 5 to 10 times lower than those observed in groups of mice treated with reference LNP1, LNP2, or 3. Overall, all mice tested in this study showed sufficient tolerance to these LNPs and developed healthily.

[0271] In summary, these data suggest that the lipids of this disclosure can be used to successfully formulate therapeutic nucleic acids, including large, rigid DNA molecules such as ceDNA, with high levels of homogeneity and encapsulation, resulting in optimal, or even superior, ability to deliver therapeutic nucleic acids to target cells in vivo.

[0272] References and Equivalents All patents and other publications cited throughout this application, including references to literature, issued patents, published patent applications, and pending patent applications, are expressly incorporated herein by reference for the purpose of explaining and disclosing methodologies described in such publications that may be used, for example, 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 acknowledgment by the inventor that there is no prior right to such disclosure, either due to prior invention or for any other reason. All statements regarding dates or expressions regarding the content of these documents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or content of these documents.

[0273] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. While specific embodiments and examples of the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be apparent to those skilled in the art. For example, while the steps or functions of a method are 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 may be applied to other procedures or methods as needed. The various embodiments described herein may be combined to provide further embodiments. The aspects of the disclosure may be modified as needed to provide further embodiments of the disclosure using the compositions, functions, and concepts of the above-mentioned references and applications. Furthermore, several modifications to the protein structure may be made without affecting the type or amount of biological or chemical action, taking into consideration the equivalence of biological function. These and other modifications may be made in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0274] Certain elements of any of the embodiments described above can be combined with or replaced by elements of other embodiments. Furthermore, while advantages related to certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments necessarily need to demonstrate such advantages in order to be within the scope of the present disclosure.

[0275] The techniques described herein are further illustrated by the following examples and should not be construed as further limiting them. It should be understood that the present invention is not limited in any way to the specific methodologies, protocols, reagents, etc., described herein, and may be modified accordingly. The terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of the present invention as defined solely by the claims. In certain embodiments, for example, the following items are provided: (Item 1) Ionizable lipids of formula (I): [ka] 、 , or a pharmaceutically acceptable salt thereof, in the formula, R 1 and R 1 ' are each independent of R a (C) is optionally replaced with one or more groups selected from 1 ~C 6 ) is alkylene, R 2 and R 2 ' are each independent of (C 1~ C 2 ) is alkylene, R 3 and R 3 ' are each independent of R b (C) is optionally replaced with one or more groups selected from 1 ~C 6 ) is alkyl, Alternatively, R 2 and R 3 and / or R 2 'and R 3 ', together with the intervening N atoms, form 4- to 7-membered heterocyclines. R 4 and R 4 Each of the 's is interrupted by -C(O)O- (C 2 ~C 6 ) is alkylene, R 5 and R 5 ' are each independent of (C 2 ~C 30 ) Alkyl or (C 2 ~C 30 ) are alkenyls, and each of these can optionally be -C(O)O- or (C 3 ~C 6 ) Interrupted by cycloalkyl, R a and R b These are ionizable lipids, or pharmaceutically acceptable salts thereof, which are halo or cyano, respectively. (Item 2) R 1 and R 1 ', each independently, (C 1 ~C 6 ) an ionizable lipid as described in item 1, which is alkylene, or a pharmaceutically acceptable salt thereof. (Item 3) R 1 and R 1 ', each independently, (C 1 ~C 3 ) an ionizable lipid as described in item 1 or 2, which is alkylene, or a pharmaceutically acceptable salt thereof. (Item 4) The aforementioned lipid is given by formula (II):

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Claims

1. Ionizable lipids represented by formula (X): 【Chemistry 9】 , or a pharmaceutically acceptable salt thereof, in the formula, R ５ is -(CH₂)₃C(O)O(CH₂)₈CH₃, -(CH₂)₅C(O)O(CH₂)₈CH₃, -(CH₂)₇C(O)O(CH₂)₈CH₃, -(CH₂)₇C(O)OCH[(CH₂)₇CH₃]₂, -(CH₂)₇-C₃H₆-(CH₂)₇CH₃, -(CH₂)₇CH₃, -(CH₂)₉CH₃, -(CH₂)₁₆CH₃, -(CH₂)₇CH=CH(CH₂)₇CH₃, or -(CH₂)₇CH=CHCH₂CH=CH(CH₂)₄CH₃, R ５ ’ is -(CH ２ )) ５ C(O)OCH[(CH ２ )) ７ CH ３ , -(CH ２ )) ２ ), -(CH ７ )) ２ C(O)OCH[(CH ７ CH ３ , -(CH ２ )) ２ ), -(CH ５ )) ２ C(O)OCH(CH ２ )[(CH ２ )) ７ CH ３ , -(CH ２ )) ２ ), -(CH ７ )) ２ C(O)OCH(CH ２ )[(CH ２ )) ７ CH ３ , or -(CH ２ )) ２ C(O)O(CH ７ )) ２ CH ３ CH ３ is an ionizable lipid or a pharmaceutically acceptable salt thereof.

2. R ５ 'が、-(CH ２ ) ５ C(O)OCH[(H ２ ) ７ CH ３ ] ２ ,-(EH ２ ) ７ C(O)OCH[(H ２ ) ７ CH ３ ] ２、 - (CH ２ ) ５ C(O)OCH(CH ２ ) ２ [(CH ２ ) ７ CH ３ ] ２ , or - (CH ２ ) ７ C(O)OCH(CH ２ ) ２ [(CH ２ ) ７ CH ３ ] ２ The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

3. The ionizable lipids mentioned above are 【Chemistry 14-1】 【Chemistry 14-2】 An ionizable lipid according to claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of the above.

