Novel lipids and their nanoparticle compositions

Abstract

A lipid having the chemical formula (I) and a pharmaceutically acceptable salt thereof are provided herein: (I) [In the formula, R1, R2, a and b are as defined herein]. A lipid nanoparticle (LNP) composition comprising a lipid having the formula (I) and a capsid-free nonviral vector (e.g., ceDNA) is also provided herein. In any one aspect of the embodiments or examples thereof, such LNPs may be used to deliver a capsid-free nonviral DNA vector to a target site of interest (e.g., a cell, tissue, organ, etc.).

Description

Technology Field

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 000,990 filed March 27, 2020, the full text of which is incorporated herein by reference.

[0003] Sequence list

[0004] The present application includes a sequence list submitted as an electronic document in ASCII format, the entirety of which is incorporated herein by reference. The name of the said ASCII copy created on March 18, 2021, is 131698_07720_SL.txt, and its size is 417 bytes. Background Technology

[0005] Gene therapy aims to improve clinical outcomes for patients suffering from genetic disorders or acquired diseases caused by abnormal gene expression profiles. To date, various types of gene therapy have been developed to deliver therapeutic nucleic acids to patients' cells as drugs to treat these diseases.

[0006] The delivery and expression of a correction gene into a patient's target cells can be performed through various methods, including the use of engineered viral gene delivery vectors and potentially plasmids, minigenes, oligonucleotides, minicircles, or various closed DNAs. Among the many available viral-derived vectors (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated virus (rAAV) is accepted as a versatile and relatively reliable vector in gene therapy. However, viral vectors such as adeno-associated vectors can be highly immunogenic and can induce humoral and cell-mediated immunity that may impair efficacy, particularly in relation to re-administration.

[0007] Non-viral gene transfer has certain disadvantages associated with viral transduction, particularly humoral and cellular immune responses to viral structural proteins forming vector particles, and any new ( de novo It avoids the disadvantages caused by viral gene expression. One of the non-viral gene delivery technologies is to use cationic lipids as a carrier.

[0008] Ionizable lipids are largely composed of amine moiety and lipid moiety, and cationic amine moiety and polyvalent anionic nucleic acid electrostatically interact to form a positively charged liposome or lipid membrane structure. Therefore, absorption into the cell is facilitated, and nucleic acid is delivered into the cell.

[0009] Some widely used ionizable lipids are cationic lipids such as CLinDMA, DLinDMA (also known as DODAP), and DOTAP. For reference, these lipids have been used for siRNA delivery to the liver, but there have been problems with hepatotoxicity at high doses and suboptimal delivery efficiency. Given the current disadvantages of cationic lipids, there is a need in the industry to provide lipid scaffolds that not only demonstrate enhanced efficacy along with reduced toxicity, but also improve pharmacokinetics and intracellular kinetics, such as cellular uptake and nucleic acid release from the lipid carrier.

[0010] In one embodiment, chemical formula ( I Ionizable lipids having ) and pharmaceutically acceptable salts thereof are provided herein:

[0011]

[0012] [In the middle of the meal, R 1 , R 2 , a and b are as defined herein].

[0013] A pharmaceutical composition comprising a disclosed ionizable lipid or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier is also provided.

[0014] Another aspect of the present disclosure relates to a composition comprising lipid nanoparticles (LNPs) comprising a nucleic acid and an ionizable lipid or a pharmaceutically acceptable salt thereof as described herein. In one embodiment of any of the aspects or embodiments of the present invention, the nucleic acid is encapsulated in an ionizable lipid. In a specific embodiment, the nucleic acid is closed DNA (ceDNA).

[0015] According to some embodiments of any of the aspects or embodiments of the present invention, the LNP further comprises a sterol. According to some embodiments of any of the aspects or embodiments of the present invention, the sterol may be cholesterol or beta-sitosterol.

[0016] According to some embodiments of any of the aspects or embodiments of the present invention, cholesterol is present in a molar percentage 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 a molar percentage 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 of any of the aspects or embodiments of the present invention, cholesterol is present in a molar percentage of about 20% to about 40%, e.g., about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%, and ionizable lipids are present in a molar percentage of about 80% to about 60%, e.g., about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about It is present in molar percentages of 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 embodiment of any of the aspects or embodiments of the present invention, cholesterol is present in a molar percentage of about 40%, and ionizable lipids are present in a molar percentage of about 50%.

[0017] According to some embodiments of any of the aspects or embodiments of the present invention, the composition further comprises cholesterol or a PEG-lipid conjugate and a non-cationic lipid. According to some embodiments of any of the aspects or embodiments of the present invention, the PEG-lipid conjugate is present in an amount of about 1.5% to about 3%, e.g., about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%, about 2.25% to about 3%, about 2.25% to about 2.75%, or about 2.25% to about 2.5%. According to some embodiments of any of the aspects or embodiments of the present invention, the PEG-lipid conjugate is present in an amount of about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3%. According to some embodiments of any of the aspects or embodiments of the present invention, cholesterol is present in a molar percentage of about 30% to about 50%, e.g., about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 20% to about 40%, about 40% to about 50%, or about 45% to about 50%. According to some embodiments of any of the aspects or embodiments of the present invention, cholesterol is present in a molar percentage of about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%.

[0018] According to some embodiments of any of the aspects or embodiments of the present invention, the LNP further comprises a polyethylene glycol (PEG)-lipid. According to some embodiments of any of the aspects or embodiments of the present invention, the PEG-lipid is 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG). According to some embodiments of any of the aspects or embodiments of the present invention, the LNP further comprises a non-cationic lipid.According to some embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid is 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), distearoylphosphatidylethanolamine (DSPE), Monomethylphosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethylphosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dielucoyylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-dipitanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); selected from the group consisting of 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 of any of the aspects or embodiments of the present invention, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

[0019] According to some embodiments of any of the aspects or embodiments of the present invention, the PEG-lipid conjugate is present in an amount of about 1.5% to about 4%, e.g., about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, and about 2% to about 2.25%. According to some embodiments of any of the aspects or embodiments of the present invention, the PEG-lipid conjugate is present in an amount of about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3%. According to some embodiments of any of the aspects or embodiments of the present invention, the ionizable lipid is present in a molar percentage of about 42.5% to about 62.5%. According to some embodiment of any of the aspects or embodiments of the present invention, the ionizable lipid is about 42.5%, about 43%, about 43.5%, about 44%, about 44.5%, about 45%, about 45.5%, about 46%, about 46.5%, about 47%, about 47.5%, about 48%, about 48.5%, about 49%, about 49.5%, about 50%, about 50.5%, about 51%, 51.5%, about 52%, about 52.5%, about 53%, about 53.5%, about 54%, about 54.5%, about 55%, about 55.5%, about 56%, about 56.5%, about 57%, 57.5%, about 58%, about It is present in molar percentages of 58.5%, about 59%, about 59.5%, about 60%, about 60.5%, about 61%, about 61.5%, about 62%, or about 62.5%. According to some embodiments of any of the aspects or embodiments of the present invention, the non-cationic lipid is about 2.5% to about 12%.It is present in a molar percentage of 5%. According to some embodiments of any of the aspects or embodiments of the present invention, cholesterol is present in a molar percentage of about 40%, ionizable lipids are present in a molar percentage of about 52.5%, non-cationic lipids are present in a molar percentage of about 7.5%, and PEG-lipids are present in a molar percentage of about 3%.

[0020] According to some embodiment of any of the aspects or embodiments of the present invention, the LNP composition further comprises dexamethasone palmitate.

[0021] According to some embodiment of any aspect or embodiment of the present invention, the size of the LNP has a diameter of about 50 nm to about 110 nm, e.g., about 50 nm to about 100 nm, about 50 nm to about 95 nm, about 50 nm to about 90 nm, about 50 nm to about 85 nm, about 50 nm to about 80 nm, about 50 nm to about 75 nm, about 50 nm to about 70 nm, about 50 nm to about 65 nm, about 50 nm to about 60 nm, about 50 nm to about 55 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 95 nm, about 60 nm to about 90 nm, about 60 nm to about 85 nm, about 60 nm to about 80 nm, about 60 nm to about 75 nm, about The range is 60 nm to about 70 nm, about 60 nm to about 65 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 nm to about 95 nm, about 70 nm to about 90 nm, about 70 nm to about 85 nm, about 70 nm to about 80 nm, about 70 nm to about 75 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, about 80 nm to about 95 nm, about 80 nm to about 90 nm, about 80 nm to about 85 nm, about 90 nm to about 110 nm, or about 90 nm to about 100 nm.According to some embodiment of any of the aspects or embodiments of the present invention, the size of the LNP is less than about 100 nm, e.g., less than about 105 nm, less than about 100 nm, less than about 95 nm, less than about 90 nm, less than about 85 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm, less than about 15 nm, or less than about 10 nm. According to some embodiments of any of the aspects or embodiments of the present invention, the size of the LNP is less than about 70 nm, e.g., less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm, less than about 15 nm, or less than about 10 nm. According to some embodiments, the size of the LNP is less than about 60 nm, e.g., less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm, less than about 15 nm, or less than about 10 nm.

[0022] According to some embodiment of any of the embodiments or types of the present invention, the ratio of total lipids to nucleic acids of the LNP composition is about 10:1. According to some embodiment of any of the embodiments or types of the present invention, the ratio of total lipids to nucleic acids of the LNP composition is about 20:1. According to some embodiment of any of the embodiments or types of the present invention, the ratio of total lipids to nucleic acids of the composition is about 30:1. According to some embodiment of any of the embodiments or types of the present invention, the ratio of total lipids to nucleic acids of the composition is about 40:1. According to some embodiment of any of the embodiments or types of the present invention, the ratio of total lipids to nucleic acids of the composition is about 50:1.

[0023] According to some embodiments of any of the embodiments of the present invention, the LNP further comprises a tissue targeting moiety. The tissue targeting moiety may be a peptide, oligosaccharide, etc., which can be used for the delivery of the LNP to one or more specific tissues, such as cancer, liver, CNS, or muscle. According to some embodiments of any of the embodiments of the present invention, the tissue targeting moiety is linked to a PEG-lipid conjugate. According to some embodiments of any of the embodiments of the present invention, the tissue targeting moiety is a ligand for a liver-specific receptor. According to some embodiments of any of the embodiments of the present invention, the ligand for a liver-specific receptor used for liver targeting is an oligosaccharide such as N-acetylgalactosamine (GalNAc).

[0024] According to some embodiment of any of the aspects or embodiments of the present invention, GalNAc-linked GalNAc-linked PEG-lipid conjugates are present in lipid nanoparticles at a molar percentage of 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%. According to some embodiment of any of the aspects or embodiments of the present invention, GalNAc-linked PEG-lipid conjugates are present in LNPs at a molar percentage of 0.2%. According to some embodiment of any of the aspects or embodiments of the present invention, GalNAc-linked PEG-lipid conjugates are present in LNPs at a molar percentage of 0.3%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of 0.4%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of 0.5%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of 0.6%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of 0.7%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of 0.8%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of 0.9%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of 1.0%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP at a molar percentage of about 1.It is present in a molar percentage of 5%. According to some embodiment of any of the aspects or embodiments of the present invention, the GalNAc-linked PEG-lipid conjugate is present in LNP in a molar percentage of 2.0%.

[0025] According to some embodiments of any of the aspects or embodiments of the present invention, the LNP composition is prepared in a buffer such as malic acid. In some embodiments of any of the aspects and embodiments of the present invention, the composition is prepared in about 10 mM to about 30 mM, e.g., about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 25 mM malic acid. According to some embodiments of any aspect or embodiment of the present invention, the composition is prepared from 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, about 21 mM malic acid, about 22 mM malic acid, about 23 mM malic acid, about 24 mM malic acid, about 25 mM malic acid, about 26 mM malic acid, about 27 mM malic acid, about 28 mM malic acid, about 29 mM malic acid, or about 30 mM malic acid. According to some embodiments of any aspect or embodiment of the present invention, the composition comprises about 20 mM malic acid.

[0026] According to some embodiments of any of the aspects or embodiments of the present invention, the LNP composition is prepared in a solution containing about 30 mM to about 50 mM NaCl, e.g., about 30 mM to about 45 mM NaCl, about 30 mM to about 40 mM NaCl, about 30 mM to about 35 mM NaCl, about 35 mM to about 45 mM NaCl, about 35 mM to about 40 mM NaCl, or about 40 mM to about 45 mM NaCl. According to some embodiments of any of the aspects or embodiments of the present invention, the LNP composition is prepared in a solution containing about 30 mM NaCl, about 35 mM NaCl, about 40 mM NaCl, or about 45 mM NaCl. According to some embodiments of any of the aspects or embodiments of the present invention, the LNP composition is prepared in a solution having about 40 mM NaCl.

[0027] According to some embodiments of any aspect or embodiment of the present invention, the LNP composition comprises about 20 mM to about 100 mM MgCl2, e.g., 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, about 20 mM to about 30 mM MgCl2, about 320 mM to about 90 mM MgCl2, about 30 mM to about 80 mM MgCl2, about 30 mM to about 70 mM MgCl2, about 30 mM to about 60 mM MgCl2, about 30 mM to about 50 mM MgCl2, about 30 mM to about 40 mM MgCl2, about 40 mM to about 90 mM MgCl2, about 40 mM to about 80 mM MgCl2, about 40 mM to about 70 mM MgCl2, about 40 mM to about 60 mM MgCl2, about 40 mM to about 50 mM MgCl2, about 50 mM to about 90 mM MgCl2, about 50 mM to about 80 mM MgCl2, about 50 mM to about 70 mM MgCl2, about 50 mM to about 60 mM MgCl2, about 60 mM to about 90 mM MgCl2, about 60 mM to about 80 mM MgCl2, about 60 mM to about 80 mM MgCl2, about 60 mM to about 70 mM MgCl2, about 70 mM to about 90 mM MgCl2, about 70 mM to about 90 mM MgCl2, about 70 mM to about 80 mM MgCl2, or about 80 mM to about 90 mM MgCl2 It is manufactured in a solution containing

[0028] According to some embodiment of any of the aspects or embodiments of the present invention, ceDNA is a closed linear dimer DNA. According to some embodiment of any of the aspects or embodiments of the present invention, ceDNA comprises an expression cassette comprising a promoter sequence and a transposable gene.

[0029] According to some embodiment of any of the aspects or embodiments of the present invention, ceDNA comprises an expression cassette containing a polyadenylation sequence.

[0030] According to some embodiment of any of the embodiments of the present invention, ceDNA comprises at least one inverted terminal repeat (ITR) that flanks the 5' end or the 3' end of the expression cassette. According to some embodiment of any of the embodiments of the present invention, the expression cassette is flanked by two ITRs, wherein the two ITRs comprise one 5' ITR and one 3' ITR. According to some embodiment of any of the embodiments of the present invention, the expression cassette is connected to the ITR at the 3' end (3' ITR). According to some embodiment of any of the embodiments of the present invention, the expression cassette is connected to the ITR at the 5' end (5' ITR). According to some embodiment of any of the embodiments of the present invention, at least one of the 5' ITR and the 3' ITR is a wild-type AAV ITR. According to some embodiment of any of the aspects or embodiments of the present invention, at least one of the 5' ITR and the 3' ITR is a modified ITR. According to some embodiment of any of the aspects or embodiments of the present invention, ceDNA further comprises a spacer sequence between the 5' ITR and the expression cassette.

[0031] According to some embodiment of any of the embodiments or types of the present invention, ceDNA further comprises a spacer sequence between the 3' ITR and the expression cassette. According to some embodiment of any of the embodiments or types of the present invention, the length of the spacer sequence is at least 5 base pairs. According to some embodiment of any of the embodiments or types of the present invention, the length of the spacer sequence is 5 to 100 base pairs. According to some embodiment of any of the embodiments or types of the present invention, the length of 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. According to some embodiments of any of the aspects or embodiments of the present invention, the length of the spacer sequence is 5 to 500 base pairs.According to some embodiment of any of the aspects or embodiments of the present invention, the length of the spacer sequence is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, The length is 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, or 495 base pairs.

[0032] According to some embodiments of any of the aspects or embodiments of the present invention, ceDNA has a nick or a gap.

[0033] According to some embodiments of any of the aspects or embodiments of the present invention, the ITR is an ITR derived from an AAV serotype, an ITR derived from a goose virus ITR, an ITR derived from a B19 virus ITR, and a wild-type ITR derived from a parvovirus. According to some embodiments of any of the aspects or embodiments of the present invention, the AAV serotype is selected from the group comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0034] According to some embodiment of any of the aspects or embodiments of the present invention, the ITR is a mutant ITR, and ceDNA optionally comprises an additional ITR different from the first ITR. According to some embodiment of any of the aspects or embodiments of the present invention, ceDNA comprises two mutant ITRs at both the 5' and 3' ends of the expression cassette, optionally wherein the two mutant ITRs are symmetric mutants. According to some embodiment of any of the aspects or embodiments of the present invention, ceDNA is CELiD, a DNA-based minicircle, MIDGE, ministring DNA, dumbbell-shaped linear dichotomous closed DNA comprising two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette, or doggybone™ DNA. According to some embodiment of any of the aspects or embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0035] According to some embodiments, the present disclosure provides a method for treating a genetic disorder in a subject suffering from a genetic disorder, comprising administering an effective amount of a pharmaceutical composition according to any of the embodiments or examples of the present invention to a subject suffering from a genetic disorder. According to some embodiments of any of the embodiments or examples of the present invention, the subject is a human. According to some embodiments of any of the aspects or embodiments of the present invention, genetic 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 defect), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary hepatic metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharide storage disorders (e.g., Hurler syndrome (MPS type I), Scheie syndrome (MPS type I S), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo syndrome types A, B, C and D (MPS type III A, B, C and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS type VI), Sly syndrome (MPS type VII), Hyaluronidase deficiency (MPS type IX), Niemann-Pick disease types A / B, C1 and C2, Fabry disease, Schindler disease,GM2-gangliosidosis type II (Sandhoff Disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III and IV, sialic acidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neurogenic ceroid lipofuscinoses (CLN1-8, INCL and LINCL), sphingolipidosis, galactosialic acidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy dystrophy)(ABCA4),It is selected from the group consisting of ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency. According to some embodiments of any of the embodiments or examples of the present invention, the genetic disorder is Leber congenital amaurosis (LCA). According to some embodiments of any of the embodiments or examples of the present invention, the LCA is LCA10. According to some embodiments of any of the embodiments or examples of the present invention, the genetic disorder is Niemann-Pick disease. According to some embodiments of any of the embodiments or examples of the present invention, the genetic disorder is Stargardt macular dystrophy. According to some embodiments of any of the embodiments or types of the present invention, the genetic disorder is glucose-6-phosphatase (G6Pase) deficiency (glycogen storage disease type I) or Pompe disease (glycogen storage disease type II). According to some embodiments of any of the embodiments or types of the present invention, the genetic disorder is hemophilia type A (factor VIII deficiency). According to some embodiments of any of the embodiments or types of the present invention, the genetic disorder is hemophilia type B (factor IX deficiency). According to some embodiments of any of the embodiments or types of the present invention, the genetic disorder is Hunter syndrome (mucopolysaccharidosis type II). According to some embodiments of any of the embodiments or types of the present invention, the genetic disorder is cystic fibrosis. According to some embodiment of any of the aspects or embodiments of the present invention, the genetic disorder is dystrophic epidermolysis bullosa (DEB). According to some embodiment of any of the aspects or embodiments of the present invention,The genetic disorder is phenylketonuria (PKU). According to some embodiment of any of the embodiments or examples of the present invention, the genetic disorder is progressive familial intrahepatic cholestasis (PFIC). According to some embodiment of any of the embodiments or examples of the present invention, the genetic disorder is Wilson's disease. According to some embodiment of any of the embodiments or examples of the present invention, the genetic disorder is Gaucher disease types I, II, and III. According to some embodiment of any of the embodiments or examples of the present invention, the genetic disorder is age-related macular degeneration. According to some embodiment of any of the embodiments or examples of the present invention, the genetic disorder is ornithine transcarbamylase deficiency. According to some embodiment of any of the embodiments or examples of the present invention, the genetic disorder is retinitis pigmentosa (RP1). According to some embodiment of any of the embodiments or examples of the present invention, the genetic disorder is Usher syndrome. According to some embodiment of any of the aspects or embodiments of the present invention, the genetic disorder is lysosomal acid lipase (LAL) deficiency. Brief explanation of the drawing

[0036] Embodiments of the present disclosure, briefly outlined above and discussed in more detail below, may be understood by reference to exemplary embodiments of the present disclosure illustrated in the accompanying drawings. However, the accompanying drawings are merely illustrative of typical embodiments of the present disclosure and should not be construed as limiting the scope of the present disclosure, and the present disclosure may allow for other equally effective embodiments. Fig. 1 It shows the improvement in ceDNA-luc expression achieved using the disclosed lipid nanoparticles (e.g., LNP 5 containing lipid 1 and LNP 6 containing lipid 3) compared to SS-OP (e.g., LNP 1, LNP 2, and LNP 7 to LNP 12), as observed in Study A. Fig. 2It shows the improvement in ceDNA-luc expression achieved using the disclosed lipid nanoparticles (e.g., LNP 16 containing lipid 2, LNP 17 containing lipid 1, and LNP 18 containing lipid 3) compared to SS-OP (i.e., LNP 13), as observed in Study B. Fig. 3 This shows an improvement in responsiveness to increased dosage levels, where the increased dosage administered to mice induces a greater increase in ceDNA-luc expression achieved using the initiated lipid nanoparticles (e.g., LNP 20 containing lipid 1) compared to SS-OP (i.e., LNP 19), as observed in Study C. Fig. 4a It shows the improvement in ceDNA-luc expression achieved using the disclosed lipid nanoparticles (e.g., LNP 24 containing lipid 6, LNP 25 containing lipid 7, and LNP 26 containing lipid 8) compared to SS-OP (i.e., LNP 23), as observed in Study D. Fig. 4b It shows improved tolerability (measured by changes in body weight) in mice when using the disclosed lipid nanoparticles (e.g., LNP 24 containing lipid 6, LNP 25 containing lipid 7, and LNP 26 containing lipid 8) compared to ionizable lipid A (i.e., LNP 22) used as a control. Fig. 5a It shows the improvement in ceDNA-luc expression achieved using the disclosed lipid nanoparticles (e.g., LNP 28 containing lipid 9 and LNP 29 containing lipid 10) compared to SS-OP (i.e., LNP 27). Fig. 5b Is, Fig. 5a It shows that the improvement in ceDNA-luc expression described in [figure] did not impair the tolerability of lipid nanoparticles initiated in mice. Specific details for implementing the invention

[0037] The present disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs), such as viral or non-viral vectors (e.g., closed DNA), which can move from the cytoplasm to the nucleus of a cell and maintain high levels of expression. For example, the immunogenicity associated with viral vector-based gene therapy limits the number of patients who can be treated due to pre-existing background immunity, and also prevents re-administration to patients to titrate to an effective level for each patient or to maintain efficacy over a long period. Furthermore, other nucleic acid modalities have significant immunogenicity issues due to innate DNA or RNA sensing mechanisms that trigger a series of immune responses. Due to the lack of pre-existing immunity, the TNA lipid particles (e.g., lipid nanoparticles) described herein allow for additional doses of TNA, such as mRNA, siRNA, or ceDNA, when necessary, and further expand patient access, including for pediatric populations that may require subsequent doses during tissue growth. Furthermore, the present disclosure has found that TNA lipid particles (e.g., lipid nanoparticles) comprising a lipid composition, in particular one comprising one or more tertiary amino groups and a disulfide bond, 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 packaging constraints imposed by space within the viral capsid, theoretically, the only size limitation of the TNA lipid particles (e.g., lipid nanoparticles) lies in the efficiency of expression (e.g., DNA replication or RNA translation) in the host cell.

[0038] One of the biggest obstacles in the development of treatments for rare diseases, in particular, is the large number of individual conditions. Approximately 350 million people on Earth live with rare disorders, defined by the National Institutes of Health as disorders or conditions with fewer than 200,000 diagnosed individuals. About 80% of these rare disorders are of genetic origin, and about 95% of them have no FDA-approved treatment (rarediseases.info.nih.gov / diseases / pages / 31 / faqs-about-rare-diseases). One of the benefits of the TNA lipid particles (e.g., lipid nanoparticles) described herein is that they provide an approach that can be rapidly applied to many diseases that can be treated in a specific way with TNA, particularly rare monogenetic diseases that could significantly change the current state of treatment for many genetic disorders or diseases.

[0039] I. Definition

[0040] The term "alkyl" refers to a monovalent saturated, straight-chain (i.e., unbranched) or branched-chain hydrocarbon radical. Exemplary alkyl groups include, but are not limited to, ethyl, propyl, isopropyl, 2-methyl-1-butyl, 3-methyl-2-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decanyl, undecanyl, dodecanyl, tridecanyl, tetradecanyl, pentadecanyl, hexadecanyl, heptadecanyl, octadecanyl, nonadecanyl, eicosanyl, etc.

[0041] The term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon radical having one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl radical is " Sis " and " Trans It includes radicals having "E" and "Z" orientations according to the orientation, or alternative nomenclature.

[0042] As used herein, the term “pharmaceuticalally acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt of the ionizable lipid of the present invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisinates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates "mesylates", ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoate (i.e., 1,1'-methylenebis(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., Magnesium salts and ammonium salts are included. Pharmaceutically acceptable salts may include the incorporation of another molecule, such as acetate ions, succinate ions, or other counterions. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, they may have multiple counterions. Thus, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0043] The term “about” as used in this specification and the appended claims is intended to include a difference 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 when indicating a measurable value such as a quantity, a temporal duration, etc., because such a difference is suitable for carrying out the method disclosed herein.

[0044] As used herein, “comprising,” “including,” and “consisting of” mean synonymous with “to be included,” “including,” or “contain,” and are inclusive or open terms specifying the presence of what follows, e.g., components, and do not exclude or exclude the presence of additional unquoted components, features, elements, members, or steps known in the art or disclosed herein.

[0045] The term “consisting of” refers to a composition, method, process, and each component thereof as described herein, excluding any element not cited in the description of the embodiments.

[0046] As used herein, the term “essentially composed of” indicates elements necessary for a given embodiment. The term allows for the presence of additional elements that do not substantially affect the basic and novel or functional feature(s) of the embodiment of the invention.

[0047] As used herein, the terms “administration,” “administering,” and variations thereof indicate the introduction of a composition or agent (e.g., nucleic acids, particularly ceDNA) to a subject, and include the simultaneous and sequential introduction of one or more compositions or agents. “Administration” may refer, for example, to treatment, pharmacokinetics, diagnosis, research, placebo, and experimental methods. “Administration” also In vitro and In vitroIncluding treatment. Introduction of the composition or agent into the target is made by any suitable route, including oral, pulmonary, nasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, lymphatic, intratumoral, or local. Administration includes self-administration and other administration by means thereof. Administration may be performed by any suitable route. A suitable route of administration enables the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the target's vein. In any one aspect of the embodiments or examples thereof, "administration" refers to therapeutic administration.

[0048] As used herein, phrases such as "anti-therapeutic nucleic acid immune response," "anti-delivery vector immune response," "immune response to therapeutic nucleic acid," and "immune response to delivery vector" refer to any unintended immune response to therapeutic nucleic acids of viral or non-viral origin. In some embodiments of any of the aspects and embodiments of the present invention, the unintended immune response is an antigen-specific immune response to the viral delivery vector itself. In some embodiments of any of the aspects and embodiments of the present invention, the immune response is specific to the delivery 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 delivery vector. In other embodiments, the immune response is specific to the CpG content of the delivery vector.

[0049] As used herein, the terms “carrier” and “excipient” include any and all solvents, dispersion media, vehicles, coating agents, diluents, antimicrobial and antifungal agents, isotonic agents and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is known in the art. Additional active ingredients may also be incorporated into the composition. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce toxic, allergic, or similar adverse reactions when administered to a host.

[0050] As used herein, the term “ceDNA” refers to capsid-free closed linear double-stranded (ds) dimer DNA for synthetic or other non-viral gene delivery. A detailed description of ceDNA is provided in International Application PCT / US2017 / 020828 filed March 3, 2017, the full contents of which are expressly incorporated herein by reference. Specific methods for producing ceDNA, including various reverse end repeat (ITR) sequences and configurations using cell-based methods, are provided in Example 1 of International Application PCT / US18 / 49996 filed September 7, 2018 and PCT / US2018 / 064242 filed December 6, 2018, the full text of which is incorporated herein by reference. Specific methods for producing synthetic ceDNA vectors comprising various ITR sequences and configurations are described, for example, in International Application PCT / US2019 / 14122 filed January 18, 2019, the entire contents of which are incorporated herein by reference. The terms “ceDNA vector” and “ceDNA” as used herein are used interchangeably. According to some embodiments of any of the embodiments or examples thereof, ceDNA is a closed linear dimer (CELiD) CELiD DNA. According to some embodiments of any of the embodiments or examples thereof, ceDNA is a DNA-based minicircle. According to some embodiments of any of the embodiments or examples thereof, ceDNA is a minimalistic immunological-defined gene expression (MIDGE) vector. According to some embodiments of any of the embodiments or examples thereof, ceDNA is a ministring DNA. According to some embodiment of any of the aspects or embodiments of the present invention, ceDNA is a dumbbell-shaped linear dichotomous closed DNA comprising two hairpin structures of ITRs at the 5' and 3' ends of an expression cassette.According to some embodiments of any of the aspects or embodiments of the present invention, ceDNA is doggybone™ DNA.

[0051] The term "ceDNA-vacmid" as used herein refers to an infectious vacurovirus genome containing a ceDNA genome as an intermolecular duplex that can replicate in E. coli as a plasmid and thus act as a shuttle vector for vacuroviruses.

[0052] The term "ceDNA-vacurovirus" as used herein refers to a vacurovirus containing a ceDNA genome as an intermolecular duplex within the vacurovirus genome.

[0053] The terms “ceDNA-vacurovirus-infected insect cells” and “ceDNA-BIIC” as used herein are used interchangeably and refer to invertebrate host cells infected with ceDNA-vacurovirus (including, but not limited to, insect cells (e.g., Sf9 cells)).

[0054] As used herein, the term “ceDNA genome” refers to an expression cassette further comprising at least one reverse-terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments of any of the aspects and embodiments of the present invention, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular dimer polynucleotide of DNA.

[0055] The terms “DNA regulatory sequence,” “control element,” and “regulatory element” as used herein are used interchangeably and refer to transcription and translation control sequences, such as promoters, enhancers, polyadenylation signals, termination factors, proteolysis signals, etc., that provide a non-coding sequence (e.g., DNA targeting RNA) or a coding sequence (e.g., site-specific modification polypeptide or Cas9 / Csn1 polypeptide) and / or regulate the transcription thereof, or regulate the transcription of an encoded polypeptide.

[0056] As used herein, the term “exogenous” refers to a substance present in a cell that is not of its original source. As used herein, the term “exogenous” may refer to 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 process involving human hands, which is not typically found, and which humans intend to introduce into such a cell or organism. Alternatively, “exogenous” may refer to a nucleic acid or polypeptide introduced into a biological system, such as a cell or organism, by a process involving human hands, which is found at relatively low levels, and which humans intend to increase the amount of the nucleic acid or polypeptide within such cell or organism, for example, to produce ectopic expression or levels. In contrast, as used herein, the term “endogenous” refers to a substance inherent to a biological system or cell.

[0057] As used herein, the term “expression” refers to cellular processes involved in the production of RNA and proteins, and, where appropriate, protein secretion, including, where applicable and, but not limited to, transcription, transcriptome processing, translation and protein folding, modification and processing. As used herein, the phrase “expression product” includes RNA transcribed from a gene (e.g., transgenic), and polypeptides obtained by the translation of mRNA transcribed from a gene.

[0058] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence may often, but not necessarily, be heterogeneous to the host cell. The expression vector may contain additional elements, and, for example, because the expression vector may have two replication systems, it may be maintained in two organisms, for example, for expression in human cells and for cloning and amplification in a prokaryotic host. The expression vector may be a recombinant vector.

[0059] As used herein, the terms “expression cassette” and “expression unit” are used interchangeably and refer to a heterogeneous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct the transcription of a transgene of a DNA vector, e.g., a synthetic AAV vector. Suitable promoters include, e.g., tissue-specific promoters. The promoter may also be of AAV origin.

[0060] As used herein, the term “planking” refers to the relative position of one nucleic acid sequence to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked by A and C. This applies equally to the array AxBxC. Thus, the flanking sequence may be located before or after the flanked sequence, but it does not need to be adjacent to or immediately adjacent to the flanked sequence. In any embodiment of the aspects or embodiments of the present invention, the term “planking” refers to the terminal repeat sequences at each end of a linear single-stranded synthetic AAV vector.

[0061] The term "gene" as used in this document is, In vitro or In vivo at It is widely used to represent any segment of nucleic acid associated with the expression of a given RNA or protein. Thus, a gene includes a region encoding the expressed RNA (typically containing a polypeptide-coding sequence) and often regulatory sequences necessary for its expression. Genes can be obtained from various sources, including cloning from sources of interest or synthesis from known or predicted sequence information, and may specifically contain sequences designed to have desired parameters.

[0062] As used herein, the phrases “genetic disease” or “genetic disorder” refer to a disease caused by one or more abnormalities within the genome, either partially or completely, directly or indirectly, including, in particular, pathological conditions present at birth. The abnormalities may be mutations, insertions, or deletions in genes. The abnormalities may affect the coding sequence of a gene or its regulatory sequence.

[0063] As used herein, the term “heterogeneous” refers to a nucleotide or polypeptide sequence that is not found in natural nucleic acids or proteins, respectively. A heterogeneous nucleic acid sequence may be linked to a naturally occurring nucleic acid sequence (or a variant thereof) (e.g., by genetic engineering) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterogeneous nucleic acid sequence may be linked to a variant polypeptide (e.g., by genetic engineering) to generate a nucleotide sequence encoding a fusion variant polypeptide.

[0064] As used herein, the term “host cell” refers to any cell type sensitive to transformation, transfection, transduction, etc. using the nucleic acid therapeutic agents of this disclosure. Non-limiting examples include isolated primary cells, pluripotent stem cells, CD34 + It may be a cell, induced pluripotent stem cell, or any of a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell is of a tissue, organ, or organism. In place or In vivo It can be a cell. Furthermore, the host cell can be a target cell of, for example, a mammalian subject (e.g., a human patient requiring gene therapy).

[0065] As used herein, the "inducible promoter" indicates that it is characterized by initiating or enhancing transcriptional activity in the presence of, being affected by, or coming into contact with an inducer or inducible agent. As used herein, the "inducer" or "inducible agent" may be an endogenous, or typically exogenous, compound or protein administered in a manner that is active in inducing transcriptional activity from the inducible promoter. In some embodiments of any of the aspects and embodiments of the present invention, the inducer or inducible agent, i.e., a chemical, compound, or protein, may be itself the result of transcription or expression of a nucleic acid sequence (i.e., the inducer may be an inducible protein expressed by another component or module), and may be under the control of the inducible promoter. In some embodiments of any of the aspects and embodiments of the present invention, the inducible promoter is induced in the absence of a specific agent, such as an inhibitor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter; and mouse mammary tumor virus long terminal repeat sequence (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters, etc.

[0066] "used in this document" In vitro The term "indicates and methods that do not require the presence of cells having intact membranes, such as cell extracts, and may refer to the introduction of programmable synthetic biological circuits into non-cellular systems, such as cell-free media, or cellular systems, such as cell extracts."

[0067] "used in this document" In vivoThe term "indicates a test or process occurring in or within an organism, such as a multicellular animal. In some embodiments described herein, the method or use, when a single-celled organism, such as bacteria, is used, " In vivo It can be said that it occurs in. In vitro The term "methods and uses" refers to methods and uses performed using living cells having intact membranes in vitro from multicellular animals or plants, for example, particularly explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells).

[0068] As used herein, the term “lipid” refers to a group of organic compounds characterized by including, but not limited to, esters of fatty acids, being insoluble in water but soluble in many organic solvents. These are typically classified into at least three classes: (1) “simple lipids” including fats and oils as well as waxes; (2) “complex lipids” including phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0069] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other phosphorus-deficient compounds, such as sphingolipids, the glycosphingolipid family, diacylglycerols, and β-acyloxy acids, also belong to the group designated as amphiphilic lipids. Additionally, the amphiphilic lipids described above may be mixed with other lipids, including triglycerides and sterols.

[0070] In one embodiment of any of the aspects or embodiments of the present invention, the lipid composition comprises one or more tertiary amino groups, one or more phenyl ester bonds and disulfide bonds.

[0071] As used herein, the term "lipid conjugate" refers to a conjugated lipid that inhibits the aggregation of lipid particles (e.g., lipid nanoparticles). These lipid conjugates include, but are not limited to, PEG-lipid conjugates 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 PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugate; see, for example, U.S. Provisional Application No. 61 / 294,828 filed January 13, 2010 and U.S. Provisional Application No. 61 / 295,140 filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Further examples of POZ-lipid conjugates are described in International Publication WO 2010 / 006282. PEG or POZ may be directly conjugated to lipids or linked to lipids via a linker moiety. Any linker moiety suitable for coupling PEG or POZ to lipids may be used, including, for example, non-ester-containing linker moiety and ester-containing linker moiety. In certain preferred embodiments, a non-ester-containing linker moiety, such as an amide or carbamate, is used. The disclosure of each of the above patent documents is incorporated herein by reference in its entirety for all purposes. The lipid conjugates described herein (e.g., PEG-lipids or PEGylated lipids) may be further covalently linked to useful tissue-targeting moiety known in the art (e.g., N-acetylgalactosamine (GalNAc; 1, 2, 3, or 4-antennary GalNAc)).

[0072] As used herein, the term “lipid-encapsulated” refers to a lipid particle in which an active agent or therapeutic agent, such as a nucleic acid (e.g., ASO, mRNA, siRNA, ceDNA, viral vector), is fully encapsulated, partially encapsulated, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., forming a nucleic acid-containing lipid particle).

[0073] As used herein, the terms “lipid particle” or “lipid nanoparticle” refer to a lipid formulation (referred to as “TNA lipid particle”, “TNA lipid nanoparticle”, or “TNA LNP”) that can be used to deliver a therapeutic agent, such as a nucleic acid therapeutic agent (TNA), to a target site of interest (e.g., a cell, tissue, organ, etc.). In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle of the present invention is a therapeutic nucleic acid-containing lipid particle formed typically of an ionizable lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents the aggregation of the particle. In another preferred embodiment, a therapeutic agent, such as a therapeutic nucleic acid, may be encapsulated in the lipid portion of the particle to be protected from enzymatic degradation. In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle comprises a nucleic acid (e.g., ceDNA) and a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds, and disulfide bonds.

[0074] The average diameter of the lipid particles of the present invention is typically about 20 nm to about 120 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, It is about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm.

[0075] As used herein, the term “hydrophobic lipid” refers, but is not limited to, compounds having non-polar groups comprising long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N,N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0076] As used herein, the term “ionizable lipid” refers to a lipid having at least one protonable or deprotonable group, e.g., a cationic lipid, such that the lipid becomes positively charged at a physiological pH (e.g., pH 7.4) or lower and neutral at a second pH, preferably above the physiological pH. Those skilled in the art will understand that the addition or removal of protons as a function of pH is an equilibrium process, that references to charged or neutral lipids indicate the nature of the dominant species, and that not all lipids need to exist in a charged or neutral form. Generally, the pKa of the protonable group of an ionizable lipid is in the range of about 4 to about 7. In some embodiments of any of the aspects and embodiments of the present invention, the ionizable lipid may include a “cleavable lipid” or an “SS-cleavable lipid.” Accordingly, as used herein, the term “ionizable lipid” includes both the ionized (or charged) and neutral forms of the lipids of the present invention.

[0077] As used herein, the term “neutral lipids” refers to any lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, these lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

[0078] 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, ricylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifiers linked to neutral lipids.

[0079] The term "non-cationic lipid" as used herein refers to any amphiphilic lipid, as well as any other neutral lipid or anionic lipid.

[0080] As used herein, the terms “cleavable lipid” or “SS-cleavable lipid” refer to lipids comprising a disulfide bond cleavable unit. In one embodiment of any of the embodiments or examples thereof, the cleavable lipid comprises a tertiary amine that reacts with an acidic compartment, e.g., an endosome or lysosome, for membrane destabilization, and a disulfide bond that can be cleaved in a reducing environment, such as the cytoplasm. In one embodiment of any of the embodiments or examples thereof, the cleavable lipid is an ionizable lipid. In one embodiment of any of the embodiments or examples thereof, the cleavable lipid is a cationic lipid. In one embodiment of any of the embodiments or examples thereof, the cleavable lipid is an ionizable cationic lipid. The cleavable lipid is described in more detail herein.

[0081] As used herein, the term "organic lipid solution" refers to a composition comprising, wholly or partially, an organic solvent having lipids.

[0082] As used herein, the term “liposome” refers to a lipid molecule assembled into a spherical configuration that encapsulates an internal aqueous volume separated from an aqueous exterior. A liposome is a vesicle containing at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of drug development. They operate by fusing with a cell membrane and rearranging their lipid structure to deliver a drug or active pharmaceutical ingredient. Liposome compositions for such delivery are typically composed of phospholipids, particularly compounds having phosphatidylcholine groups, but these compositions may also include other lipids.

[0083] As used herein, the term “local delivery” refers to the direct delivery of an activator, such as interfering RNA (e.g., siRNA), to a target site within an organism. For example, the activator may be delivered locally by direct injection to a diseased site, such as a tumor, or other target sites, such as a site of inflammation, or to target organs, such as the liver, heart, pancreas, kidney, etc.

[0084] The terms “neDNA” or “nicked ceDNA” as used herein refer to closed DNA having 2 to 100 base pairs of nicks or gaps upstream of the stem region or spacer region of an open reading frame (e.g., a promoter and a transgene to be expressed).

[0085] As used herein, the term “nucleic acid” refers to a polymer containing at least two nucleotides in a single-stranded or double-stranded form (i.e., deoxyribonucleotide or ribonucleotide), including DNA, RNA, and hybrids thereof. DNA may be in the form, for example, an antisense molecule, plasmid DNA, DNA-DNA dimer, pre-condensed DNA, PCR product, vector (P1, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of a minicircle, plasmid, vacmid, minigene, ministring DNA (a DNA vector closed by linear covalent bonds), closed linear dimer DNA (CELiD or ceDNA), doggybone™ DNA, dumbbell DNA, minimally immunologically defined gene expression (MIDGE) vector, viral vector, or non-viral vector. RNA may 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 known nucleotide analogs, or nucleic acids containing modified backbone residues or linkages that are synthetic, naturally occurring, and non-natural, and have binding properties similar to the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholino), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, lock nucleic acids (LNA™), and peptide nucleic acids (PNA). Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides having binding properties similar to the reference nucleic acid.Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly presented sequence.

[0086] As used herein, the terms “nucleic acid therapeutics,” “therapeutic nucleic acids,” and “TNA” are used interchangeably and refer to any therapeutic mode using nucleic acids as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutics and DNA-based therapeutics. 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 nonviral DNA vectors, closed linear divalent DNA (ceDNA / CELiD), plasmids, vacmids, doggybone™ DNA vectors, minimally immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (DNA vectors closed by linear covalent bonds), and dumbbell DNA minimal vectors (“Dumbbell DNA”). As used herein, the term “TNA LNP” refers to a lipid particle containing at least one of the TNAs described above.

[0087] The "nucleotide" as used herein contains the sugar oxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together through the phosphate group.

[0088] As used herein, "operably connected" refers to a juxtaposition in which the components described herein are in a relationship that enables them to function in an intended manner. For example, if a promoter influences transcription or expression, the promoter is operably connected to the coding sequence. The promoter may be said to induce the expression or transcription of the nucleic acid sequence it regulates. The phrases "operably connected," "operably positioned," "operably connected," "under control," and "under transcriptional control" indicate that the promoter exists at the correct functional location and / or orientation relative to the regulated nucleic acid sequence to control the initiation of transcription and / or expression of the sequence. As used herein, "reverse promoter" refers to a promoter in which the nucleic acid sequence exists in a reverse orientation such that the coding strand becomes the non-coding strand and vice versa. The reverse promoter sequence may be used to control the state of the switch in various embodiments. Additionally, in various embodiments, the promoter may be used in conjunction with an enhancer.

[0089] As used herein, the term “promoter” refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence in a manner that induces the transcription of a nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. A promoter may be constitutive, inducible, repressive, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence in which the transcription initiation and rate of the rest of the nucleic acid sequence are controlled. A promoter may also contain genetic elements capable of binding to regulatory proteins and molecules, such as RNA polymerases and other transcription factors. Within the promoter sequence, a protein binding domain responsible for the binding of RNA polymerases will be found as well as a transcription initiation site. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to induce the expression of the transgenic gene of the synthetic AAV vector disclosed herein. The promoter sequence can be coupled to the 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at a level detectable on the background.