4. Lipid nanoparticles (LNPs) comprising an ionizable lipid according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, and a nucleic acid.

5. The lipid nanoparticle according to claim 4, wherein the nucleic acid is encapsulated within the lipid nanoparticle.

6. Lipid nanoparticles according to claim 4 or 5, wherein the nucleic acid is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNA (siRNA), antisense oligonucleotides (ASOs), ribozymes, closed-end DNA (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.

7. The lipid nanoparticle according to claim 6, wherein the nucleic acid is siRNA or mRNA.

8. Lipid nanoparticles according to any one of claims 4 to 7, further comprising sterols.

9. The lipid nanoparticle according to claim 8, wherein the sterol is cholesterol or beta-sitosterol.

10. Lipid nanoparticles according to any one of claims 4 to 9, further comprising PEGylated lipids.

11. The lipid nanoparticle according to claim 10, wherein the PEGylated lipid is 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

12. Lipid nanoparticles according to any one of claims 4 to 11, further comprising a noncationic lipid.

13. The aforementioned noncationic lipids are distearoyl-sn-glycerophosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and distearoylphosphatidylethanolamine (DSPE). , monomethylphosphatidylethanolamine (16-O-monomethylPE, etc.), dimethylphosphatidylethanolamine (16-O-dimethylPE, etc.), 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), diylcoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielideylphosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,Lipid nanoparticles according to claim 12, selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

14. The lipid nanoparticles according to claim 13, wherein the noncationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

15. The lipid nanoparticles according to any one of claims 11 to 14, wherein the PEGylated lipid is present in the lipid nanoparticles in a molar percentage of about 1.5% to about 4%.

16. Lipid nanoparticles according to any one of claims 9 to 15, wherein the sterol is present in the lipid nanoparticles in a molar percentage of about 20% to about 40%, and the lipid is present in the lipid nanoparticles in a molar percentage of about 80% to about 60%, or the sterol is present in the lipid nanoparticles in a molar percentage of about 40%, and the lipid is present in the lipid nanoparticles in a molar percentage of about 50%.

17. Lipid nanoparticles according to any one of claims 4 to 7, further comprising cholesterol, PEG-modified lipids, and noncationic lipids.

18. Lipid nanoparticles according to any one of claims 4 to 17, further comprising a tissue-specific targeting ligand.

19. The lipid nanoparticle according to claim 18, wherein the tissue-specific targeting ligand is conjugated to a PEG-modified lipid.

20. The lipid nanoparticle according to claim 18 or 19, wherein the tissue-specific targeting ligand is N-acetylgalactosamine (GalNAc), or a derivative thereof selected from the group consisting of mono-antennary GalNAc, tri-antennary GalNAc, and tetra-antennary GalNAc.

21. The lipid nanoparticles according to claim 19, wherein the PEGylated lipid conjugated with the tissue-specific targeting ligand is present in the lipid nanoparticles in a molar percentage of approximately 1.5%, approximately 1.4%, approximately 1.3%, approximately 1.2%, approximately 1.1%, approximately 1.0%, approximately 0.9%, approximately 0.8%, approximately 0.7%, approximately 0.6%, approximately 0.5%, approximately 0.4%, approximately 0.3%, approximately 0.2%, or approximately 0.1%.

22. Lipid nanoparticles according to any one of claims 4 to 21, having an average diameter of approximately 50 nm to approximately 110 nm, less than approximately 100 nm, less than approximately 75 nm, less than approximately 70 nm, or less than approximately 65 nm.

23. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 4 to 22, and a pharmaceutically acceptable excipient.

24. A pharmaceutical composition comprising a lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable excipient.

25. A composition comprising lipid nanoparticles according to any one of claims 4 to 22, or a pharmaceutical composition according to claim 23 or 24, for use in the treatment of a disorder in a subject.

26. The composition for use according to claim 25, wherein the disorder is a hereditary disorder.

27. The composition for use according to claim 25 or 26, wherein the subject is a human.

28. The aforementioned hereditary disorders include melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency), hemophilia B (coagulation factor IX (FIX) deficiency), cystic fibrosis (CFTR deficiency), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary hepatic metabolic disorders, Lesch-Neyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis, and Niemann-Pick disease. A / B type, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler's disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis type I, mucolipidosis type II / III, mucolipidosis type IV, sialidosis type I, sialidosis type II, glycogen storage disorder type I, glycogen storage disorder type II (Pompe disease), Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Fabry disease, cystinosis, Batten disease, aspartylglucosamineuria, Salah 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, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophy of epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, all infancy The following conditions are selected from the group consisting of holistic arterial calcification (GACI), Leber congenital amaurosis (LCA), Stargardt macular dystrophy (ABCA4 deficiency), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), progressive familial intrahepatic cholestasis type II (ABCB11 deficiency), progressive familial intrahepatic cholestasis type III (ABCB4 deficiency), progressive familial intrahepatic cholestasis type IV (TJP2 deficiency), and cathepsin A deficiency.A composition for use according to claim 26 or 27.

29. The composition for use according to claim 28, wherein the mucopolysaccharidosis is selected from the group consisting of Hurler syndrome (MPS I), Schayet syndrome (MPS I-S), Hurler-Scheyet syndrome (MPS I H-S type), Hunter syndrome (MPS II), Sanfilippo syndrome type A (MPS IIIIA), Sanfilippo syndrome type B (MPS IIIB), Sanfilippo syndrome type C (MPS IIIC), Sanfilippo syndrome type D (MPS IIID), Morquio syndrome A (MPS IVA), Morquio syndrome type B (MPS IVB), Maloto-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), and hyaluronidase deficiency (MPS IX).

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