[0090] A promoter may be naturally associated with a gene or sequence, such as by isolating a coding segment of a given gene or sequence and / or a 5' non-coding sequence located upstream of an exon. Such a promoter may be referred to as "endogenous." Similarly, in some embodiments of any of the aspects and embodiments of the present invention, an enhancer may be naturally associated with a nucleic acid sequence located downstream or upstream of said sequence. In some embodiments of any of the aspects and embodiments of the present invention, a coding nucleic acid segment is placed under the control of a "recombinant promoter" or a "heterogeneous promoter," wherein both promoters represent promoters that are not typically associated with an encoded nucleic acid sequence linked operably in a natural environment. Similarly, a "recombinant or heterogeneous enhancer" represents an enhancer that is not typically associated with a given nucleic acid sequence in a natural environment. Such a promoter or enhancer is a promoter or enhancer of another gene; It may include a promoter or enhancer isolated from any other prokaryotic, viral, or eukaryotic cell; and a synthetic promoter or enhancer comprising a mutation that is not "naturally occurring," i.e., a different element of a different transcriptional regulatory region, and / or a mutation that alters expression through genetic engineering methods known in the art. In addition to synthetically generating the nucleic acid sequences of the promoter and enhancer, the promoter sequence may be generated using recombinant cloning and / or nucleic acid amplification techniques (including PCR) in connection with the synthetic biological circuits and modules disclosed herein (see, for example, U.S. Patent No. 4,683,202 and U.S. Patent No. 5,928,906, the full text of which is incorporated herein by reference). Furthermore, it is considered that control sequences directing the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc. may also be utilized.

[0091] As used herein, the terms “Rep binding site” (“RBS”) and “Rep binding element” (“RBE”) are used interchangeably and refer to a binding site for a Rep protein (e.g., AAV Rep 78 or AAV Rep 68) that, when bound by the Rep protein, enables the Rep protein to perform its site-specific endonuclease activity at a sequence containing an RBS. The RBS sequence and its reverse complement together form a single RBS. RBS sequences are widely known in the art and include, for example, 5’-GCGCGCTCGCTCGCTC-3’, the RBS sequence identified in AAV2.

[0092] The phrase "recombinant vector" as used herein refers to a heterogeneous nucleic acid sequence, or In vivo Represents a vector comprising an expressible "transgenic gene." It should be understood that in some embodiments of any of the aspects and embodiments of the present invention, the vector described herein may be combined with other suitable compositions and therapies. In some embodiments of any of the aspects and embodiments of the present invention, the vector is an episome. The use of a suitable episome vector provides a means to maintain a high copy number of the nucleotide of interest in the target on extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0093] As used herein, the term "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally generate 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 observed.

[0094] The terms "sense" and "antisense" as used herein refer to the orientation of structural elements in polynucleotides. The sense and antisense versions of the elements are inverse complements to each other.

[0095] 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 (see the work of Needleman and Wunsch (1970)) as implemented in the Needle program (preferably version 3.0.0 or higher) of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, see the work of Rice et al. (2000) above). The optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the needle-labeled "longest identity" (obtained using the -nobrief option) is used as identity %, which is calculated as follows: (identical deoxynucleotides x 100) / (alignment length - 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.

[0096] As used herein, the term “spacer region” refers to an interpositional sequence that separates functional elements from a vector or genome. In some embodiments of any of the embodiments and examples of the present invention, an AAV spacer region maintains two functional elements at a desired distance for optimal function. In some embodiments of any of the embodiments and examples of the present invention, a spacer region provides or adds genetic stability to the vector or genome. In some embodiments of any of the embodiments and examples of the present invention, a spacer region facilitates prepared genetic manipulation of the genome by providing a convenient location for a cloning site and a gap in the design number of base pairs. For example, in certain embodiments, an oligonucleotide “polylinker” or “multicloning site” containing several restriction endonuclease sites, or a non-open reading frame sequence designed not to have a known protein (e.g., transcription factor) binding site, Sis - To isolate the action factor, it can be placed in a vector or genome by inserting, for example, a hexamer, a dotmer, an octamer, a 24mer, a 48mer, an 86mer, a 176mer, etc.

[0097] As used herein, the term “subject” refers to a human or animal to which treatment (including prophylactic treatment) is provided with the therapeutic nucleic acid according to the present invention. Typically, the animal is a vertebrate, such as primates, rodents, domestic animals, or game animals, but not limited to them. Primates include, but not limited to, chimpanzees, cynomylgus monkeys, spider monkeys, and macaques, e.g., rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but not limited to, cattle, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canids (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostriches), and fish (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 a child. In some embodiments of any of the aspects and embodiments of the present invention, the subject may be a newborn or an unborn subject, for example, a subject in the womb. Preferably, the subject is a mammal. The mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Non-human mammals may be advantageously used as subjects representing animal models of diseases and disorders. Additionally, the methods and compositions described herein may be used for livestock and / or pets. The human subject may be of any age, sex, race, or ethnicity, e.g., Caucasian, Asian, African, Black, African American, African European, Hispanic, Middle Eastern, etc. In some embodiments of any of the aspects and embodiments of the present invention, the subject may be a patient or other subject in a clinical setting. In some embodiments of any of the aspects and embodiments of the present invention, the subject is already receiving treatment. In some embodiments of any of the aspects and embodiments of the present invention, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments of any of the aspects and embodiments of the present invention, the subject is a human fetus, a human newborn, a human infant, a human child, a human adolescent, or a human adult. In some embodiments of any of the aspects and embodiments of the present invention, the subject is an animal embryo, a non-human embryo, or a non-human primate embryo. In some embodiments of any of the aspects and embodiments of the present invention, the subject is a human embryo.

[0098] The phrase “subjects requiring this” as used herein refers, unless otherwise indicated by the context and usage of such phrase, to (i) a subject who is to be administered TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) according to the invention described herein; (ii) a subject who is currently being administered TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) according to the invention described herein; or (iii) a subject who has previously been administered TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) according to the invention described herein.

[0099] As used herein, the terms “inhibit,” “reduce,” “interfer,” “inhibit,” and / or “reduce” (and similar terms) generally indicate an action that reduces, directly or indirectly, a concentration, level, function, activity, or behavior compared to an intrinsic value, predicted value, or mean value, or compared to control conditions.

[0100] As used herein, the terms "synthetic AAV vector" and "synthetic production of an AAV vector" refer to an AAV vector and a method for its synthetic production in a completely cell-free environment.

[0101] As used herein, the term “systemic delivery” refers to the delivery of lipid particles that induce extensive biodistribution of an activator, such as interfering RNA (e.g., siRNA), within an organism. Some administration techniques may lead to systemic delivery of a specific agent, while others do not. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most of the body. To achieve extensive biodistribution, a blood shelf life is generally required to ensure that the agent is not rapidly degraded or eliminated (e.g., by primary passage organs (liver, lungs, etc.) or by rapid and non-specific cell binding) before reaching a disease site far from the site of administration. Systemic delivery of lipid particles (e.g., lipid nanoparticles) may be achieved through any means known in the art, including, for example, intravenously, subcutaneously, and intraperitoneally. In a preferred embodiment, systemic delivery of lipid particles (e.g., lipid nanoparticles) is achieved by intravenous delivery.

[0102] The terms “terminal degradation site” and “TRS” as used herein are used interchangeably and refer to a region in which Rep forms a tyrosine-phosphodiester bond with 5’ thymidine to produce a 3’-OH that acts 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 participate in a coordinated ligation reaction.

[0103] The terms “therapeutic dose,” “therapeutic effective dose,” “effective dose,” “effective dose,” or “pharmaceutical effective dose” of the activator used herein (e.g., TNA lipid particles as described herein) are used interchangeably to indicate an amount sufficient to provide the intended benefit or effect of treatment, e.g., inhibition of the expression of a target sequence, compared to the expression level detected in the absence of therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include tests for protein or RNA levels using phenotypic assays known to those skilled in the art, as well as techniques known to those skilled in the art, such as dot blot, northern blot, hybridization in situ, ELISA, immunoprecipitation, and enzymatic function. Dose levels are based on various factors, including the type of disease, the patient’s age, weight, sex and medical condition, the severity of the condition, the route of administration, and the specific activator used. Accordingly, the dosage regimen may vary widely, but can typically be determined by a physician using standard methods. Additionally, the terms “therapeutic dose,” “therapeutic effective dose,” and “pharmaceutical effective dose” include the prophylactic or preventive amounts of the compositions of the invention described herein. In the prophylactic or preventive application of the invention described herein, the pharmaceutical composition or agent is administered to a patient who is susceptible to or otherwise at risk of a disease, disorder, or condition, in an amount sufficient to eliminate or reduce the risk of, reduce the severity of, or delay the onset of a disease, disorder, or condition, including the disease, disorder, or condition, its complications, and biochemical, histological, and / or behavioral symptoms of an intermediate pathological phenotype that appear during the development of the disease, disorder, or condition. It is generally desirable to use the maximum dose, that is, the safest dose according to some medical judgment. The terms “dose” and “administrative amount” are used interchangeably herein.In any one of the embodiments or examples of the present invention, the “therapeutic dose,” “therapeutic effective dose,” and “pharmaceutical effective dose” are for non-preventive or non-preventive application.

[0104] As used herein, the term "therapeutic effect" refers to a therapeutic outcome deemed desirable and beneficial. A therapeutic effect may include, directly or indirectly, the inhibition, reduction, or elimination of the manifestation of the disease. A therapeutic effect may also include, directly or indirectly, the inhibition, reduction, or elimination of the progression of the disease.

[0105] For any therapeutic agent described herein, the therapeutically effective dose may be determined initially in preliminary in vitro studies and / or animal models. The therapeutically effective dose may also be determined from human data. The applicable dose may be adjusted based on the relative bioavailability and efficacy of the administered compound. It is within the capability of those skilled in the art to adjust the dose to achieve maximum efficacy based on the methods described above and other widely known methods. The general principles for determining therapeutic effects, which can be identified in Chapter 1 of the literature [Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001)] (which is incorporated herein by reference), are summarized below.

[0106] Pharmacokinetic principles provide a basis for modifying the administration regimen to achieve the desired level of therapeutic efficacy while minimizing unacceptable side effects. In situations where the plasma concentration of a drug can be measured and this is related to the therapeutic range, additional guidance for changing the dosage can be obtained.

[0107] As used herein, the terms “treat,” “treating,” and / or “treatment” include obtaining beneficial or desired clinical outcomes by stopping, inhibiting, slowing, or reversing the progression of a condition; improving the clinical symptoms of a condition; or preventing the appearance of clinical symptoms of a condition. Treatment further indicates achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of characteristic symptoms of the disorder(s) to be treated; (c) limiting the worsening of characteristic symptoms of the disorder(s) to be treated; (d) limiting the recurrence of the disorder(s) in patients who have previously suffered from the disorder(s); and (e) limiting the recurrence of symptoms in patients who have previously been asymptomatic for the disorder(s). In any one of the embodiments or features of the present invention, the terms “treat,” “treating,” and / or “treat” include stopping, inhibiting, slowing, or reversing the progression of a disease condition, or improving the clinical symptoms of a disease condition.

[0108] Beneficial or intended clinical outcomes, such as pharmacological and / or physiological effects, include, without limitation, preventing the onset of the disease, disorder, or condition in subjects who may be susceptible to the disease, disorder, or condition but have not yet experienced or exhibited symptoms of the disease (preventive treatment); 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., not exacerbating it); preventing the spread of the disease, disorder, or condition; delaying or slowing the progression of the disease, disorder, or condition; improving or alleviating the disease, disorder, or condition; and combinations thereof, as well as extending survival time compared to the survival time expected without treatment.

[0109] As used herein, the terms “vector” or “expression vector” refer to a replicon, such as a plasmid, vacmid, phage, virus, virion, or cosmid, which may be attached to another DNA segment, namely an “insert,” “transgenic,” or “expression cassette,” to cause the expression or replication of the segment attached within the cell (“expression cassette”). A vector may be a nucleic acid construct designed for delivery to a host cell or between different host cells. As used herein, a vector may be viral or non-viral in its final form of origin. However, for the purposes of this disclosure, “vector” generally refers to a synthetic AAV vector or a nicked ceDNA vector. Accordingly, the term “vector” comprises any genetic element capable of replicating when combined with a suitable control element and capable of delivering a gene sequence to a cell. In some embodiments of any of the aspects and embodiments of this invention, a vector may be a recombinant vector or an expression vector.

[0110] The grouping of alternative elements or embodiments of the invention disclosed herein shall not be construed as a limitation. Each member of a group may be referred to and claimed individually or in any combination with other members of the group or other elements identified herein. One or more members of a group may be included in or omitted from a group for reasons of convenience and / or patentability. Where any such inclusion or omission occurs, this specification is deemed to include the groups modified herein and satisfies the written description of all Markush groups used in the appended claims.

[0111] In some embodiments of any mode, the disclosures described herein do not relate to methods for cloning a human, methods for modifying the genetic identity of a human germline, the use of a human embryo for industrial or commercial purposes, or methods for modifying the genetic identity of an animal that may cause suffering without any substantial medical benefit to a human or animal, nor to animals as a result of such methods.

[0112] Other terms are defined herein within the description of various aspects of the invention.

[0113] All patents and other publications cited throughout this application (including references, registered patents, published patent applications, and concurrently 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. Such publications are provided only for disclosures prior to the filing date of this application. In this regard, it should not be construed that the inventors are not qualified to precede such disclosures due to prior art or for any other reason. Any representations regarding dates or expressions of the contents of such documents are based on information available to the applicant and are not to be construed as an endorsement of the accuracy of the dates or contents of such documents.

[0114] The description of embodiments of the present disclosure is not intended to limit the present disclosure to the exact form disclosed or to be complete. Specific embodiments of the present disclosure and examples thereof are described herein for illustrative purposes only, and as understood by those skilled in the art, various equivalent variations may be made within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order or may perform functions substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods where appropriate. Various embodiments described herein may be combined to provide additional embodiments. Aspects of the present disclosure may be modified to utilize the compositions, functions, and concepts of the references and applications, where necessary, to provide additional embodiments of the present disclosure. Furthermore, with regard to biological functional equivalence, minor modifications to the protein structure may be made so as not to affect the type or amount of biological or chemical action. Such and other modifications may be made in consideration of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0115] Certain elements of any of the aforementioned embodiments may be combined or substituted with elements of other embodiments. Furthermore, while the advantages associated with a specific embodiment of the present disclosure are described in the context of such embodiment, other embodiments may also exhibit such advantages, and not all embodiments are required to exhibit such advantages to fall within the scope of the present disclosure.

[0116] The technology described herein is further illustrated by the following examples, which should not be construed as any additional limitation. It should be understood that the invention is not limited in any way to the specific methodologies, protocols, and reagents described herein, and may vary. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is defined solely by the claims.

[0117] II. Geology

[0118] In the first chemical embodiment, chemical formula ( I An ionizable lipid of ) or a pharmaceutically acceptable salt thereof is provided:

[0119] ( I )

[0120] [During the meal,

[0121] a is an integer in the range of 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20);

[0122] b is an integer in the range of 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0123] R 1 is not present, or (C2-C 20 )Alkenyl, -C(O)O(C2-C 20 )alkyl, and (C2-C 20 Selected from cyclopropyl substituted with alkyl;

[0124] R 2 is (C2-C 20 )alkyl is].

[0125] In the second chemical embodiment, chemical formula ( I The ionizable lipid of ) is the chemical formula ( II ) or a pharmaceutically acceptable salt thereof:

[0126] ( II )

[0127] [In the formula, c and d are each independently integers in the range of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and the remaining variables are the formula ( I As stated in )].

[0128] In the third chemical embodiment, chemical formula ( I ) or chemical formula ( II In the ionizable lipid of ) or a pharmaceutically acceptable salt thereof, c and d are each independently integers ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7, or 6 to 8, and the remaining variables are the chemical formula ( I ) or chemical formula ( II As stated in ).

[0129] In the fourth chemical embodiment, chemical formula ( I ) or chemical formula ( II In the ionizable lipids of ), c ​​is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are the chemical formula ( I ), or as described in the second or third chemical embodiment. Alternatively, as part of the fourth chemical embodiment, chemical formula ( I ) or chemical formula ( II In the ionizable lipid of ) or its pharmaceutically acceptable salt, c and d are each independently 1, 3, 5, or 7, and the remaining variables are the chemical formula ( I ), or as described in the second or third chemical embodiment.

[0130] In the fifth chemical embodiment, chemical formula ( I ) or chemical formula ( IIIn the ionizable lipids of ), d is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are the chemical formula ( I ), or as described in the second, third, or fourth chemical embodiment. Alternatively, as part of the fifth chemical embodiment, the formula ( I ) or chemical formula ( II In the ionizable lipid of ) or a pharmaceutically acceptable salt thereof, at least one of c and d is 7, and the remaining variables are the chemical formula ( I ), or as described in the second, third, or fourth chemical embodiment.

[0131] In the 6th chemical embodiment, chemical formula ( I The ionizable lipid of ) is the chemical formula ( III ) or a pharmaceutically acceptable salt thereof:

[0132] ( III )

[0133] [In the formula, the remaining variables are chemical formulas ( I As stated in )].

[0134] In the 7th chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III In the ionizable lipid of ) or a pharmaceutically acceptable salt thereof, b is an integer in the range of 3 to 9, and the remaining variables are the chemical formula ( I ), or as described in the second, third, fourth, or fifth chemical embodiments. Alternatively, as part of the seventh chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( IIIIn the ionizable lipid of ) or a pharmaceutically acceptable salt thereof, b is an integer in the range of 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, and the remaining variables are in the chemical formula ( I ), or as described in the second, third, fourth, or fifth chemical embodiments. In another alternative, as part of the seventh chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III In the ionizable lipid of ) or a pharmaceutically acceptable salt thereof, b is 3, 4, 5, 6, 7, 8, or 9, and the remaining variables are in the chemical formula ( I ), or as described in the second, third, fourth, or fifth chemical embodiments.

[0135] In the eighth chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III In the ionizable lipid of ) or a pharmaceutically acceptable salt thereof, a is an integer in the range of 2 to 18, and the remaining variables are the chemical formula ( I ), or as described in the second, third, fourth, fifth, or seventh chemical embodiments. Alternatively, as part of the eighth embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( IIIIn the ionizable lipid of ) or a pharmaceutically acceptable salt thereof, a is 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 25 to 8, 5 to 7, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 11 to 18, 11 to 17, 11 to 16, 11 to 15, 11 to 14, 10 to 13, 11 to 18, 11 to 17, 11 to 16, 11 to 15, 11 to 14, 11 to 13, 12 to 18, 12 to 17, 12 to 16,The integer is in the range of 12 to 15, 12 to 14, 13 to 18, 13 to 17, 13 to 16, 13 to 15, 14 to 18, 14 to 17, 14 to 16, 15 to 18, 15 to 17, or 16 to 18, and the remaining variable is a chemical formula (, I ), or as described in the second, third, fourth, fifth, or seventh chemical embodiments. In another alternative, as part of the eighth embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III In the ionizable lipid of ), a is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and the remaining variables are the chemical formula ( I ), or as described in the second, third, fourth, fifth, or seventh chemical embodiments.

[0136] In the ninth chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III R in ionizable lipids or pharmaceutically acceptable salts thereof 1 is not present, or (C5-C 15 )Alkenyl, -C(O)O(C4-C 18 )alkyl, and (C4-C 16 Selected from cyclopropyl substituted with alkyl, and the remaining variables are chemical formulas ( I ), or as described in the second, third, fourth, fifth, seventh, or eighth chemical embodiments. Alternatively, as part of the ninth chemical embodiment, the formula ( I ), chemical formula ( II ) or chemical formula ( III R in ionizable lipids or pharmaceutically acceptable salts thereof 1 is not present, or (C5-C 15 )Alkenyl, -C(O)O(C4-C 16 )alkyl, and (C4-C16 Selected from cyclopropyl substituted with alkyl, and the remaining variables are chemical formulas ( I ), or as described in the second, third, fourth, fifth, seventh, or eighth chemical embodiments. Alternatively, as part of the ninth chemical embodiment, the formula ( I ), chemical formula ( II ) or chemical formula ( III R in ionizable lipids or pharmaceutically acceptable salts thereof 1 is not present, or (C5-C 12 )Alkenyl, -C(O)O(C4-C 12 )alkyl, and (C4-C 12 Selected from cyclopropyl substituted with alkyl, and the remaining variables are chemical formulas ( I ), or as described in the second, third, fourth, fifth, seventh, or eighth chemical embodiments. In another alternative, as part of the ninth chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III R in ionizable lipids or pharmaceutically acceptable salts thereof 1 is not present, or (C5-C 10 )Alkenyl, -C(O)O(C4-C 10 )alkyl, and (C4-C 10 Selected from cyclopropyl substituted with alkyl, and the remaining variables are chemical formulas ( I ), or as described in the second, third, fourth, fifth, seventh, or eighth chemical embodiments.

[0137] In the 10th chemical embodiment, R 1 C 10 It is alkenyl, and the remaining variables are as described in any one of the aforementioned implementations.

[0138] In the 11th chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( IIIR in ionizable lipids or pharmaceutically acceptable salts thereof 1 of C(O)O(C2-C 20 )alkyl, -C(O)O(C4-C 18 )alkyl, -C(O)O(C4-C 12 )alkyl or -C(O)O(C4-C 10 ) The alkyl in the alkyl group is an unbranched alkyl, and the remaining variables are as described in any one of the aforementioned embodiments. In one chemical embodiment, R 1 is -C(O)O(C9alkyl). Alternatively, in the 11th chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III R in ionizable lipids or pharmaceutically acceptable salts thereof 1 -C(O)O(C4-C 18 )alkyl, -C(O)O(C4-C 12 )alkyl or -C(O)O(C4-C 10 ) The alkyl in the alkyl group is a branched alkyl, and the remaining variables are as described in any one of the aforementioned chemical embodiments. In one chemical embodiment, R 1 -C(O)O(C 17 alkyl), and the remaining variables are as described in any one of the aforementioned chemical embodiments.

[0139] In the 12th chemical embodiment, chemical formula ( I ), chemical formula ( II ) or chemical formula ( III R in ionizable lipids or pharmaceutically acceptable salts thereof 1 is selected from any group listed in Table 1 below, where the wavy bond in each group represents the attachment point of the corresponding group to the rest of the lipid molecule, and the remaining variables are the chemical formula ( I ), or as described in the second, third, fourth, fifth, seventh, or eighth chemical embodiments. The present disclosure is R of Table 1. 1Any one of the instruments and R in Table 2 2 Consider an additional combination of any one of the groups, and the remaining variables are chemical formulas ( I ), or as described in the second, third, fourth, fifth, seventh, or eighth chemical embodiments.

[0140]

[0141] In the 13th chemical embodiment, chemical formula ( I R in ionizable lipids or pharmaceutically acceptable salts thereof 2 is selected from any group listed in Table 2 below, where the wavy bond in each group represents the attachment point of the corresponding group to the rest of the lipid molecule, and the remaining variables are the chemical formula ( I ), or as described in the 7th, 8th, 9th, 10th, or 11th chemical embodiments.

[0142]

[0143] A specific example is of the example section below. Table 3 It is provided in, which is the chemical formula ( I ) is included as part of the 14th chemical embodiment of the present invention for ionizable lipids. It includes pharmaceutically acceptable salts, as well as ionized and neutral forms.

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] In an additional embodiment, chemical formula ( Ia ), chemical formula ( Ib) or chemical formula ( Ic Lipids of ) or pharmaceutically acceptable salts thereof are considered herein:

[0152] ( Ia );

[0153] ( Ib );

[0154] ( Ic )

[0155] [In the middle of the meal, R q and R z [’’ is each independently an aliphatic group (including alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl) or an aryl group, and the remaining variables are as described above in any one of the aforementioned chemical embodiments]. In one embodiment, R q and R z Each is independently hydrogen or a C1-C6 alkyl, and the remaining variables are as described above in any one of the aforementioned chemical embodiments. The disclosed LNP, composition, method of use, etc. also includes the chemical formula ( Ia ), chemical formula ( Ib ) or chemical formula ( Ic It also applies to lipids of ). Chemical formula ( Ia ), chemical formula ( Ib ) or chemical formula ( Ic The lipids of ) are, for example, chemical formula ( I It can be manufactured by treating the lipids of ) with chloromethane (CH3Cl) in acetonitrile (CH3CN) and chloroform (CHCl3).

[0156] Furthermore, chemical formula ( II ) or chemical formula ( III A lipid of ) or any of the exemplary lipids disclosed herein, for example, of the formula ( IThe lipids of ) can be converted into corresponding quaternary lipids (all considered in the present disclosure) by treating them with chloromethane (CH3Cl) in acetonitrile (CH3CN) and chloroform (CHCl3).

[0157] Lipid nanoparticles (LNPs) or pharmaceutical compositions thereof comprising ionizable lipids and capsid-free nonviral vectors (e.g., ceDNA) as described herein can be used to deliver capsid-free nonviral DNA vectors to target sites of interest (e.g., cells, tissues, organs, etc.).

[0158] In one embodiment of any of the aspects or embodiments of the present invention, a lipid particle (e.g., lipid nanoparticle) formulation is prepared and loaded using TNA. In one embodiment, a lipid particle (e.g., lipid nanoparticle) formulation is prepared and loaded with ceDNA obtained by a method as disclosed in International Application PCT / US2018 / 050042 filed September 7, 2018, the full text of which is incorporated herein by reference. This can be achieved by a high-energy mixture of an ethanolic lipid at a low pH and an aqueous TNA such as ceDNA, which protonates the lipid and provides energy useful for ceDNA / lipid association and particle nucleation. The particles may be further stabilized through aqueous dilution and removal of organic solvents. The particles may be concentrated to a desired level.

[0159] 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 of any of the aspects and embodiments of the present invention, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) is 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; The ratio may be in the range of about 30:1 to about 60:1. According to some embodiments of any of the embodiments or examples of the present invention, lipid particles (e.g., lipid nanoparticles) are prepared with a ratio of nucleic acid (mass or weight) to total lipid of about 60:1. According to some embodiments of any of the embodiments or examples of the present invention, lipid particles (e.g., lipid nanoparticles) are prepared with a ratio of nucleic acid (mass or weight) to total lipid of about 30:1. The amounts of lipids and nucleic acids may be adjusted to provide a desired N / P ratio, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or higher. Generally, the total lipid content of the lipid particle formulation may be in the range of about 5 mg / ml to about 30 mg / mL.

[0160] In some embodiments of any of the aspects and embodiments of the present invention, lipid nanoparticles comprise an agent that condenses and / or encapsulates nucleic acid cargoes such as ceDNA. Such agents are also referred to herein as condensing agents or encapsulating agents. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acids may be used, provided that it is non-fusional. That is, an agent that can condense and / or encapsulate nucleic acid cargoes such as ceDNA but has little or no fusional activity. Without being bound by theory, the condensing agent may exhibit some degree of fusional activity when not condensing / encapsulating nucleic acids such as ceDNA, but the nucleic acid-encapsulated lipid nanoparticles formed using said condensing agent may be non-fusional.

[0161] Generally, ionizable lipids or cationic lipids are typically used to condense nucleic acid cargoes, e.g., ceDNA, at low pH and to induce membrane association and fusogenicity. Generally, cationic lipids are lipids containing at least one amino group that becomes positively charged or protonated under acidic conditions, e.g., at a pH of 6.5 or lower. Cationic lipids may also be ionizable lipids, e.g., ionizable cationic lipids. "Non-fusible ionizable lipids" refers to ionizable lipids that can condense and / or encapsulate nucleic acid cargoes such as ceDNA but have little to no fusion activity.

[0162] In one embodiment of any of the aspects or embodiments of the present invention, the ionizable lipid may account for 20% (mol) to 90% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). For example, the molar content of the ionizable lipid may be 20% (mol) to 70% (mol), 30% (mol) to 60% (mol), 40% (mol) to 60% (mol), 40% (mol) to 55% (mol), or 45% (mol) to 55% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments of the present invention, the ionizable lipid accounts for about 50 mol% to about 90 mol% of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0163] In one embodiment of any of the aspects or embodiments of the present invention, the lipid particles (e.g., lipid nanoparticles) may further comprise non-cationic lipids. Non-cationic lipids may serve to increase fusion potential and also increase the stability of LNPs during formation. Non-cationic lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Thus, non-cationic lipids may be neutral uncharged lipids, zwitterionic lipids, or anionic lipids. Non-cationic lipids are typically used to enhance fusion potential.

[0164] Exemplary non-cationic lipids include, to a non-limited extent, 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), distearoylphosphatidylethanolamine (DSPE). Monomethylphosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethylphosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dielucoyylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-dipitanoyl-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 are included. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups within these lipids are preferably C 10 -C24 It is an acyl group derived from fatty acids having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0165] Other examples of non-cationic lipids suitable for use in lipid particles (e.g., lipid nanoparticles) include, for example, phosphorus-free lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolicinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymer, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amide, dioxadecyldimethylammonium bromide, ceramide, sphingomyelin, etc.

[0166] In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid is a phospholipid. In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments of any of the aspects and embodiments of the present invention, the non-cationic lipid is DSPC. In another embodiment, the non-cationic lipid is DOPC. In another embodiment, the non-cationic lipid is DOPE.

[0167] In some embodiments of any of the aspects and embodiments of the present invention, the non-cationic lipid may account for 0% (mol) to about 20% (mol) of the total lipid present in the lipid nanoparticles. In some embodiments of any of the aspects and embodiments of the present invention, the non-cationic lipid content is 0.5% (mol) to 15% (mol) of the total lipid present in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments of the present invention, the non-cationic lipid content is 5% (mol) to 12% (mol) of the total lipid present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid content is 5% (mol) to 10% (mol) of the total lipid present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid content is about 6% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid content is about 7.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments of the present invention, 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 of any of the aspects or embodiments of the present invention, the non-cationic lipid content is about 8.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid content is about 9.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid content is about 10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles).In one embodiment of any of the aspects or embodiments of the present invention, the non-cationic lipid content is about 11% (mol) of the total lipid present in the lipid particles (e.g., lipid nanoparticles).

[0168] Exemplary non-cationic lipids are described in International Publication WO2017 / 099823 and U.S. Patent Application Publication US2018 / 0028664, the contents of which are all incorporated herein by reference in their entirety.

[0169] In one embodiment of any of the aspects or embodiments of the present invention, lipid particles (e.g., lipid nanoparticles) may further comprise a component such as a sterol to provide membrane integrity and stability of the lipid particles. In one embodiment of any of the aspects or embodiments of the present invention, an exemplary sterol that may be used in the lipid particles is cholesterol or a derivative thereof. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)ethyl ether, cholesteryl-(4'-hydroxy)butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestan, cholestanone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments of any of the aspects and embodiments of the present invention, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)butyl ether. In some embodiments of any of the aspects and embodiments of the present invention, the cholesterol derivative is cholesteryl hemisuccinate (CHEMS).

[0170] Exemplary cholesterol derivatives are described in PCT Publication WO2009 / 127060 and U.S. Patent Publication US2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0171] In one embodiment of any of the aspects or embodiments of the present invention, a component providing membrane integrity, such as a sterol, may account for 0% (mol) to 50% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments of the present invention, this component is 20% (mol) to 50% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments of the present invention, this component is 30% (mol) to 40% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments of the present invention, this component is 35% (mol) to 45% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments of the present invention, these components are 38% (mol) to 42% (mol) of the total lipid content of lipid particles (e.g., lipid nanoparticles).

[0172] In one embodiment of any of the aspects or embodiments of the present invention, the lipid particles (e.g., lipid nanoparticles) may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. Generally, these are used to inhibit the aggregation of the lipid particles (e.g., lipid nanoparticles) and / or to provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (e.g., ATTA-lipid conjugates), cationic polymer-lipid (CPL) conjugates, and mixtures thereof. In some embodiments of any of the aspects and embodiments of the present invention, the conjugated lipid molecule is a PEG-lipid conjugate, e.g., (methoxypolyethylene glycol)-conjugated lipid. In some other embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, e.g., PEG 2000 It is DMG (Dimiristoylglycerol).

[0173] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxypolyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonylmethoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium Salts, or mixtures thereof, are included. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Patents No. 5,885,613 and 6,287,591, U.S. Patent Application Publications US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224 and US2017 / 0119904, the contents of all said documents are incorporated herein by reference in their entirety.

[0174] In one embodiment of any of the aspects or embodiments of the present invention, the PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. PEG-lipids may be one or more of PEG-DMG, PEG-Dilaurylglycerol, PEG-Dipalmitoylglycerol, PEG-Disterylglycerol, PEG-Dilaurylglycamide, PEG-Dimiristylglycamide, PEG-Dipalmitoylglycamide, PEG-Disterylglycamide, PEG-Cholesterol (1-[8'-(Cholester-5-N-3[Beta]-Oxy)Carboxamido-3',6'-Dioxaoctanyl]carbamoyl-[Omega]-Methyl-Poly(Ethylene Glycol), PEG-DMB (3,4-Ditetradecoxybenzyl-[Omega]-Methyl-Poly(Ethylene Glycol)Ether), and 1,2-Dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy(Polyethylene Glycol)-2000]. There are. In any one embodiment of the aspects or embodiments of the present invention, the PEG lipid may be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0175] In any one embodiment of the aspects or embodiments of the present invention, a lipid conjugated with a molecule other than PEG may also be used instead of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (e.g., ATTA-lipid conjugates), and cationic polymer-lipid (CPL) conjugates may be used instead of or in addition to PEG-lipid. Exemplary conjugated lipids, namely PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are PCT patent applications WO1996 / 010392, WO1998 / 051278, WO2002 / 087541, WO2005 / 026372, WO2008 / 147438, WO2009 / 086558, WO2012 / 000104, WO2017 / 117528, WO2017 / 099823, WO2015 / 199952, WO2017 / 004143, WO2015 / 095346, WO2012 / 000104, WO2012 / 000104, and WO2010 / 006282; As described in U.S. patent applications US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2013 / 0303587, US2018 / 0028664, US2015 / 0376115, US2016 / 0376224, US2016 / 0317458, US2013 / 0303587, US2013 / 0303587 and US20110123453; and U.S. patents US5,885,613, US6,287,591, US6,320,017 and US6,586,559, the contents of all said documents are incorporated herein by reference in their entirety.

[0176] In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate is present in lipid nanoparticles at a molar ratio of about 0% to about 20%. In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate content is 0.5% (mol) to 10% (mol) in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate content is 1% (mol) to 5% (mol) in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate content is 1% (mol) to 3% (mol) in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the embodiments or examples of the present invention, the PEG-lipid conjugate content is about 1.5% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate content is about 2% (mol) of the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate content is about 2.5% (mol) of the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate content is about 3% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the embodiments and examples of the present invention, the PEG-lipid conjugate content is about 3% (mol) of the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments of the present invention, the PEG-lipid conjugate content is about 3.5% (mol) in lipid particles (e.g., lipid nanoparticles).

[0177] In some embodiments of any of the aspects and embodiments of the present invention, the conjugated lipid, such as a PEG-lipid conjugate or a PEGylated lipid, is greater than about 2.0% of the total lipid in the lipid nanoparticles, e.g., about 2.1% or 2.2% or 2.3% or 2.4% or about 2.5% to about 10%; or about 2.1% or 2.2% or 2.3% or 2.4% or about 2.5% to about 7.5%; about 2.1% or 2.2% or 2.3% or 2.4% or about 2.5% to about 5%; about 3% to about 5%; about 3% to about 4.5%; about 3% to about 4%; about 3.5% to about 5%; about 3.5% to about 4.5%, about 2.5% to about 4%; It exists in a molar percentage of about 2.5% to about 3.5%, or about 2.5% to about 3%.

[0178] It is understood that the molar ratios of the disclosed ionizable lipids, non-cationic lipids, sterols, and PEG-conjugated lipids may vary as needed. For example, lipid particles (e.g., lipid nanoparticles) may comprise 30% to 70% lipids based on the mole or total weight of the composition, 0% to 60% cholesterol based on the mole or total weight of the composition, 0% to 30% non-cationic lipids based on the mole or total weight of the composition, and 1% to 10% PEG-conjugated lipids based on the mole or total weight of the composition. In one embodiment of any of the aspects or embodiments of the present invention, the composition comprises 40% to 60% of ionizable lipids based on the mole or total weight of the composition, 30% to 50% of cholesterol based on the mole or total weight of the composition, 5% to 15% of non-cationic lipids based on the mole or total weight of the composition, and 1% to 5% of PEG-conjugated lipids based on the mole or total weight of the composition. In one embodiment of any of the aspects or embodiments of the present invention, the composition comprises 40% to 60% of ionizable lipids based on the mole or total weight of the composition, 30% to 40% of cholesterol based on the mole or total weight of the composition, 5% to 10% of non-cationic lipids based on the mole or total weight of the composition, and 1% to 5% of PEG-conjugated lipids based on the mole or total weight of the composition. The above composition may contain 60% to 70% of ionizable lipids based on the mole or total weight of the composition, 25% to 35% of cholesterol based on the mole or total weight of the composition, 5% to 10% of non-cationic lipids based on the mole or total weight of the composition, and 0% to 5% of PEG-conjugated lipids based on the mole or total weight of the composition.The above composition may also contain up to 45% to 55% of ionizable lipids based on the mole or total weight of the composition, 35% to 45% of cholesterol based on the mole or total weight of the composition, 2% to 15% of non-cationic lipids based on the mole or total weight of the composition, and 1% to 5% of PEG-conjugated lipids based on the mole or total weight of the composition. The formulation may also, for example, 8% to 30% of ionizable lipids based on the mole or total weight of the composition, 5% to 15% of non-cationic lipids based on the mole or total weight of the composition, and 0% to 40% of cholesterol based on the mole or total weight of the composition; 4% to 25% of ionizable lipids based on the mole or total weight of the composition, 4% to 25% of non-cationic lipids based on the mole or total weight of the composition, 2% to 25% of cholesterol based on the mole or total weight of the composition, 10% to 35% of conjugated lipids based on the mole or total weight of the composition, and 5% of cholesterol based on the mole or total weight of the composition; or 2% to 30% of ionizable lipids based on the mole or total weight of the composition, 2% to 30% of non-cationic lipids based on the mole or total weight of the composition, 1% to 15% of cholesterol based on the mole or total weight of the composition, 2% to 35% of PEG-conjugated lipids based on the mole or total weight of the composition, and 1% to 20% of cholesterol based on the mole or total weight of the composition; Or it may be a lipid nanoparticle formulation comprising up to 90% ionizable lipids based on the mole or total weight of the composition and 2% to 10% non-cationic lipids based on the mole or total weight of the composition, or even 100% ionizable lipids based on the mole or total weight of the composition.In some embodiments of any of the aspects and embodiments of the present invention, the lipid particle formulation comprises ionizable lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:10:38.5:1.5. In some embodiments of any of the aspects and embodiments of the present invention, the lipid particle formulation comprises ionizable lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:10:38:2. In some embodiments of any of the aspects and embodiments of the present invention, the lipid particle formulation comprises ionizable lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:10:37:3. In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle (e.g., lipid nanoparticle) formulation comprises ionizable lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:7:40:3. In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle (e.g., lipid nanoparticle) formulation comprises ionizable lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:8:40:2. In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle (e.g., lipid nanoparticle) formulation comprises ionizable lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:9:39:2. In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle (e.g., lipid nanoparticle) formulation comprises ionizable lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of about 50:9:38:3.

[0179] In one embodiment of any of the aspects or embodiments of the present invention, lipid particles (e.g., lipid nanoparticles) comprise ionizable lipids, non-cationic lipids (e.g., phospholipids), sterols (e.g., cholesterol), and PEGylated lipids (conjugated lipids), wherein the molar ratio of ionizable lipids is in the range of 20 mol% to 70 mol% (wherein the target is 30 mol% to 60 mol%), the molar percentage of non-cationic lipids is in the range of 0 mol% to 30 mol% (wherein the target is 0 mol% to 15 mol%), the molar percentage of sterols is in the range of 20 mol% to 70 mol% (wherein the target is 30 mol% to 50 mol%), and the molar percentage of PEGylated lipids (conjugated lipids) is in the range of 1 mol% to 6 mol% (wherein the target is 2 mol% to 5 mol%).

[0180] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in international application PCT / US2018 / 050042 filed on September 7, 2018, the entirety of which is incorporated herein by reference and is considered for use in methods and compositions as disclosed herein.

[0181] The size of lipid particles (e.g., lipid nanoparticles) can be determined by quasi-elastic light scattering using the Malvern Zetasizer Nano ZS (Malvern, UK), and the diameter is approximately 50 nm to 150 nm, approximately 55 nm to 95 nm, or approximately 70 nm to 90 nm.

[0182] The pKa of formulated ionizable lipids may correlate with the effect of LNPs for nucleic acid delivery (Reference [Jayaraman et al[., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533]; see reference [Semple et al., Nature Biotechnology 28, 172-176 (2010)], all of which are incorporated herein by reference in their entirety). In any one embodiment of the aspects or embodiments of the present invention, the pKa of each ionizable lipid is determined in lipid nanoparticles using a assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS). Lipid nanoparticles comprising ionizable lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 (mol%)) at a total lipid concentration of 0.4 mM in PBS can be prepared using an in-line process as described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. The vesicle can be diluted with 24 mM lipids in 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, where the pH ranges from 2.5 to 11. An aliquot of the TNS solution is added to provide a final concentration of 1 mM, and after vortexing mixing, the fluorescence intensity is measured at room temperature using an SLM Aminco Series 2 emission spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. Sigmoidal best fit analysis can be applied to the fluorescence data, and pKa is measured at the pH at which half the maximum fluorescence intensity is achieved.

[0183] In any one embodiment of the aspects or embodiments of the present invention, 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 mg ceDNA / kg and 1.0 mg ceDNA / kg and expressed as luciferase ng / liver g measured 4 hours after administration.

[0184] Non-limitingly, the lipid particles (e.g., lipid nanoparticles) of the present disclosure comprise a lipid formulation that can be used to deliver a capsid-free nonviral DNA vector to a target site of interest (e.g., a cell, tissue, organ, etc.). Generally, the lipid particles (e.g., lipid nanoparticles) comprise a capsid-free nonviral DNA vector and an ionizable lipid or a salt thereof.

[0185] In one embodiment of any of the aspects or embodiments of the present invention, lipid particles (e.g., lipid nanoparticles) comprise ionizable lipids / non-cationic lipids / sterols / conjugated lipids in a molar ratio of 50:10:38.5:1.5.

[0186] In one embodiment of any of the aspects or embodiments of the present invention, the present disclosure provides a lipid particle (e.g., lipid nanoparticle) formulation comprising a phospholipid, lecithin, phosphatidylcholine and phosphatidylethanolamine.

[0187] III. Therapeutic Nucleic Acids (TNA)

[0188] The present disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNA). 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 nonviral DNA vectors, closed linear divalent DNA (ceDNA / CELiD), plasmids, vacmids, doggybone™ DNA vectors, minimally immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (DNA vectors closed by linear covalent bonds), or dumbbell DNA minimal vectors ("Dumbbell DNA"). As such, aspects of the present disclosure generally provide ionizable lipid particles (e.g., lipid nanoparticles) comprising TNA.

[0189] Therapeutic nucleic acids

[0190] Exemplary therapeutic nucleic acids of the present disclosure may comprise, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA (“ministring”), doggybone™, protelomere closed DNA or dumbbell linear DNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, and DNA viral vectors, viral RNA vectors, and any combination thereof.

[0191] siRNA or miRNA capable of downregulating the intracellular level of a specific protein through a process called RNA interference (RNAi) is also considered as a nucleic acid therapeutic agent in the present invention. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA structures can bind to a protein called RISC. The sense strand of the siRNA or miRNA is removed by the RISC complex. When combined with complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strand. RNAi induces the specific destruction of mRNA, thereby downregulating the corresponding protein.

[0192] Antisense oligonucleotides (ASOs) and ribozymes that inhibit mRNA translation into proteins can be nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxynucleotides have a sequence complementary to the sequence of the target protein mRNA and can bind to the mRNA through Watson-Crick base pairing. This binding prevents the translation of the target mRNA and / or triggers RNaseH degradation of the mRNA transcript. As a result, antisense oligonucleotides have increased specificity of action (i.e., downregulation of specific disease-related proteins).

[0193] In any method and composition provided herein, the therapeutic nucleic acid (TNA) may be a therapeutic RNA. The therapeutic RNA may be an inhibitor of mRNA translation, an RNA interference agent (RNAi), a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), an RNA that binds to an mRNA transcript (ASO), or an RNA that binds to a protein or other molecular ligand (aptamer). In any method provided herein, the RNAi agent may be double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, short hairpin RNA, or a trivalent oligonucleotide.

[0194] In any method and composition provided herein, the therapeutic nucleic acid (TNA) may be therapeutic DNA such as closed double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA (“ministring”), doggybone™, protelomere closed DNA, dumbbell linear DNA, plasmid, minicircle, etc.). Some embodiments of the present disclosure are based on methods and compositions comprising a closed linear double (ceDNA) capable of expressing a transgenic gene (e.g., therapeutic nucleic acid). The ceDNA vector as described herein is not subject to packaging constraints imposed by the limited space within the viral capsid. The ceDNA vector represents a viable eukaryotic alternative to plasmid DNA vectors produced in prokaryotes.

[0195] The ceDNA vector is preferably a linear and continuous structure rather than a discontinuous structure. Linear and continuous structures are considered to be not only more stable against attack by cellular endonucleases but also less likely to undergo recombination and induce mutations. Therefore, a ceDNA vector of a linear and continuous structure is a preferred embodiment. A continuous, linear, single-stranded intramolecular dimer ceDNA vector may have covalently bonded ends without a sequence encoding an AAV capsid protein. Such ceDNA vectors are structurally distinguished from plasmids (including the ceDNA plasmid described herein), which are circular dimer nucleic acid molecules of bacterial origin. While the complementary strand of a plasmid separates after denaturation to produce two nucleic acid molecules, in contrast, a ceDNA vector having a complementary strand remains a single molecule even after denaturation because it is a single DNA molecule. In some embodiments of any of the aspects and embodiments of the present invention, the ceDNA vector may be produced without prokaryotic-type DNA base methylation, unlike a plasmid. Therefore, ceDNA vectors and ceDNA-plasmids differ in terms of structure (particularly, linear versus circular), in terms of the methods used to produce and purify these different targets, and in terms of DNA methylation, which is prokaryotic in the case of ceDNA-plasmids and eukaryotic in the case of ceDNA vectors.

[0196] A nonviral capsid-free ceDNA molecule (ceDNA) having covalently closed ends is provided herein. Such nonviral capsid-free ceDNA molecule may be produced in an accepting host cell from an expression construct (e.g., ceDNA-plasmid, ceDNA-vacmid, ceDNA-vacurovirus, or integrated cell line) containing a heterogeneous gene (e.g., a transgenic gene, particularly a therapeutic transgenic gene) placed between two different reverse end repeat (ITR) sequences, wherein the ITRs are different from each other. In some embodiments of any of the aspects and embodiments of the present invention, one of the ITRs is modified by deletion, insertion, and / or substitution relative to a wild-type ITR sequence (e.g., AAV ITR); and at least one of the ITRs comprises a functional end degradation site (TRS) and a Rep binding site. The ceDNA vector is preferably a dimer and is self-complementary across at least a portion of the molecule, such as the expression cassette (e.g., ceDNA is not a double-stranded circular molecule). Because the ceDNA vector has covalently closed ends, it is resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) for more than 1 hour at 37°C, for example.

[0197] In any one embodiment of the aspects or embodiments of the present invention, the ceDNA vector comprises, in the 5'→3' direction, a first adeno-associated virus (AAV) reverse end repeat sequence (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein) and a second AAV ITR. In any one embodiment of the aspects or embodiments of the present invention, the first ITR (5' ITR) and the second ITR (3' ITR) are asymmetric to each other, that is, have different 3D spatial configurations. As an exemplary embodiment, the first ITR may be a wild-type ITR and the second ITR may be a mutated or modified ITR, or conversely, the first ITR may be a mutated or modified ITR and the second ITR may be a wild-type ITR. In any one embodiment of the aspects or embodiments of the present invention, the first ITR and the second ITR are both modified, but have different sequences, different modifications, or are not the same modified ITR, and have different 3D spatial configurations. In other words, a ceDNA vector having an asymmetric ITR has any change in one ITR for the WT-ITR that is not reflected in the other ITR; or alternatively, has an ITR that has a pair of asymmetric ITRs modified from the asymmetric ITR and has different sequences and different 3D shapes relative to each other.

[0198] In any one embodiment of the aspects or embodiments of the present invention, the ceDNA vector comprises, in the 5'→3' direction, a first adeno-associated virus (AAV) inverse terminal repeat sequence (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein) and a second AAV ITR, wherein the first ITR (5' ITR) and the second ITR (3' ITR) are symmetric or substantially symmetric with respect to each other; that is, the ceDNA vector may comprise an ITR sequence having a symmetric three-dimensional spatial configuration such that its structure has the same shape in geometric space or has the same A, CC' and BB' loops in 3D space. In such an embodiment, the symmetric ITR pair or the substantially symmetric ITR pair may be a modified ITR (e.g., mod-ITR) rather than a wild-type ITR. The mod-ITR pair may have one or more modifications from the wild-type ITR and may have the same sequence that is inverse complement (inverse phase) to each other. In one embodiment of any of the aspects or embodiments of the present invention, the modified ITR pair is substantially symmetric as defined herein, that is, the modified ITR pair may have different sequences but may have a corresponding or identical symmetric three-dimensional shape. In some embodiments of any of the aspects and embodiments of the present invention, the symmetric ITR or the substantially symmetric ITR may be a wild type (WT-ITR) as described herein. That is, the two ITRs may have wild type sequences but do not necessarily have to be WT-ITRs of the same AAV serotype. In one embodiment of any of the aspects or embodiments of the present invention, one WT-ITR may be derived from one AAV serotype and the other WT-ITR may be derived from a different AAV serotype. In such an embodiment, the WT-ITR pair is substantially symmetric as defined herein, that is, they may have one or more conservative nucleotide modifications while still maintaining a symmetric three-dimensional spatial configuration.

[0199] The wild-type, mutated, or otherwise modified ITR sequences provided herein represent DNA sequences contained in expression constructs (e.g., ceDNA-plasmid, ceDNA-vacuol, ceDNA-vacurovirus) for the production of ceDNA vectors. Accordingly, the ITR sequence actually contained in a ceDNA vector produced from a ceDNA-plasmid or other expression construct may or may not be the same as the ITR sequence provided herein as a result of natural changes (e.g., replication errors) occurring during the production process.

[0200] In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector described herein, comprising an expression cassette having a transgenic gene which is a therapeutic nucleic acid sequence, may be operatively connected to one or more regulatory sequence(s) that enable or control the expression of the transgenic gene. In one embodiment of any of the aspects or embodiments of the present invention, the polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is flanked by the first ITR sequence and the second ITR sequence, and the first ITR sequence and the second ITR sequence are asymmetric or symmetric to each other.

[0201] In one embodiment of any of the aspects or embodiments of the present invention, the expression cassette is positioned between two ITRs comprising, in this order, a promoter operably linked to the transposable gene, a post-transcriptional regulatory element, and one or more of polyadenylation and termination signals. In one embodiment of any of the aspects or embodiments of the present invention, the promoter is regulated, i.e., inducible or repressive. The promoter may be any sequence that facilitates the transcription of the transposable gene. In one embodiment of any of the aspects or embodiments of the present invention, the promoter is a CAG promoter or a variant thereof. The post-transcriptional regulatory element is any sequence that regulates the expression of the transposable gene, and, as a non-limiting example, a therapeutic nucleic acid sequence, which forms a tertiary structure that enhances the expression of the transposable gene.

[0202] In one embodiment of any of the aspects or embodiments of the present invention, the post-transfer control element comprises WPRE. In one embodiment of any of the aspects or embodiments of the present invention, the polyadenylation and serial signal comprises BGHpolyA. Any of which is known in the art Sis Regulatory elements or combinations thereof, e.g., SV40 late polyA signal upstream enhancer sequence (USE), or, but not limited to, thymidine kinase genes of herpes simplex virus, or other post-transcriptional processing elements including hepatitis B virus (HBV) may be further used. In one embodiment of any of the aspects or embodiments of the present invention, the expression cassette length in the 5'→3' direction is greater than the maximum length known to be capsidized to an AAV virion. In one embodiment of any of the aspects or embodiments of the present invention, 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.

[0203] In one embodiment of any of the aspects or embodiments of the present invention, the expression cassette may comprise 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 about 4,000 to 10,000 nucleotides, or any range between 10,000 and 50,000 nucleotides, or more than 50,000 nucleotides.

[0204] In one embodiment of any of the aspects or embodiments of the present invention, the expression cassette may also include an internal ribosome entry site (IRES) and / or 2A element. Sis Regulatory elements include, but are not limited to, promoters, riboswitches, isolating factors, mir-regulatory elements, post-transcriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments of any of the aspects and embodiments of the present invention, the ITR may act as a promoter for a transgenic gene. In some embodiments of any of the aspects and embodiments of the present invention, the ceDNA vector may include additional components for regulating the expression of the transgenic gene, for example, a regulatory switch for controlling and regulating the expression of the transgenic gene, and, if desired, may include a regulatory switch that is a death switch enabling controlled apoptosis of the cell containing the ceDNA vector.

[0205] In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector may be obtained from a plasmid that does not contain a capsid and encodes a first ITR, an expressible transgenic cassette, and a second ITR in this order, wherein at least one of the first ITR sequence and / or the second ITR sequence is mutated for the corresponding wild-type AAV2 ITR sequence.

[0206] In any one embodiment of the aspects or embodiments of the present invention, the ceDNA vector disclosed herein is used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary purposes) or for immunogenic polypeptides.

[0207] The expression cassette may include any transgenic gene that is a therapeutic nucleic acid sequence. In a specific embodiment, the ceDNA vector includes any gene of interest in the target, and such gene of interest includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0208] In any one embodiment of the aspects or embodiments of the present invention, the sequence provided in the expression cassette, expression construct, or donor sequence of the ceDNA vector described herein may be codon-optimized for host cells. As used herein, the terms “codon-optimized” or “codon-optimized” refer to a process of modifying a nucleic acid sequence for expression enhancement in the cells of a vertebrate of interest, e.g., mouse or human, by replacing at least one, more than one, or a significant number of codons in a natural sequence (e.g., a prokaryotic sequence) with codons that are used more frequently or most frequently in the genes of the vertebrate. Various species exhibit specific biases toward specific codons of specific amino acids.

[0209] Typically, codon optimization does not alter the amino acid sequence of the originally translated protein. Optimized codons are, for example, It may be determined using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171), or another publicly available database.

[0210] Many organisms exhibit a bias in using specific codons to encode the insertion of particular amino acids in growing peptide chains. Codon preference, or codon bias—differences in codon usage among organisms—is conferred by the degeneracy of the genetic code and is well documented in many organisms. Codon bias is often correlated with the translation efficiency of messenger RNA (mRNA), which is believed to depend particularly on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be tuned for optimal gene expression based on codon optimization in a given organism.

[0211] Considering the numerous gene sequences available for various animal, plant, and microbial species, it is possible to calculate the relative frequency of codon usage (Reference [Nakamura, Y., et al . "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000)]).

[0212] Inverse terminal repeat sequence (ITR)

[0213] As described herein, a ceDNA vector is a capsid-free linear dimer DNA molecule (linear, continuous, and uncapsidized structure) formed from a continuous strand of complementary DNA having covalently closed ends, comprising a 5' inverse terminal repeat (ITR) sequence and a 3' ITR sequence that are different from or asymmetric to each other. At least one of the ITRs comprises a functional terminal degradation site and a replication protein binding site (RPS) (sometimes referred to as a replication protein binding site), e.g., a Rep binding site. Generally, the ceDNA vector contains at least one modified AAV inverse terminal repeat sequence (ITR), i.e., a deletion, insertion, and / or substitution of another ITR, and an expressible transgene.

[0214] In one embodiment of any of the aspects or embodiments of the present invention, at least one of the ITRs is an AAV ITR, e.g., a wild-type AAV ITR. In one embodiment of any of the aspects or embodiments of the present invention, at least one of the ITRs is an ITR modified with respect to another ITR, i.e., ceDNA comprises ITRs that are asymmetric with respect to each other. In one embodiment of any of the aspects or embodiments of the present invention, at least one of the ITRs is a non-functional ITR.

[0215] In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector comprises (1) an expression cassette comprising a cis-regulatory element, a promoter, and at least one transposable gene; or (2) a promoter operably linked to at least one transposable gene; and (3) two self-complementary sequences, e.g., ITR, flanking the expression cassette, wherein the ceDNA vector is not bound to a capsid protein. In some embodiments of any of the aspects and embodiments of the present invention, the ceDNA vector comprises two self-complementary sequences found in the AAV genome (wherein at least one comprises the operatory Rep-binding element (RBE) and terminal degradation site (TRS) of the AAV, or a functional variant of the RBE) and one or more cis-regulatory elements operably linked to a transposable gene. In some embodiments of any of the aspects and embodiments of the present invention, the ceDNA vector may include additional components for regulating the expression of a transgenic gene, for example, a regulatory switch for controlling and regulating the expression of a transgenic gene, and may include a regulatory switch that is a death switch enabling controlled cell death of a cell containing the ceDNA vector.

[0216] In one embodiment of any of the aspects or embodiments of the present invention, the two self-complementary sequences may be ITR sequences derived from any known parvovirus, e.g., defendoviruses such as AAV (e.g., AAV1 to AAV12). In addition to variable palindromic sequences that enable the formation of a hairpin secondary structure, any AAV serotype may be used, but not limited to, a modified AAV2 ITR sequence having a Rep-binding site (RBS) and a terminal degradation site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3'. In some embodiments of any of the aspects or embodiments of the present invention, the ITR may be synthetic. In one embodiment of any of the aspects or embodiments of the present invention, the synthetic ITR is based on ITR sequences derived from more than one AAV serotype. In another embodiment, the synthetic ITR does not contain an AAV-based sequence. In another embodiment, the synthetic ITR has no AAV-derived sequence or only a portion thereof, but preserves the ITR structure described above. 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 but only by mutated Rep. In some embodiments of any of the embodiments and examples of the present invention, the ITR is a synthetic ITR sequence having a functional Rep-binding site (RBS) and a terminal degradation site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to a variable palindromic sequence that enables the formation of a hairpin secondary structure. In some examples, the modified ITR sequence has the structure and location of a Rep-binding element forming a terminal loop portion of one of the ITR hairpin secondary structures derived from the sequences of the RBS and TRS, and the corresponding sequence of the wild-type AAV2 ITR.Exemplary ITR sequences for use in ceDNA vectors are disclosed in Tables 2 through 9, Table 10A and Table 10B, SEQ ID NO. 2, SEQ ID NO. 52, SEQ ID NO. 101 through SEQ ID NO. 449, and SEQ ID NO. 545 through SEQ ID NO. 547 of International Application PCT / US 18 / 49996 filed September 7, 2018, and partial ITR sequences are shown in FIG. 26a and FIG. 26b. In some embodiments of any of the aspects and embodiments of the present invention, the ceDNA vector may comprise an ITR having a modification in the ITR corresponding to any modification in the ITR sequence or any modification in the ITR partial sequence presented in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A and 10B of International Application PCT / US 18 / 49996 filed on September 7, 2018.

[0217] In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector may be produced in an expression construct further comprising a specific combination of cis-regulatory elements. Sis Regulatory elements include, but are not limited to, promoters, riboswitches, isolating factors, mir-regulatory elements, post-transcriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments of any of the aspects and embodiments of the present invention, the ITR may act as a promoter for a transgenic gene. In some embodiments of any of the aspects and embodiments of the present invention, the ceDNA vector comprises additional components for regulating the expression of the transgenic gene, for example, a regulatory switch as described in International Application PCT / US 18 / 49996 filed September 7, 2018, for regulating the expression of the transgenic gene, or a death switch capable of killing a cell containing the ceDNA vector.

[0218] In one embodiment of any of the aspects or embodiments of the present invention, the expression cassette may also include a post-transcriptional element to increase the expression of the transposable gene. In one embodiment of any of the aspects or embodiments of the present invention, a Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase the expression of the transposable gene. Other post-transcriptional processing elements, such as the thymidine kinase gene of herpes simplex virus or a post-transcriptional element from hepatitis B virus (HBV), may be used. Secretion sequences, for example, VH-02 and VK-A26 sequences, may be linked to the transposable gene. The expression cassette may include a polyadenylation sequence known in the art or a variant thereof, such as a naturally occurring sequence isolated from bovine BGHpA or viral SV40pA, or a synthetic sequence. Some expression cassettes may also include an SV40 late polyA signal upstream enhancer (USE) sequence. The USE can be used in combination with SV40pA or heterogeneous polyA signals.

[0219] FIGS. 1a through 1c of International Application PCT / US2018 / 050042 filed September 7, 2018 (the entirety thereof is incorporated herein by reference) illustrate a non-limiting and exemplary ceDNA vector or a corresponding sequence of a ceDNA plasmid. The ceDNA vector may be obtained from a plasmid that encodes a first ITR, an expressible transgenic cassette, and a second ITR in this order, without containing a capsid, wherein at least one of the first ITR sequence and / or the second ITR sequence is mutated for the corresponding wild-type AAV2 ITR sequence. The expressible transgenic cassette preferably comprises one or more of an enhancer / promoter, an ORF reporter (transgenic), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA) in this order.

[0220] Promoter

[0221] Suitable promoters comprising those described above may be derived from viruses and thus referred to as viral promoters, or may be derived from any organism including prokaryotic or eukaryotic organisms. Suitable promoters may be used to induce expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); These include herpes simplex virus (HSV) promoters, cytomegalovirus (CMV) promoters, e.g., CMV ultra-early promoter region (CMVTE), rous sarcoma virus (RSV) promoters, human U6 small nuclear promoters (U6, e.g., Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), augmented U6 promoters (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), human H1 promoters (H1), CAG promoters, human alpha 1-antitrypsin (HAAT) promoters (and others). In any one embodiment of the aspects or embodiments of the present invention, these promoters are modified at their downstream intron-containing ends to include one or more nuclease cleavage sites. Aspects or embodiments of the present invention In any one embodiment of the above, the DNA containing the nuclease cleavage site(s) is heterogeneous to the promoter DNA.

[0222] In any one embodiment of the aspects or embodiments of the present invention, the promoter may include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial expression and / or temporal expression. The promoter may also include distal enhancer or inhibitor elements located thousands of base pairs away from the transcription start site. The promoter may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter may constitutively or differentially regulate the expression of a gene component with respect to the cell, tissue, or organ where expression occurs, with respect to the developmental stage where expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter, as well as the promoters listed below. These promoters and / or enhancers may be used for the expression of any gene of interest (e.g., a therapeutic protein). For example, a vector may include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein.In any one embodiment of the aspects or embodiments of the present invention, a promoter operably linked to a therapeutic protein coding sequence may be a promoter from monkey virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as a cytomegalovirus (CMV) very early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. In one embodiment of any of the aspects or embodiments of the present invention, the promoter may also be a promoter of human gene origin, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a liver-specific promoter, such as natural or synthetic human alpha 1-antitrypsin (HAAT) or transthyretin (TTR). In one embodiment of any of the aspects or embodiments of the present invention, delivery to the liver may be achieved by using endogenous ApoE-specific targeting of a composition comprising a ceDNA vector to hepatocytes via low-density lipoprotein (LDL) receptors present on the surface of hepatocytes.

[0223] In any one embodiment of the aspects or embodiments of the present invention, the promoter used is a natural promoter of a gene encoding a therapeutic protein. The promoter and other regulatory sequences for each gene encoding a therapeutic protein are known and characterized. The promoter region used may further include one or more additional regulatory sequences (e.g., natural), such as enhancers known in the art (e.g., Serpin Enhancers).

[0224] Non-limiting examples of promoters suitable for use according to the present invention include, for example, a CAG promoter, a HAAT promoter, a fragment of a human EF1-α promoter or EF1-α promoter, and a rat EF1-α promoter.

[0225] Polyadenylation sequence

[0226] To stabilize mRNA expressed in the ceDNA vector and to aid in nuclear release and translation, a sequence encoding a polyadenylation sequence may be included in the ceDNA vector. In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector does not include a polyadenylation sequence. In another embodiment, the vector comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenines or dinucleotides. In some embodiments of any of the aspects and embodiments of the present invention, the polyadenylation sequence comprises about 43 nucleotides, about 40 to 50 nucleotides, about 40 to 55 nucleotides, about 45 to 50 nucleotides, about 35 to 50 nucleotides, or any range of nucleotides between the disclosed ranges.

[0227] In one embodiment of any of the aspects or embodiments of the present invention, ceDNA may be obtained from a vector polynucleotide encoding a heterogeneous nucleic acid operatively positioned between two different reverse end repeat sequences (ITRs) (e.g., AAV ITRs), wherein at least one of the ITRs comprises 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 comprises a deletion, insertion, or substitution for the other ITR, e.g., a functional ITR.

[0228] In one embodiment of any of the aspects or embodiments of the present invention, the host cell does not express a viral capsid protein, and the polynucleotide vector template does not contain any viral capsid coding sequence. In one embodiment of any of the aspects or embodiments of the present invention, the polynucleotide vector template does not contain the AAV capsid gene as well as the capsid gene of another virus. In one embodiment of any of the aspects or embodiments of the present invention, the nucleic acid molecule also does not contain the AAV Rep protein coding sequence. Therefore, in some embodiments of any of the aspects and embodiments of the present invention, the nucleic acid molecule of the present invention does not contain both the functional AAV cap gene and the AAV rep gene.

[0229] In any one embodiment of the aspects or embodiments of the present invention, the ceDNA vector does not have a modified ITR.

[0230] In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector comprises a control switch as disclosed in the present invention (or international application PCT / US 18 / 49996 filed September 7, 2018).

[0231] IV. Production of ceDNA Vector

[0232] A method for producing a ceDNA vector as described herein, comprising an asymmetric ITR pair or a symmetric ITR pair as defined herein, is described in Section IV of International Application PCT / US 18 / 49996 filed September 7, 2018, the entirety of which is incorporated herein by reference. As described herein, a ceDNA vector may be obtained, for example, by a process comprising the following steps: a) incubating a population of host cells (e.g., insect cells) possessing a polynucleotide expression structure template (e.g., ceDNA-plasmid, ceDNA-vacmid and / or ceDNA-vacurovirus) that lacks a viral capsid-coding sequence for a sufficient time under conditions effective for inducing the production of a ceDNA vector within the host cells in the presence of a Rep protein, wherein the host cells do not contain a viral capsid-coding sequence; and b) collecting and isolating the ceDNA vector from the host cells. The presence of Rep protein induces the replication of a vector polynucleotide containing a modified ITR, producing a ceDNA vector in the host cell.

[0233] However, viral particles (e.g., AAV virions) are not expressed. Therefore, there are no size limitations like those naturally imposed on AAV or other virus-based vectors.

[0234] 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, analyzing the digested DNA material on an undenatured gel, and confirming the presence of characteristic bands of linear and continuous DNA by comparing them with linear and discontinuous DNA.

[0235] In one embodiment of any of the aspects or embodiments of the present invention, the present invention is, for example, described in the literature [Lee, L. et al.As described in

[2013] Plos One 8(8): e69879, the use of a host cell line in which a DNA vector polynucleotide expression template (ceDNA template) is stably incorporated into its own genome in the production of a nonviral DNA vector is provided. Preferably, Rep is added to the host cell at an MOI of about 3. If the host cell line is a mammalian cell line, e.g., HEK293 cell, said cell line may have a stably incorporated polynucleotide vector template and may enable the excision and amplification of ceDNA in the presence of Rep and a helper virus by introducing the Rep protein into the cell using a second vector such as a herpesvirus.

[0236] In one embodiment of any of the aspects or embodiments of the present invention, the host cell used to produce the ceDNA vector described herein is an insect cell, and a vacurovirus is used to deliver both a polynucleotide encoding the Rep protein and a non-viral DNA vector polynucleotide expression construct template for ceDNA. In some embodiments of any of the aspects and embodiments of the present invention, the host cell is engineered to express the Rep protein.

[0237] Next, the ceDNA vector is collected and isolated from the host cells. The time for collecting and harvesting the ceDNA vector described herein from the cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the collection time can be selected in terms of cell viability, cell morphology, cell growth, etc. In one embodiment of any of the aspects or embodiments of the present invention, cells are grown under sufficient conditions and collected for a sufficient time after vacurovirus infection to produce the ceDNA vector, but before most of the cells begin to die due to vacurovirus toxicity. The DNA vector can be isolated using a plasmid purification kit, such as the Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation may also be applied to the DNA vector. In general, any nucleic acid purification method may be adopted.

[0238] DNA vectors may be purified by any means known to those skilled in the art regarding the purification of DNA. In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector is purified into DNA molecules. In one embodiment of any of the aspects or embodiments of the present invention, the ceDNA vector is purified into exosomes or microparticles. The presence of the ceDNA vector may be confirmed by digesting the vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector, and analyzing the digested and micronized DNA material using gel electrophoresis to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[0239] V. Preparation of lipid particles

[0240] Lipid particles (e.g., lipid nanoparticles) can be spontaneously formed upon mixing TNA (e.g., ceDNA) and lipid(s). Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane using a thermobarrel extruder, such as the Lipex extruder (Northern Lipids, Inc.) (e.g., 100 nm cutoff). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration.

[0241] Generally, lipid particles (e.g., lipid nanoparticles) may be formed according to any method known in the art. For example, lipid particles (e.g., lipid nanoparticles) may be prepared according to the methods described, for example, in U.S. Patent Applications Published in US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129, and US2010 / 0130588, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments of any of the aspects and embodiments of the present invention, lipid particles (e.g., lipid nanoparticles) may be prepared using a continuous mixing method, a direct dilution method, or an inline dilution method. A process and apparatus for producing lipid nanoparticles using direct dilution and inline dilution methods is described in U.S. Patent Application Publication US2007 / 0042031, the contents of which are incorporated herein by reference in their entirety. A process and apparatus for producing lipid nanoparticles using a stepwise dilution method is described in U.S. Patent Application Publication US2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0242] In one embodiment of any of the aspects or embodiments of the present invention, lipid particles (e.g., lipid nanoparticles) may be prepared according to an impinging jet method. Generally, lipids dissolved in an alcohol (e.g., ethanol) are mixed with ceDNA dissolved in a buffer, e.g., citrate buffer, sodium acetate buffer, sodium acetate and magnesium chloride buffer, malate buffer, malate and sodium chloride buffer, or sodium citrate and sodium chloride buffer to form particles. The mixing ratio of lipids to ceDNA is about 45% to 55% lipids and about 65% to 45% ceDNA.

[0243] The lipid solution may contain the disclosed ionizable lipids, non-cationic lipids (e.g., phospholipids, e.g., DSPC, DOPE, and DOPC), PEG-lipid conjugated molecules (e.g., PEG-lipids), and sterols (e.g., cholesterol) in alcohol, e.g., ethanol, at a total lipid concentration of 5 mg / mL to 30 mg / mL, more possibly 5 mg / mL to 15 mg / mL, and most possibly 9 mg / mL to 12 mg / mL. In the lipid solution, the molar ratio of lipids is about 25% to 98%, preferably about 35% to 65%, for cationic lipids; about 0% to 15%, preferably about 0% to 12%, for non-cationic lipids; and about 0% to 15%, preferably about 1% to 6%, for PEG-lipid conjugated lipid molecules. In the case of sterols, the range may be about 0% to 75%, preferably about 30% to 50%.

[0244] The ceDNA solution may contain ceDNA at a concentration of 0.3 mg / mL to 1.0 mg / mL, preferably 0.3 mg / mL to 0.9 mg / mL, in a buffer solution having a pH in the range of 3.5 to 5.

[0245] For LNP formation, in one exemplary non-limiting embodiment, two lipids are heated to a temperature in the range of about 15°C to 40°C, preferably about 30°C to 40°C, and then mixed, for example, in a collision jet mixer to immediately form LNP. The mixing flow rate may be in the range of 10 mL / min to 600 mL / min. The tube ID may be in the range of 0.25 mm to 1.0 mm, and the total flow rate may be 10 mL / min to 600 mL / min. The combination of flow rate and tube ID may have the effect of controlling the particle size of the LNP to 30 nm to 200 nm. Subsequently, the solution may be mixed with a buffer solution at a higher pH in a mixing ratio in the range of 1:1 to 1:3 vol:vol, preferably about 1:2 vol:vol. If necessary, this buffer solution may be at a temperature in the range of 15°C to 40°C or 30°C to 40°C. Next, the mixed LNP can be applied to an anion exchange filtration step. Before anion exchange, the mixed LNP can be incubated for a certain period of time, for example, from 30 minutes to 2 hours. The temperature during incubation may be in the range of 15°C to 40°C or 30°C to 40°C. After incubation, the solution is filtered through a filter, for example, a 0.8 μm filter (including an anion exchange separation step). This process may use a tube ID in the range of 1 mm ID to 5 mm ID and a flow rate of 10 mL / min to 2000 mL / min.

[0246] After formation, the LNP can be dialysis-filtered through an ultrafiltration process in which the LNP is concentrated, the alcohol is removed, and the buffer solution is exchanged with a final buffer solution, for example, phosphate-buffered saline (PBS), with a pH of about 7, e.g., about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, or about 7.4.

[0247] The ultrafiltration process may utilize a tangential flow filtration (TFF) format using a membrane nominal molecular weight cutoff in the range of 30 kD to 500 kD. The membrane format is a hollow fiber or a flat cassette. A TFF process using an appropriate molecular weight cutoff may retain LNP in the residue, and the filtrate or permeate contains alcohol; citrate buffer and final buffer waste. The TFF process is a multi-step process from an initial concentration to a ceDNA concentration of 1 mg / mL to 3 mg / mL. After concentration, the LNP solution is dialyzed to 10 to 20 volumes relative to the final buffer to remove alcohol and perform buffer exchange. Subsequently, the material may be further concentrated by 1 to 3 times. The concentrated LNP solution may be sterile filtered.

[0248] VI. Pharmaceutical Composition and Formulation

[0249] A pharmaceutical composition comprising TNA lipid particles and a pharmaceutically acceptable carrier or excipient is also provided herein.

[0250] In one embodiment of any of the aspects or embodiments of the present invention, TNA lipid particles (e.g., lipid nanoparticles) provide complete or partial encapsulation of therapeutic nucleic acids. In one embodiment of any of the aspects or embodiments of the present invention, a nucleic acid therapeutic agent is completely encapsulated in lipid particles (e.g., lipid nanoparticles) to form nucleic acid-containing lipid particles. In one embodiment of any of the aspects or embodiments of the present invention, the nucleic acid is encapsulated in the lipid portion of the particle and can be protected from enzymatic degradation.

[0251] In one embodiment of any of the aspects or embodiments of the present invention, the average diameter of the lipid particles ensuring effective delivery is about 20 nm to about 100 nm, 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, The dimensions are 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 the same are disclosed, for example, in International Application PCT / US18 / 50042, U.S. Patent Application Publications 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In any one embodiment of the aspects or embodiments of the present invention, the size of the lipid particles (e.g., lipid nanoparticles) may be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) system.

[0252] Generally, the lipid particles of the present invention (e.g., lipid nanoparticles) have an average diameter selected to provide an intended therapeutic effect.

[0253] Depending on the intended use of (e.g., lipid nanoparticles), the ratio of components may vary, and the delivery efficiency of a specific formulation can be measured, for example, using an endosome release parameter (ERP) assay.

[0254] In one embodiment of any of the aspects or embodiments of the present invention, lipid particles (e.g., lipid nanoparticles) may be bonded to other moiety to prevent aggregation. These lipid conjugates include, but are not limited to, PEG-lipid conjugates 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), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugate; see, for example, U.S. Provisional Application No. 61 / 294,828 filed January 13, 2010 and U.S. Provisional Application No. 61 / 295,140 filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Further examples of POZ-lipid conjugates are described in International Publication WO 2010 / 006282. PEG or POZ may be directly conjugated to a lipid or linked to a lipid via a linker moiety. Any linker moiety suitable for coupling PEG or POZ to a lipid may be used, including, for example, non-ester-containing linker moiety and ester-containing linker moiety. In certain preferred embodiments, a non-ester-containing linker moiety, such as an amide or carbamate, is used. The disclosure of each of the above patent documents is incorporated herein by reference in its entirety for all purposes.

[0255] In one embodiment of any of the aspects or embodiments of the present invention, ceDNA may be complexed with the lipid portion of the particle or encapsulated at the lipid location of the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments of the present invention, ceDNA may be completely encapsulated at the lipid location of the lipid particle (e.g., lipid nanoparticle) so as to be protected from degradation by nuclease, for example, in an aqueous solution. In one embodiment of any of the aspects or embodiments of the present invention, ceDNA within the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after the lipid particle (e.g., lipid nanoparticle) is exposed to nuclease at 37°C for at least about 20, 30, 45, or 60 minutes. In some embodiments of any of the aspects and embodiments of the present invention, ceDNA in lipid particles (e.g., lipid nanoparticles) is not substantially degraded after incubating the particles in serum at 37°C for at least about 30 minutes, 45 minutes, or 60 minutes, or at least about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, or 36 hours.

[0256] In any one embodiment of the aspects or embodiments of the present invention, the lipid particles (e.g., lipid nanoparticles) are substantially non-toxic to the target, e.g., mammals such as humans.

[0257] In one embodiment of any of the aspects or embodiments of the present invention, 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 of any of the aspects and embodiments of the present invention, 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 non-cationic lipids. In a preferred embodiment, the lipid particle of the present invention is a nucleic acid-containing lipid particle formed from the disclosed ionizable lipid comprising a therapeutic nucleic acid 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 linear divalent DNA (ceDNA / CELiD), plasmids, vacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (DNA vectors closed by linear covalent bonds), or dumbbell DNA minimal vectors ("Dumbbell DNA").

[0258] In another preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles formed from non-cationic lipids and optionally conjugated lipids that prevent particle aggregation.

[0259] In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle formulation is an aqueous solution. In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle (e.g., lipid nanoparticle) formulation is a freeze-dried powder.

[0260] According to some embodiments, the present disclosure provides a lipid particle formulation further comprising one or more pharmaceutical excipients. In one embodiment of any of the embodiments or examples of the present invention, the lipid particle (e.g., lipid nanoparticle) formulation further comprises sucrose, tris, trehalose and / or glycine.

[0261] In any one embodiment of the aspects or embodiments of the present invention, the lipid particles disclosed herein (e.g., lipid nanoparticles) are used to enter the target cells, tissues, or organs In vivo For delivery, it may be incorporated into a pharmaceutical composition suitable for administration to a target. Typically, the pharmaceutical composition comprises the TNA lipid particles (e.g., lipid nanoparticles) disclosed herein and a pharmaceutically acceptable carrier. In one embodiment of any of the aspects or embodiments of the present invention, the TNA lipid particles (e.g., lipid nanoparticles) of the present invention may be incorporated into a pharmaceutical composition suitable for the intended route of therapeutic administration (e.g., parenteral administration). Passive tissue delivery via intracellular injection, such as intranuclear microinjection or intracytoplasmic injection, as well as high-pressure intravenous or intra-arterial infusion, is also considered. The pharmaceutical composition for therapeutic purposes may be formulated as a solution, microemulsion, dispersion, liposome, or other aligned structure suitable for high ceDNA vector concentrations. A sterile injectable solution may be prepared by incorporating the required amount of the ceDNA vector compound into a suitable buffer having one or a combination of the components listed above, and then, if necessary, filtering and sterilizing.

[0262] Lipid particles as disclosed herein may be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intradural, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intradural, intravesical, conjunctival (e.g., extraorbital, intraorbital, posterior orbital, intraretinal, subretinal, choroidal, subchoroidal, interstitial, anterior chamber, and vitreous), intracochlear, and mucosal (e.g., oral, sublingual, nasal) administration. Passive tissue delivery via intracellular injection, such as intranuclear microinjection or intracytoplasmic injection, as well as high-pressure intravenous or intra-arterial infusion, is also considered.

[0263] A pharmaceutically active composition comprising TNA lipid particles (e.g., lipid nanoparticles) can be formulated to deliver a transgene of nucleic acid to a receptor cell to induce therapeutic expression of the transgene therein. The composition may also comprise a pharmaceutically acceptable carrier.

[0264] Pharmaceutical compositions for therapeutic purposes are typically sterile and stable under manufacturing and storage conditions. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other aligned structure suitable for high ceDNA vector concentrations. A sterile injectable solution may be prepared by incorporating the required amount of the ceDNA vector compound into a suitable buffer having one or a combination of the components listed above, and then, if necessary, filtering and sterilizing.

[0265] In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle (e.g., lipid nanoparticle) is a solid core particle having at least one lipid bilayer. In one embodiment of any of the aspects or embodiments of the present invention, the lipid particle (e.g., lipid nanoparticle) has a non-bilayer structure, that is, a non-lamellar (i.e., non-bilayer) form. Non-limitingly, the non-bilayer form may include, for example, a three-dimensional tube, a rod, cubic symmetry, etc. The non-lamellar form (i.e., non-bilayer structure) of the lipid particle (e.g., lipid nanoparticle) can be determined using analytical techniques known to and used by those skilled in the art. Such techniques include, but are not limited to, cryo-transmission electron microscopy ("Cryo-TEM"), differential scanning calorimetry ("DSC"), X-ray diffraction, etc. For example, the morphology of lipid particles (lamellar versus non-lamellar) can be easily evaluated and characterized using cryo-TEM analysis, such as that described in, for example, U.S. Patent Application Publication US2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0266] In any one embodiment of the aspects or embodiments of the present invention, lipid particles having a non-lamellar form (e.g., lipid nanoparticles) have a high electron density.

[0267] In one embodiment of any of the aspects or embodiments of the present invention, the present disclosure provides lipid particles (e.g., lipid nanoparticles) having a monolamellar or multilamellar structure. In some aspects, the present disclosure provides a formulation of lipid particles (e.g., lipid nanoparticles) comprising polysaccharidic particles and / or foam-based particles. By controlling the composition and concentration of lipid components, the rate at which lipid conjugates are exchanged from lipid particles (e.g., lipid nanoparticles), and consequently, the rate at which lipid nanoparticles become fused, can be controlled. Additionally, by using other variables, such as pH, temperature, or ionic strength, the rate at which lipid particles (e.g., lipid nanoparticles) become fused, can be varied and / or controlled. Other methods that may be used to control the rate at which lipid particles (e.g., lipid nanoparticles) become fused will be apparent to those skilled in the art based on the present disclosure. It will also be apparent that lipid particle size can be controlled by controlling the composition and concentration of lipid conjugates.

[0268] In one embodiment of any of the embodiments or examples of the present invention, the pKa of the formulated ionizable lipid may be correlated with the effect 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)], all of which are incorporated herein by reference in their entirety). In one embodiment of any of the embodiments or examples of the present invention, the preferred pKa range is about 5 to about 8. In one embodiment of any of the embodiments or examples of the present invention, the preferred pKa range is about 6 to about 7. In one embodiment of any of the embodiments or examples of the present invention, the preferred pKa is about 6.5. In one embodiment of any of the aspects or embodiments of the present invention, the pKa of an ionizable lipid can be determined in lipid particles (e.g., lipid nanoparticles) using a assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS).

[0269] In one embodiment of any of the aspects or embodiments of the present invention, the encapsulation of ceDNA within lipid particles (e.g., lipid nanoparticles) may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, e.g., the Oligreen® assay or the PicoGreen® assay, using a dye whose fluorescence is enhanced upon association with nucleic acid. Generally, encapsulation is determined by adding the dye to the lipid particle formulation, measuring the generated fluorescence, and comparing it to the fluorescence observed upon the addition of a small amount of nonionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ceDNA, enabling interaction with the membrane-impermeable dye. The encapsulation of ceDNA can be calculated as follows: E = (I0 - I) / I0 (where I and I0 represent the fluorescence intensity before and after the addition of detergent).

[0270] Unit dosage

[0271] In one embodiment of any of the embodiments or examples of the present invention, the pharmaceutical composition may exist in a unit dosage form. The unit dosage form may typically be adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments of any of the embodiments and examples of the present invention, the unit dosage form is adapted to administration by inhalation. In some embodiments of any of the embodiments and examples of the present invention, the unit dosage form is adapted to administration by a vaporizer. In some embodiments of any of the embodiments and examples of the present invention, the unit dosage form is adapted to administration by a nebulizer. In some embodiments of any of the embodiments and examples of the present invention, the unit dosage form is adapted to administration by an aerosol generator. In some embodiments of any of the embodiments and examples of the present invention, the unit dosage form is adapted to oral administration, buccal administration, or sublingual administration. In some embodiments of any of the embodiments and examples of the present invention, the unit dosage form is adapted to intravenous, intramuscular, or subcutaneous administration. In some embodiments of any of the aspects and embodiments of the present invention, the unit dosage form is adapted for intrathecal or intraventricular administration. In some embodiments of any of the aspects and embodiments of the present invention, the pharmaceutical composition is formulated for topical administration. The amount of the active ingredient that may be combined with a carrier material to produce a single dosage form may generally be the amount of the compound that produces a therapeutic effect.

[0272] VII. Treatment Methods

[0273] The ionizable lipid compositions and methods described herein (e.g., TNA lipid particles as described herein (e.g., lipid nanoparticles)) can be used to introduce nucleic acid sequences (e.g., therapeutic nucleic acid sequences) into host cells. In one embodiment of any of the aspects or embodiments of the present invention, the introduction of nucleic acid sequences into host cells using TNA LNPs (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) can be monitored using appropriate biomarkers from treated patients to evaluate gene expression.

[0274] The LNP compositions provided herein may be used to deliver transgenic genes (nucleic acid sequences) for various purposes. In one embodiment of any of the aspects or embodiments of the present invention, a ceDNA vector (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) is, for example, Outside the site , In vitro and In vivo It can be used in various ways, including applications, methodologies, diagnostic procedures, and / or gene therapy regimens.

[0275] A method for treating a disease or disorder in a subject is provided herein, comprising introducing a therapeutically effective amount of TNA LNP (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)), which is optionally accompanied by a pharmaceutically acceptable carrier, into the subject's target cells that require it (e.g., hepatocytes, muscle cells, kidney cells, neurons, or other affected cell types). The implemented TNA LNP (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) comprises a nucleotide sequence of interest useful for treating the disease. In particular, the TNA may comprise a target exogenous DNA sequence operably linked to a control element capable of directing the transcription of a target polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into the subject. TNA LNPs (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) may be administered via any suitable route as described herein and known in the art. In one embodiment of any of the aspects or embodiments of the present invention, the target cells are human subjects.

[0276] A method is provided herein for providing a diagnostically or therapeutically effective amount of TNA LNP (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) to a subject in need thereof, comprising providing a certain amount of TNA LNP (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) to the cell, tissue, or organ of the subject in need thereof for a time effective for enabling the expression of a transgenic gene from the TNA LNP, thereby providing the subject with a diagnostically or therapeutically effective amount of protein, peptide, or nucleic acid expressed by the TNA LNP (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)). In one embodiment of any of the aspects or embodiments of the present invention, the subject is a human.

[0277] A method for diagnosing, preventing, treating, or improving at least one symptom of disease, disorder, dysfunction, injury, abnormal pathological condition, or trauma in a subject is provided herein. Generally, the method comprises the step of administering at least TNA LNP (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) to a subject in need of such a method for a sufficient amount and for a sufficient period of time to diagnose, prevent, treat, or improve one or more symptoms of disease, disorder, dysfunction, injury, abnormal pathological condition, or trauma in the subject. In any one embodiment of the aspects or embodiments of the present invention, the subject is a human.

[0278] A method of using TNA LNPs as a tool to treat one or more symptoms of a disease or disease state is provided herein. There exist numerous known genetic diseases involving defective genes, which are typically classified into two classes: deficiencies of enzymes that are generally inherited in a recessive manner, and disequilibrium states in which regulatory or structural proteins may be involved and are typically inherited in a dominant manner, but not always in a dominant manner. In the case of deficiency states, TNA LNPs (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) may be used not only to deliver transgenes to bring normal genes into affected tissues for replacement therapy, but also, in some embodiments of any of the aspects and embodiments of the present invention, to generate animal models of the disease using antisense mutations. In the case of disequilibrium disease states, TNA LNPs (e.g., ceDNA vector lipid particles) may be used to generate a disease state in a model system, which may be used as an effort to address the disease state. Accordingly, the TNA LNP (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) and method disclosed herein enable the treatment of genetic diseases. The disease state used herein is treated in a manner that partially or wholly improves the deficiency or imbalance that causes the disease or makes it more severe.

[0279] Generally, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) may be used to deliver any transgene as described above to treat, prevent, or improve symptoms associated with any disorder related to gene expression. Exemplary disease states include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer, diabetes mellitus vera, muscular dystrophy (e.g., Duchenne type, Becker type), Hurler's disease, adenosine deaminase deficiency, metabolic defects, retinal degenerative diseases (and other eye diseases), mitochondrial disorders (e.g., Leber's hereditary optic neuropathy (LHON), Leigh syndrome and subacute sclerosing encephalopathy), myopathy (e.g., facialoscapulohumeral myopathy (FSHD) and cardiomyopathy), and diseases of solid organs (e.g., brain, liver, kidney, heart). In some embodiments of any of the aspects and embodiments of the present invention, a ceDNA vector as disclosed herein may be advantageously used to treat individuals suffering from metabolic disorders (e.g., ornithine transcarbamylase deficiency).

[0280] In one embodiment of any of the aspects or embodiments of the present invention, the TNA LNPs described herein may be used to treat, improve, and / or prevent diseases or disorders caused by mutations in genes or gene products. Exemplary diseases or disorders that may be treated with TNA LNPs (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage diseases); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Hunter syndrome)); Liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia A and B, thalassemia and anemia); cancers and tumors, and genetic diseases or disorders (e.g., cystic fibrosis).

[0281] In one embodiment of any of the aspects or embodiments of the present invention, a TNA LNP (e.g., ceDNA vector lipid particle) may be used to deliver a heterogeneous nucleotide sequence in a situation where it is desirable to regulate the expression level of a transgene (e.g., a transgene encoding a hormone or growth factor).

[0282] In one embodiment of any of the aspects or embodiments of the present invention, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) may be used to correct abnormal levels and / or functions (e.g., absence of a protein or defect thereof) of gene products causing disease or disorder. TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) may generate functional proteins and / or alter protein levels to alleviate or reduce symptoms caused by specific diseases or disorders resulting from the absence or defect of proteins, or to confer benefits upon such diseases or disorders. For example, treatment of OTC deficiency may be achieved by generating functional OTC enzymes; treatment of hemophilia A and B may be achieved by altering the levels of Factor VIII, Factor IX, and Factor X; and treatment of PKU may be achieved by altering 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 aryl-sulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conduction modulators; treatment of glycogen storage disease can be achieved by restoring functional G6Pase enzyme function; and treatment of PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

[0283] In one embodiment of any of the aspects or embodiments of the present invention, a TNA LNP (e.g., ceDNA vector lipid particle (e.g., lipid nanoparticle)) is an RNA-based therapeutic agent In vitro or In vivoIt can be used to deliver to cells. 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 (e.g., lipid nanoparticles)) antisense nucleic acids In vitro or In vivo It can be used to deliver to cells. For example, if the transgene is an RNAi molecule, the expression of antisense nucleic acid or RNAi within target cells reduces the expression of a specific protein by the cell. Therefore, a transgene that is an RNAi molecule or an antisense nucleic acid can be administered to reduce the expression of a specific protein in subjects that require it. Antisense nucleic acids are also used to regulate cell physiology, for example, to optimize cell or tissue culture systems. In vitro It can be administered to cells.

[0284] In any one embodiment of the aspects or embodiments of the present invention, a TNA LNP (e.g., ceDNA vector lipid particle (e.g., lipid nanoparticle)) is a DNA-based therapeutic agent In vitro or In vivoIt can be used to deliver to cells. Examples of DNA-based therapeutics include, but are not limited to, minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or nonviral synthetic DNA vectors, closed linear divalent DNA (ceDNA / CELiD), plasmids, vacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (DNA vectors closed by linear covalent bonds) or dumbbell DNA minimal vectors ("dumbbell DNA"). For example, in any embodiment of the aspects or embodiments of the present invention, a ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) forms a minicircle In vitro or In vivo It can be used to deliver to cells. For example, if the transgene is minicircle DNA, the expression of minicircle DNA within target cells reduces the expression of a specific protein by the cell. Therefore, a transgene that is minicircle DNA can be administered to reduce the expression of a specific protein in subjects that require it. Minicircle DNA can also be used to regulate cell physiology, for example, to optimize cell or tissue culture systems. In vitro It can be administered to cells.

[0285] In any embodiment of the aspects or embodiments of the present invention, the exemplary transgenic gene encoded by a TNA vector comprising an expression cassette includes, but is not limited to, X, lysosomal enzymes (e.g., hexosaminidase A associated with Tay-Sachs disease, or iduronate sulfatase associated with Hunter syndrome / MPS 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, These include 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 factor-a and -b, etc.), and receptors (e.g., tumor necrosis factor receptor). In some exemplary embodiments, the transgenic gene encodes one or more monoclonal antibodies specific to the desired target. In some exemplary embodiments, more than one transgenic gene is encoded by a ceDNA vector. In some exemplary embodiments, the transgenic gene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects and embodiments of the present invention, the transgenic gene encodes an antibody comprising a full-length antibody or an antibody fragment as defined herein. In some embodiments of any of the aspects and embodiments of the present invention, the antibody is an antigen-binding domain or immunoglobulin variable domain sequence as defined herein.Other exemplary transgenic 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 treatment, and tumor suppressor gene products.

[0286] administration

[0287] In one embodiment of any of the aspects or embodiments of the present invention, a TNA LNP (e.g., a ceDNA vector lipid particle as described herein) is In vivo It can be administered to an organism for cell transduction. In one embodiment of any of the aspects or embodiments of the present invention, the TNA LNP (e.g., ceDNA vector lipid particle) is In vitro It can be administered to an organism for cell transduction.

[0288] Generally, administration is carried out via any route commonly used to introduce molecules into ultimate contact with blood or tissue cells. Suitable methods for administering such nucleic acids are available and widely known to those skilled in the art, and while more than one route may be used to administer a particular composition, a specific route can often provide a more immediate and more effective response than another. Exemplary modes of administration of TNA LNP (e.g., ceDNA vector lipid particles) include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intradural, intraocular, transdermal, intraendothelial, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to the skeletal, diaphragmatic, and / or cardiac muscle], intrapleural, intracerebral, and intra-articular), local (e.g., skin and mucosal surfaces including airway surfaces, and transdermal administration), intralymphatic, etc., as well as direct tissue or tracheal injection (e.g., into the liver, eye, skeletal muscle, cardiac muscle, diaphragmatic muscle, or brain).

[0289] Administration of a TNA LNP-like ceDNA vector (e.g., ceDNA LNP) may be performed, without limitation, on any site of a target selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. In one embodiment of any of the aspects or embodiments of the present invention, administration of ceDNA LNP may also be performed on a tumor (e.g., within or near a tumor or lymph node). In any given case, the most suitable route will depend on the characteristics and severity of the condition to be treated, improved, and / or prevented, and on the characteristics of the specific ceDNA LNP used. Additionally, ceDNA enables the administration of more than one transgenic gene as a single vector or multiple ceDNA vectors (e.g., a ceDNA cocktail).

[0290] In any one embodiment of the aspects or embodiments of the present invention, administration of ceDNA LNP into skeletal muscle comprises, but not limited to, administration into skeletal muscle of the limbs (e.g., upper arm, lower arm, upper leg and / or lower leg), back, neck, head (e.g., tongue), thorax, abdomen, pelvis / perineum, and / or fingers (toes). The ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) may be administered intravenously, intra-arterially, intraperitoneally, or by limb perfusion (optional, isolated limb perfusion of the legs and / or arms; e.g., [Arruda et al. [See , (2005) Blood 105: 3458-3464]), and / or it may be delivered to skeletal muscle by direct intramuscular injection. In certain embodiments, ceDNA LNP is administered to the limbs (arms and / or legs) of a subject (e.g., a subject suffering from muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-articular administration). In one embodiment of any of the aspects or embodiments of the present invention, ceDNA LNP may be administered without using "hydrodynamic" technology.

[0291] Administration of TNA LNP (e.g., ceDNA LNP) to the cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or diaphragm. TNA LNP (e.g., ceDNA LNP) may be delivered to the cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., to the left atrium, right atrium, left ventricle, and right ventricle), and / or coronary perfusion. Administration to the diaphragmatic muscle may be made by any suitable method including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to the smooth muscle may be made by any suitable method including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment of any of the aspects or embodiments of the present invention, administration may be made to the smooth muscle, to endothelial cells present near and / or above it.

[0292] In one embodiment of any of the aspects or embodiments of the present invention, TNA LNP (e.g., ceDNA LNP) is administered to skeletal muscle, diaphragmatic muscle and / or cardiac muscle (e.g., to treat, improve, and / or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure)).

[0293] TNA LNP (e.g., ceDNA LNP) can be administered to the CNS (e.g., brain or eye). TNA LNP (e.g., ceDNA LNP) can 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 hypocolon. TNA LNP (e.g., ceDNA LNP) can be administered to various areas of the eye, such as the retina, cornea and / or optic nerve. TNA LNP (e.g., ceDNA LNP) can be delivered into the cerebrospinal fluid (e.g., by lumbar puncture). TNA LNPs (e.g., ceDNA vector lipid particles) can be further administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumors or cerebral infarction).

[0294] In any embodiment of the aspects or embodiments of the present invention, TNA LNP (e.g., ceDNA LNP) may be administered to the desired region(s) of the CNS via any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's region), as well as intramuscular delivery via retrograde delivery to motor neurons.

[0295] According to some embodiments of any of the aspects or embodiments of the present invention, a TNA LNP (e.g., ceDNA LNP) is administered in a liquid formulation to a target area or compartment within the CNS via direct injection (e.g., stereotactic injection). According to other embodiments, the TNA LNP (e.g., ceDNA LNP) may be provided via topical application to a target area or intranasal administration of an aerosol formulation. Administration to the eye may be made via topical application of drops. As a further alternative, the ceDNA vector may be administered in a solid sustained-release formulation (e.g., see U.S. Patent No. 7,201,898, the entire text of which is incorporated herein by reference). In one embodiment of any of the aspects or embodiments of the present invention, TNA LNP (e.g., ceDNA LNP) may be used for retrograde transport to treat, improve, and / or prevent diseases and disorders associated with motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.). For example, TNA LNP (e.g., ceDNA LNP) may be delivered to muscle tissue capable of moving toward neurons.

[0296] In one embodiment of any of the aspects or embodiments of the present invention, repeated administration of the therapeutic agent may be performed until an appropriate expression level is achieved. Accordingly, in one embodiment of any of the aspects or embodiments of the present invention, the therapeutic nucleic acid may be administered multiple times and re-administered. For example, the therapeutic nucleic acid may be administered on Day 0. After the initial treatment on Day 0, the second administration (re-administration) is, after the initial treatment with therapeutic nucleic acid, at approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, or at approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, or at approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years, approximately 11 years, approximately 12 years, approximately 13 years, approximately 14 years, approximately 15 years, approximately 16 years, approximately 17 years, approximately 18 years, approximately 19 years, approximately 20 years, approximately 21 years, approximately 22 years, approximately 23 years, It can be carried out within about 24 years, about 25 years, about 26 years, about 27 years, about 28 years, about 29 years, about 30 years, about 31 years, about 32 years, about 33 years, about 34 years, about 35 years, about 36 years, about 37 years, about 38 years, about 39 years, about 40 years, about 41 years, about 42 years, about 43 years, about 44 years, about 45 years, about 46 years, about 47 years, about 48 years, about 49 years, or about 50 years.

[0297] In any one embodiment of the aspects or embodiments of the present invention, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be included in the lipid particles (e.g., lipid nanoparticles) of the present invention. In other words, the lipid particles (e.g., lipid nanoparticles) may contain a compound other than TNA, or at least a second TNA that is different from the first TNA. Non-limitingly, 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, peptide mimics, nucleic acids, nucleic acid analogs and derivatives, extracts prepared from biological materials, or any combination thereof.

[0298] In any one embodiment of the aspects or embodiments of the present invention, one or more additional compounds may be therapeutic agents. The therapeutic agent may be selected from any class suitable for therapeutic purposes. Accordingly, the therapeutic agent may be selected from any class suitable for therapeutic purposes. The therapeutic agent may be selected according to the therapeutic purpose and the intended biological action. For example, in one embodiment of any of the aspects or embodiments of the present invention, if the TNA in the LNP is useful for cancer treatment, the additional compound may be an anticancer agent (e.g., a chemotherapy agent, a targeted cancer treatment (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates). In one embodiment of any of the aspects or embodiments of the present invention, if the LNP containing TNA is useful for the treatment of an infection, the additional compound may be an antimicrobial agent (e.g., an antibiotic or an antiviral compound). In one embodiment of any of the aspects or embodiments of the present invention, if the LNP containing TNA is useful for the treatment of an immune disease or disorder, the additional compound may be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In one embodiment of any of the aspects or embodiments of the present invention, different cocktails of different lipid particles containing different compounds, such as TNA encoding different proteins or therapeutic agents, may be used in the compositions and methods of the present invention. In one embodiment of any of the aspects or embodiments, the additional compound is an immunomodulator. For example, the additional compound is an immunosuppressant. In some embodiments of any of the aspects and embodiments of the present invention, the additional compound is an immunostimulator.

[0299] Examples

[0300] The following examples are provided as examples, not limitations. Those skilled in the art will understand that ionizable lipids can be designed and synthesized using general synthesis methods described below.

[0301] General synthesis

[0302] chemical formula I The ionizable lipids are as follows Reaction Equation 1 It was designed and synthesized using a synthesis method similar to that described in [the document].

[0303] Reaction Equation 1

[0304]

[0305] Example 1:

[0306] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate (lipid 1)

[0307] Synthesis of a cutting and ionizing head ((disulfanediylbis(ethane-2,1-diyl))bis(piperidine-1,4-diyl))bis(ethane-2,1-diyl)bis(2-(4-hydroxyphenyl)acetate) (7)

[0308] Step-1

[0309]

[0310] Synthesis of disulfan diylbis(ethane-2,1-diyl) dimethanesulfonate (2). Commercially available 2,2'-Disulfanediylbis(ethanol) ( 1 )(15 g, 97.2 mmol) was dissolved in acetonitrile (143 ml), and then triethylamine (NEt3) (33.3 g, 328 mmol) was added. Methanesulfonyl chloride (MsCl) (34.5 g, 300 mmol) was added dropwise to the reaction mixture at 0°C. The resulting reaction mixture was stirred at room temperature for 3 hours. Ethanol (EtOH) (39 ml) was added to the reaction mixture to quench the reaction, and the mixture was filtered to remove insoluble substances. The filtrate was distributed between dichloromethane (DCM) (150 ml) and a 10% aqueous sodium bicarbonate solution (150 ml). The organic layer was washed four times with water (100 ml), dried over magnesium sulfate (MgSO4), and evaporated. 2 (25 g, 81%) was obtained as brown oil and solidified by letting it stand.1 H-NMR (300 MHz, d-chloroform): δ 4.43-4.48 (t, 4H), 3.00-3.10 (m, 10H).

[0311] Step-2

[0312]

[0313] Synthesis of 2,2'-((disulfanediylbis(ethane-2,1-diyl))bis(piperidin-1,4-diyl))bis(ethane-1-ol) (4). In acetonitrile (310 ml) 2 In a solution of (12 g, 38.7 mmol), potassium carbonate (K2CO3) (13.4 g, 96.6 mmol), followed by 2-(piperidin-4-yl)ethanol-1-ol ( 3 )(20 g, 155 mmol) was added. The resulting mixture was stirred overnight at room temperature, then filtered to remove insoluble substances. The filtrate was evaporated until dry to obtain an unpurified product, which was dissolved in DCM (100 ml), washed twice with water (50 ml), dried over MgSO4, and evaporated. 4 It was obtained as yellow oil (11.8 g, 79%). 1 H-NMR (300 MHz, d-chloroform): δ 3.63-3.68 (t, 4H), 2.78-2.90 (m, 8H), 2.62-2.65 (t, 4H), 1.94-2.02 (t, 4H), 1.70 (s, 2H), 1.65-1.70 (d, 4H), 1.27-1.48 (t, 4H), 1.40-1.50 (m, 2H), 1.23-1.27 (m, 4H).

[0314] Step 3

[0315]

[0316] Synthesis of 2-(4-((tert-butyldimethylsilyl)oxy)phenyl)acetic acid (5). 4-hydroxyphenylacetic acid in dimethylformamide (DMF) (40 ml) 5a To a stirred solution of )(10 g, 65 mmol), NET3(10 g, 100 mmol), at 0°C, then tert-Butyldimethylsilyl chloride (TBSCl) (15 g, 100 mmol) was added. The resulting reaction mixture was stirred overnight at room temperature and then treated with water (200 ml) and DCM (150 ml). The organic phase was separated. The aqueous phase was extracted with DCM (100 ml). The combined organic phase was washed with a saturated sodium bicarbonate solution and brine, and dried over sodium sulfate (Na2SO4). The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0%→10% methanol (MeOH) in DCM as the eluent. The fraction containing the target compound was pooled and evaporated 5 (4.8 g, 27%) and D- tert - Butyldimethylsilyl ether (di-TBS) byproduct (10.5 g, 42%) was obtained. 5 of 1 H-NMR(300 MHz, d-chloroform): δ 7.12 (d, 2H), 6.78 (d, 2H), 3.56 (s, 2H), 0.97 (s, 9H), 0.18 (s, 6H).

[0317]

[0318] Synthesis of ((disulfanediylbis(ethane-2,1-diyl))bis(piperidin-1,4-diyl))bis(ethane-2,1-diyl)bis(2-(4-((tert-butyldimethylsilyl)oxy)phenyl)acetate) (6). In DCM (100 ml) Step-2 Disulfide obtained from 4 (1.92 g, 5 mmol) and phenylacetic acid 5 To a stirred solution of (3.4 g, 12.8 mmol), 4-dimethylaminopyridine (DMAP) (1.5 g, 12.5 mmol) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (2.4 g, 12.5 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a saturated sodium bicarbonate solution (200 ml) and brine (150 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0%→10% MeOH in DCM as the eluent. The fraction containing the target compound was evaporated6 (4.1 g, 92%) was obtained. 6 of 1 H-NMR (300 MHz, d-chloroform): δ 7.12 (d, 4H), 6.75 (d, 4H), 4.1 (t, 4H), 3.5 (s, 4H), 2.82 (m, 8H), 2.62 (m, 4H), 1.93 (t, 4H), 1.61-1.45 (m, 8H), 1.26 (m, 6H), 0.97 (s, 18H), 0.17 (s, 4H).

[0319] Step 4

[0320]

[0321] Synthesis of ((disulfanediylbis(ethane-2,1-diyl))bis(piperidin-1,4-diyl))bis(ethane-2,1-diyl)bis(2-(4-hydroxyphenyl)acetate) (7). Disulfide in tetrahydrofuran (THF) (40 ml) 6 Pyridine hydrofluoride (1 ml, 3.8 mmol) was added to a stirred solution of (3.1 g, 3.6 mmol) at 0°C. The resulting mixture was stirred at 0°C for 2 hours, followed by an additional 2 hours at room temperature. The reaction mixture was treated with a saturated sodium bicarbonate solution (200 ml) and extracted with ethyl acetate (2 × 150 ml). The combined organic phase was washed with brine (100 ml), dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography using 0%→10% MeOH in DCM as the eluent to obtain the desired product. 7 (1.92 g, 82%) was provided. 1 H-NMR (300 MHz, d-chloroform): δ 7.13 (d, 4H), 6.70 (d, 4H), 4.1 (t, 4H), 3.5 (s, 4H), 2.89 (m, 8H), 2.70 (m, 4H), 1.95 (t, 4H), 1.48 (m, 8H), 1.17 (m, 6H).

[0322] Synthesis of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (10)

[0323]

[0324] Synthesis of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (10). Nonandioic acid in dichloromethane (1000 ml) 8 )(7.34 g, 39 mmol) and heptadecan-9-ol ( 8b DMAP (2.37 g, 19 mmol) and then EDCI (3 g, 19 mmol) were added to a stirred solution of )(5 g, 19 mmol). The resulting mixture was stirred overnight at room temperature and then washed with 1 N HCl (250 ml) and water (250 ml). The organic layer was dried over MgSO4, evaporated until dry, and purified by silica gel column chromatography using 0%→10% MeOH in DCM as the eluent. The fraction containing the target compound was pooled and evaporated 10 (6.2 g, 75%) was obtained as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.80-4.90 (m, 1H), 2.25-2.34 (m, 4H), 1.55-1.70 (m, 4H), 1.40-1.50 (m, 4H), 1.20-1.40 (m, 30H), 0.84-0.90 (t, 3H).

[0325] Synthesis of 1-(heptadecan-9-yl)9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate

[0326]

[0327] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate (11). In DMF (20 ml) Step 4 Disulfide manufactured in 7 (580 mg, 0.9 mmol) and acid 10DMAP (165 mg, 1.35 mmol) and then EDCI (258 mg, 1.35 mmol) were added to a stirred solution of (422 mg, 0.99 mmol). The resulting mixture was stirred overnight at room temperature, after which a saturated sodium bicarbonate solution (50 ml) was added. The reaction mixture was extracted with dichloromethane (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%→10% MeOH in DCM as the eluent to obtain the desired product. 11 (427 mg, 45%) was obtained. 1 H-NMR (300 MHz, d-chloroform): δ 7.27 (d, 2H), 7.11 (d, 2H), 7.03 (d, 2H), 6.69 (d, 2H), 4.85 (m, 1H), 4.1 (m, 4H), 3.56 (s, 2H), 3.48 (s, 2H), 2.92 (d, 2H), 2.85-2.69 (m, 12H), 2.71 (t, 2H), 2.28 (t, 2H), 1.95 (t, 2H), 1.52-1.01 (m, 53H), 0.85 (m, 6H).

[0328] Synthesis of Lipid 1

[0329]

[0330] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate (lipid 1). Disulfide in dichloromethane (10 ml) 11 (151 mg, 0.14 mmol) and oleic acid 12DMAP (28 mg, 0.22 mmol) and then EDCI (42 mg, 0.22 mmol) were added to a stirred solution of (61 mg, 0.22 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 1 (126 mg, 68%) was obtained. Geology 1 of 1 H-NMR (300 MHz, d-chloroform): δ 7.25 (d, 4H), 7.01 (d, 4H), 5.34 (m, 2H), 4.86 (m, 1H), 4.11 (t, 4H), 3.58 (s, 4H), 2.91-2.70 (m, 8H), 2.62 (m, 4H), 2.53 (t, 4H), 2.28 (t, 2H), 2.05-1.87 (m, 8H), 1.78-1.46 (m, 22H), 1.48-1.23 (m, 54H), 0.86 (t, 9H). MS [M+H] + 1318.

[0331] Example 2: Synthesis of 1-(heptadecan-9-yl)9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((9-(nonyloxy)-9-oxononanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate (lipid 3)

[0332] Synthesis of 9-(nonyloxy)-9-oxononanoic acid (9)

[0333]

[0334] Synthesis of 9-(nonyloxy)-9-oxononanoic acid (9). Nonandioic acid in DCM (1000 ml) 8 )(13.2 g, 0.1 mol) and nonan-1-ol ( 8aDMAP (6.1 g, 0.05 mol) and then EDCI (7.7 g, 0.05 mol) were added to a stirred solution of )(7.2 g, 0.05 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1 N hydrochloric acid (HCl) solution (500 ml) and water (500 ml). The organic layer was dried over MgSO4, evaporated until dry, and purified by silica gel column chromatography using 0%→10% MeOH in DCM as the eluent. The fraction containing the target compound was pooled and evaporated 9 (12.6 g, 81%) was obtained as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.03-4.07 (t, 2H), 2.28-2.34 (m, 4H), 1.58-1.63 (m, 6H), 1.26-1.32 (m, 18H), 0.85-0.87 (t, 3H).

[0335] Synthesis of lipid 3

[0336]

[0337] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((9-(nonyloxy)-9-oxononanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate (lipid 3). Disulfide in dichloromethane (10 ml) 11 (Step-by-step synthesis described in Example 1) (150 mg, 0.14 mmol) and acid 9 DMAP (28 mg, 0.22 mmol) and then EDCI (42 mg, 0.22 mmol) were added to a stirred solution of (62 mg, 0.22 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 3 (114 mg, 60%) was obtained. Geology 3 of 1H-NMR (300 MHz, d-chloroform): δ 7.28 (d, 4H), 7.02 (d, 4H), 4.86 (m, 1H), 4.11 (t, 4H), 4.04 (t, 2H), 3.58 (s, 4H), 2.93-2.77 (m, 8H), 2.63 (m, 4H), 2.53 (t, 4H), 2.28 (m, 4H), 1.95 (t, 4H), 1.85-1.47 (m, 24H), 1.45-1.16 (m, 54H), 0.86 (t, 9H). MS [M+H] + 1350.

[0338] Example 3: Synthesis of 1-(heptadecan-9-yl)9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((5-(nonyloxy)-5-oxopentanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate (lipid 2)

[0339]

[0340] Disulfide in DCM (10 ml) 11 (Step-by-step synthesis described in Example 1) (150 mg, 0.14 mmol) and acid 9a (mountain 9 Refer to the synthesis described in Example 1 for, wherein nonandioic acid is used as the starting material ( 8 Nonan-1-ol ( 8a react with ) 9a DMAP (28 mg, 0.22 mmol) and then EDCI (42 mg, 0.22 mmol) were added to a stirred solution of (57 mg, 0.22 mmol) (producing). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 2 (151 mg, 81%) was obtained. Geology 2 of 1H-NMR (300 MHz, d-chloroform): δ 7.26 (d, 4H), 7.01 (d, 4H), 4.86 (m, 1H), 4.10-4.02 (t, 6H), 3.57 (s, 4H), 3.01 (d, 4H), 2.83-2.72 (m, 4H), 2.34-2.21 (m, 14H), 2.15-1.91 (m, 6H), 1.74-1.41 (m, 12H), 1.39-1.16 (m, 52H), 0.86 (t, 9H). MS [M+H] + 1293.

[0341] Example 4: Synthesis of 1-(heptadecan-9-yl)9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((5-(nonyloxy)-5-oxopentanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonandioate (lipid 4)

[0342]

[0343] Disulfide in a mixture of dichloromethane (5 ml) and DMF (3 ml) 7 (Step-by-step synthesis described in Example 1) (150 mg, 0.23 mmol) and compound 9 To a stirred solution of (synthesis described in Example 2) (146 mg, 0.46 mmol), DMAP (70 mg, 0.57 mmol) was added at 0°C, followed by EDCI (109 mg, 0.57 mmol). The resulting mixture was stirred at 0°C for 15 minutes, followed by overnight at room temperature. DCM (20 ml) was added, the reaction mixture was washed with a 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 target compound was evaporated. Geology 4 (180 mg, 63%) was obtained. Geology 4 of 1H-NMR (300 MHz, d-chloroform): δ 7.28 (d, 4H), 7.02 (d, 4H), 4.11 (t, 4H), 4.04 (t, 4H), 3.58 (s, 4H), 2.93-2.67 (m, 8H), 2.63-2.55 (m, 4H), 2.53 (t, 4H), 2.29 (t, 4H), 1.94 (t, 4H), 1.85-1.47 (m, 20H), 1.45-1.16 (m, 42), 0.87 (t, 6H). MS [M+H] + 1237.

[0344] Example 5: Synthesis of O'1,O1-((((((disulfanediylbis(ethane-2,1-diyl))bis(piperidin-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(4,1-phenylene)) 9,9'-di(heptadecan-9-yl)di(nonandioate) (lipid 5)

[0345]

[0346] Disulfide in DMF (20 ml) 7 (Step-by-step synthesis described in Example 1) (580 mg, 0.9 mmol) and acid 10 To a stirred solution of (synthesis described in Example 1) (422 mg, 0.99 mmol), DMAP (164 mg, 1.35 mmol) was added at 0°C, followed by EDCI (257 mg, 1.35 mmol). The resulting mixture was stirred at 0°C for 15 minutes, followed by overnight at room temperature. DCM (60 ml) was added, the reaction mixture was washed with a 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 target compound was evaporated. Geology 5 (280 mg, 38%) was obtained. Geology 5 of 1 H-NMR(300 MHz, d-chloroform): δ 7.26 (d, 4H), 7.02 (d, 4H), 4.85 (m, 2H), 4.11 (t, 4H), 3.58 (s, 4H), 2.86-2.77 (m, 8H), 2.63 (m, 4H), 2.53 (t, 4H), 2.27 (t, 4H), 1.92 (t, 4H), 1.75-1.47 (m, 26H), 1.45-1.16 (m, 64H), 0.86 (t, 12H). MS [M+H] + 1462.

[0347] Example 6: Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(undecane-3-yl)nonandioate (lipid 6)

[0348] Synthesis of 9-oxo-9-(undecan-3-yloxy)nonanoic acid (9b)

[0349]

[0350] Synthesis of 9-oxo-9-(undecan-3-yloxy)nonanoic acid (9b). Nonandioic acid in DCM (500 ml) 8 )(10.9 g, 0.058 mol) and undecan-3-ol ( 8b DMAP (3.5 g, 0.03 mol) and then EDCI (4.5 g, 0.03 mol) were added to a stirred solution of )(5 g, 0.029 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1 N HCl solution (500 ml) and water (500 ml). The organic layer was dried over MgSO4, evaporated until dry, and purified by silica gel column chromatography using 0%→10% MeOH in DCM as the eluent. The fraction containing the target compound was pooled and evaporated 9b (6.5 g, 66%) was obtained as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.79-4.83 (t, 1 H), 2.28-2.34 (m, 4 H), 1.25-1.33 (m, 8 H), 1.26-1.32 (m, 18 H), 0.85-0.87 (t, 6 H).

[0351] Synthesis of 4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl oleate (13)

[0352]

[0353] Synthesis of 4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl oleate (13). Disulfide in DCM (200 ml) 7 (Stepwise synthesis described in Example 1) (2.0 g, 3 mmol) and oleic acid (or acid as described in Example 1) 12 DMAP (340 mg, 2.8 mmol) and then EDCI (440 mg, 2.8 mmol) were added to a stirred solution of )(0.79 g, 2.8 mmol). The resulting mixture was stirred overnight at room temperature, after which a saturated sodium bicarbonate solution (20 ml) was added. The reaction mixture was extracted with dichloromethane (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% methanol in dichloromethane as the eluent. 13 (1.6 g, 57%) was obtained as a white solid. The product was used immediately in the next step without further characterization.

[0354] Synthesis of lipid 6

[0355]

[0356] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(undecane-3-yl)nonandioate (lipid 6). Disulfide in DCM (20 ml) 13 (250 mg, 0.27 mmol) and acid 9b DMAP (40 mg, 0.33 mmol) and then EDCI (51 mg, 0.33 mmol) were added to a stirred solution of (113 mg, 0.33 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 6 (120 mg, 36%) was obtained. 1H-NMR (300 MHz, d-chloroform): δ 7.31 (d, 4 H), 7.05 (d, 4 H), 5.36-5.40 (m, 2 H), 4.86 (m, 1 H), 4.11 (t, 4 H), 3.62 (t, 4 H), 2.77-2.90 (m, 8 H), 2.55-2.71 (m, 8 H), 2.30-2.34 (m, 2 H), 1.96-2.05 (m, 8 H), 1.77 (m, 4 H), 1.58-1.67 (m, 18 H), 1.30-1.58 (m, 40 H), 0.89 (t, 9H).

[0357] Example 7: Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(tridecan-5-yl)nonandioate (lipid 7)

[0358] Synthesis of tridecanol-5-ol (8c)

[0359]

[0360] Synthesis of tridecane-5-ol (8c). Aldehyde in anhydrous THF (100 ml) 8c-1 To a solution of (7.1 g, 0.05 mol), 27 ml of a 2 M butyllithium (BuLi) solution in THF was added dropwise at -78°C. The resulting mixture was stirred at -78°C for 2 hours, followed by 2 hours at room temperature. The reaction was quenched by adding water and distributed between 1 N HCl and ether. The organic layer was collected, dried over MgSO4, and evaporated to obtain the crude 8c (10 g, 100%) was obtained as yellow oil and used immediately in the next step without further purification.

[0361] Synthesis of 9-oxo-9-(tridecane-5-yloxy)nonanoic acid (9c)

[0362]

[0363] Synthesis of 9-oxo-9-(tridecane-5-yloxy)nonanoic acid (9c). Nonandioic acid in DCM (500 ml) 8 )(9.4 g, 0.05 mol) and 8cDMAP (3.05 g, 0.025 mol) and then EDCI (3.88 g, 0.025 mol) were added to a stirred solution of (5 g, 0.025 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1 N HCl solution (500 ml) and water (500 ml). The organic layer was dried over MgSO4, evaporated until dry, and purified by silica gel column chromatography using 0%→10% MeOH in DCM as the eluent. The fraction containing the target compound was pooled and evaporated 9c (2.5 g, 27%) was obtained as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.84-4.87 (t, 1 H), 2.28-2.34 (m, 4 H), 1.58-1.63 (m, 7 H), 1.26-1.32 (m, 23 H), 0.85-0.87 (t, 6 H).

[0364] Synthesis of lipid 7

[0365]

[0366] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(tridecane-5-yl)nonandioate (lipid 7). Disulfide in DCM (20 ml) 13 (Synthesis described in Example 6) (250 mg, 0.27 mmol) and acid 9c DMAP (40 mg, 0.33 mmol) and then EDCI (51 mg, 0.33 mmol) were added to a stirred solution of (116 mg, 0.33 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 7 (160 mg, 40%) was obtained. 1H-NMR (300 MHz, d-chloroform): δ 7.29 (d, 4 H), 7.04 (d, 4 H), 5.29-5.34 (m, 2 H), 4.86 (m, 1 H), 4.11 (t, 4 H), 3.58 (t, 4 H), 2.77-2.90 (m, 8 H), 2.51-2.79 (m, 8 H), 2.28 (m, 2 H), 1.94-2.05 (m, 8 H), 1.70-1.80 (m, 4 H), 1.49-1.67 (m, 18 H), 1.10-1.40 (m, 46 H), 0.88 (t, 9H).

[0367] Example 8: Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(pentadecane-7-yl)nonandioate (lipid 8)

[0368] Synthesis of pentadecane-7-ol (8d)

[0369]

[0370] Synthesis of pentadecane-7-ol (8d). Aldehyde in anhydrous THF (100 ml) 8d-1 To a solution of (7.1 g, 0.05 mol), 27 ml of a 2 M hexyl magnesium bromide solution in THF at -78°C was added. The resulting mixture was stirred at -78°C for 2 hours, followed by overnight at room temperature. The reaction was quenched by adding water and partitioned between 1 N HCl and ether. The organic layer was collected, dried over MgSO4, and evaporated to obtain the crude 8d (11 g, 100%) was obtained as a white solid and used immediately in the next step without further purification.

[0371] Synthesis of 9-oxo-9-(pentadecan-7-yloxy)nonanoic acid (9d)

[0372]

[0373] Synthesis of 9-oxo-9-(pentadecan-7-yloxy)nonanoic acid (9d). Nonandioic acid in DCM (1000 ml) 8 )(9.4 g, 0.05 mol) and pentadecane-7-ol ( 8dDMAP (3.05 g, 0.025 mol) and then EDCI (3.88 g, 0.025 mol) were added to a stirred solution of )(5.7 g, 0.025 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1 N HCl solution (500 ml) and water (500 ml). The organic layer was dried over MgSO4, evaporated until dry, and purified by silica gel column chromatography using 0%→10% MeOH in DCM as the eluent. The fraction containing the target compound was pooled and evaporated 9d (6.2 g, 62%) was obtained as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.86 (t, 1 H), 2.28-2.34 (m, 4 H), 1.58-1.63 (m, 8 H), 1.26-1.32 (m, 27 H), 0.85-0.87 (t, 6 H).

[0374] Synthesis of lipid 8

[0375]

[0376] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(pentadecan-7-yl)nonandioate (lipid 8). Disulfide in DCM (20 ml) 13 (Synthesis described in Example 6) (250 mg, 0.27 mmol) and acid 9d DMAP (40 mg, 0.33 mmol) and then EDCI (51 mg, 0.33 mmol) were added to a stirred solution of (120 mg, 0.33 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 8 (170 mg, 40%) was obtained. 1H-NMR (300 MHz, d-chloroform): δ 7.29 (d, 4 H), 7.04 (d, 4 H), 5.29-5.34 (m, 2 H), 4.86 (m, 1 H), 4.11 (t, 4 H), 3.58 (t, 4 H), 2.80-2.93 (m, 8 H), 2.51-2.68 (m, 8 H), 2.28 (m, 2 H), 1.97-2.05 (m, 8 H), 1.70-1.80 (m, 4 H), 1.50-1.70 (m, 18 H), 1.10-1.40 (m, 58 H), 0.87 (t, 9H).

[0377] Example 9: Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(undecane-3-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonandioate (lipid 9)

[0378] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-nonyl nonandioate (14).

[0379]

[0380] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-nonyl nonandioate (14). Disulfide in dichloromethane (200 ml) 7 (Stepwise synthesis described in Example 1) (3.1 g, 4.8 mmol) and 9-(nonyloxy)-9-oxononanoic acid ( 9 To a stirred solution of )(synthesis described in Example 1) (1.51 g, 4.8 mmol), DMAP (587 mg, 4.8 mmol) was added, followed by EDCI (746 mg, 4.8 mmol). The resulting mixture was stirred overnight at room temperature, after which a saturated sodium bicarbonate solution (50 ml) was added. The reaction mixture was extracted with dichloromethane (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 the desired product. 14 (2.47 g, 55%) was obtained. The product was used immediately in the next step without further characterization.

[0381] Synthesis of lipid 9

[0382]

[0383] Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(undecane-3-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonandioate (lipid 9). Disulfide in dichloromethane (20 ml) 14 (250 mg, 0.26 mmol) and acid 9b To a stirred solution of (synthesis described in Example 6) (110 mg, 0.32 mmol), DMAP (46 mg, 0.37 mmol) was added, followed by EDCI (50 mg, 0.32 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 9 (230 mg, 68%) was obtained. 1 H-NMR (300 MHz, d-chloroform): δ 7.28 (d, 4 H), 7.04 (d, 4 H), 4.86 (m, 1 H), 4.06-4.12 (t, 4 H), 4.04 (t, 2 H), 3.59 (s, 4 H), 2.60-2.90 (m, 8 H), 2.27-2.60 (m, 10 H), 1.97 (t, 3 H), 1.52-1.80 (m, 18 H), 1.10-1.40 (m, 40 H), 0.88 (t, 9 H).

[0384] Example 10: Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(tridecane-5-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonandioate (lipid 10)

[0385]

[0386] Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(tridecane-5-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonandioate (lipid 10). Disulfide in dichloromethane (20 ml) 14 (Synthesis described in Example 9) (330 mg, 0.35 mmol) and acid 9cTo a stirred solution of (synthesis described in Example 7) (143 mg, 0.39 mmol), DMAP (47 mg, 0.39 mmol) was added, followed by EDCI (60 mg, 0.39 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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% methanol in dichloromethane as the eluent. The fraction containing the target compound was evaporated Geology 10 (150 mg, 33%) was obtained. 1 H-NMR (300 MHz, d-chloroform): δ 7.26 (d, 4 H), 7.03 (d, 4 H), 4.86 (m, 1 H), 4.05-4.11 (t, 6 H), 3.58 (s, 4 H), 2.80-2.90 (m, 8 H), 2.50-2.70 (m, 8 H), 2.26-2.29 (m, 4 H), 1.92-1.99 (m, 4 H), 1.50-1.80 (m, 24 H), 1.16-1.40 (m, 46 H), 0.87 (t, 9 H).

[0387] Example 11: Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(pentadecane-7-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonandioate (Lipid 11)

[0388]

[0389] Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(pentadecan-7-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonandioate (lipid 11). Disulfide in DCM (20 ml) 14 (Synthesis described in Example 9) (260 mg, 0.28 mmol) and acid 9dDMAP (37 mg, 0.3 mmol) and then EDCI (47 mg, 0.3 mmol) were added to a stirred solution of (synthesis described in Example 8) (122 mg, 0.3 mmol). The resulting mixture was stirred overnight at room temperature, then washed with a 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 target compound was evaporated Geology 11 (110 mg, 30%) was obtained. Geology 11 of 1 H-NMR(300 MHz, d-chloroform): δ 7.26 (d, 4 H), 7.02 (d, 4 H), 4.86 (m, 1 H), 4.05-4.11 (t, 6 H), 3.59 (s, 4 H), 2.80-2.90 (m, 8 H), 2.50-2.70 (m, 8 H), 2.27-2.29 (m, 4 H), 1.90-2.20 (t, 4 H), 1.50-1.82 (m, 24 H), 1.10-1.40 (m, 50 H), 0.87 (t, 9 H).

[0390] Table 4 doing of Geology 12 inside Geology 20 It was manufactured according to a similar procedure using suitable starting materials and other modifications that may be within the knowledge of those skilled in the art.

[0391]

[0392]

[0393] Example 2: Preparation of lipid nanoparticles

[0394] Lipid nanoparticles (LNPs) were prepared with a weight ratio of total lipid to ceDNA of approximately 10:1 to 30:1. Briefly, the ionizable lipid of the present invention, a non-cationic lipid (e.g., distearoylphosphatidylcholine (DSPC)), a component for providing membrane integrity (e.g., sterol, e.g., cholesterol), and a conjugated lipid molecule (e.g., PEG-lipid, e.g., 1-(monomethoxypolyethyleneglycol)-2,3-dimyristoylglycerol with an average PEG molecular weight of 2000 (“PEG-DMG”)) were dissolved in an alcohol (e.g., ethanol) in a molar ratio of, for example, 50:10:37:3 or 20:40:38:2. ceDNA was diluted to a desired concentration in a buffer solution. For example, ceDNA was diluted to a concentration of 0.1 mg / ml to 0.25 mg / ml in a buffer solution containing sodium acetate, sodium acetate and magnesium chloride, citrate, malic acid, or malic acid and sodium chloride. In one example, ceDNA was diluted to 0.2 mg / ml in 10 mM to 50 mM citrate buffer (pH 4). An alcoholic lipid solution was mixed with the aqueous ceDNA solution in a ratio of about 1:5 to 1:3 (vol / vol) at a total flow rate of more than 10 ml / min, for example, using a syringe pump or impingement jet mixer. In one example, the alcoholic lipid solution was mixed with the aqueous ceDNA solution in a ratio of about 1:3 (vol / vol) at a flow rate of 12 ml / min. The alcohol was removed, and the buffer was replaced with PBS via dialysis. Alternatively, the buffer was replaced with PBS using a centrifuge tube. Alcohol removal and simultaneous buffer exchange were performed, for example, via dialysis or tangential flow filtration. The obtained lipid nanoparticles were filtered through a 0.2 μm pore sterile filter.

[0395] In one study, lipid nanoparticles containing exemplary ceDNA were prepared using a lipid solution containing SS-OP, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50:10:37:3) as a control. In some examples, tissue-targeting moiety such as N-acetylgalactosamine (GalNAc) was included. GalNAc moietyes such as three-branched GalNAc (GalNAc3) or four-branched GalNAc (GalNAc4) were synthesized as known in the art (see WO2017 / 084987 and WO2013 / 166121), and as widely known in the art (Reference [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 [Reference: 276:375577-37584] It can be chemically conjugated to lipids or PEG. An aqueous solution of ceDNA in a buffer solution was prepared. The lipid solution and the ceDNA solution were mixed at a lipid-to-ceDNA ratio of 1:3 (v / v) at a total flow rate of 12 mL / min using a procedure developed in-house on the NanoAssembler.

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407] Species (number, gender, age): CD-1 mice (N = 65 mice and 5 reserve mice, male, approximately 4 weeks old upon arrival).

[0408] Cage Observation: Cage observations were performed daily.

[0409] Clinical observation: Clinical observations were performed after administering the test substance on Day 0 at approximately 1 hour, approximately 5 to 6 hours, and approximately 24 hours. Additional observations were performed exceptionally. The weight of all animals was recorded on Day 0, Day 1, Day 2, Day 3, Day 4, and Day 7 (before euthanasia), where applicable. Additional weight was recorded as necessary.

[0410] Dosage administration: On day 0, the test product (LNP:ceDNA-Luc) was administered intravenously into the lateral tail vein to groups 1 to 38 at a dose of 5 mL / kg.

[0411] In-life Imaging On day 4, luciferin was administered to all animals at a dose of 150 mg / kg (60 mg / mL) via an intraperitoneal (IP) injection of 2.5 mL / kg. Within 15 minutes of each luciferin administration; all animals were as described below In vivo The IVIS imaging session was performed according to the imaging protocol.

[0412] Anesthesia recovery: The animals were continuously monitored while under anesthesia, during recovery, and until they moved.

[0413] Intermediate blood sample: On Day 0, Administration of test substance Intermediate blood was collected from all animals after 5 to 6 hours (5.0 hours or more, 6.5 hours or less).

[0414] After collection, 0.5 mL to 1.0 mL of lactic acid Ringer's solution was administered subcutaneously to the animal.

[0415] Whole blood for serum was collected via tail vein nick, saphenous vein, or orbital sinus puncture (under inhaled isoflurane). Whole blood was collected in a serum separator tube containing a thromboactivator and processed into one serum aliquot.

[0416] In vivo imaging protocol

[0417] Luciferin stock powder was stored at nominal -20℃.

[0418] Formulated luciferin stored in 1 mL aliquots at 2°C to 8°C was protected from light.

[0419] ㆍ The formulated luciferin was stable for up to 3 weeks at 2°C to 8°C with light blocked, and stable for about 12 hours at room temperature (RT).

[0420] Luciferin was dissolved in PBS in a sufficient volume to a target concentration of 60 mg / mL, and if necessary, the pH was adjusted to 7.4 using 5 M NaOH (approx. 0.5 μL / mg luciferin) and HCl (approx. 0.5 μL / mg luciferin).

[0421] It was prepared in an appropriate amount containing at least about 50% excess according to the protocol.

[0422] Scanning and Imaging (Note: Up to 5 animals can be imaged at once)

[0423] ㆍ (If necessary) Shaved the animal's fur.

[0424] According to the protocol, 150 mg / kg of luciferin in PBS was injected via IP at 60 mg / mL.

[0425] Imaging was performed immediately after administration or within a maximum of 15 minutes after administration.

[0426] The animal was anesthetized during the imaging session by setting the isoflurane vaporizer to 1% to 3% (typically 2.5%).

[0427] ㆍ Isoflurane anesthesia during imaging sessions:

[0428] Place the animal in the isoflurane chamber and wait for about 2 to 3 minutes until the isoflurane effect appears.

[0429] ○ Check if the anesthesia level on the side of the IVIS machine is in the "on" position.

[0430] ○ Place the animal(s) in the IVIS machine.

[0431] The intended acquisition protocol was performed with the setting for maximum sensitivity.

[0432] result

[0433] Research A

[0434] Fig. 1 As shown in [figure], on day 4, lipid 1, lipid 2, lipid 3, or lipid 5 (each, Fig. 1 The group of mice treated with ceDNA-luciferase (ceDNA-luc) formulated with LNP 5, LNP 4, LNP 6, and LNP 3) showed equivalent or higher luciferase expression and / or activity compared to the group treated with the positive control ceDNA LNP used in Study A (LNP 1, LNP 2, and LNP 7 to LNP 12, each of which is ceDNA-luc formulated with SS-OP lipids), suggesting that the ionizable lipids described herein possess excellent physical properties as lipid nanoparticle delivery vehicles.

[0435] Research B

[0436] Fig. 2 As illustrated in [ ], the above study A Fig. 1 Consistent with observations at, on day 4, lipid 1, lipid 2, lipid 3, or lipid 5 (each, Fig. 2The group of mice treated with ceDNA-luc formulated with LNP 17, LNP 16, LNP 18, and LNP 15) showed equivalent or higher luciferase activity compared to the group treated with the positive control ceDNA LNP used in Study B (LNP 13, which is ceDNA-luc formulated with SS-OP lipid), suggesting that the ionizable lipids described herein possess excellent physical properties as lipid nanoparticle delivery vehicles.

[0437] Research C

[0438] Lipids 1 and 3, which showed the highest luciferase expression and / or activity in Studies A and B, were further studied for dose-response in Study C. Fig. 3 As shown in [figure], of Study A and Study B Fig. 1 and Fig. 2 Consistent with observations at, on day 4, lipid 1 or lipid 3 (each, Fig. 1 The group of mice treated with ceDNA-luc formulated with LNP 20 and LNP 21) exhibited higher luciferase expression and / or activity at both 25 mg / kg and 1 mg / kg compared to the group treated with the positive control ceDNA LNP used in Study C (LNP 19, which is ceDNA-luc formulated with SS-OP lipids), suggesting that the ionizable lipids described herein possess excellent physical properties as lipid nanoparticle delivery vehicles. Furthermore, Fig. 3The results show that when the dosage of LNP 20 was increased from 0.25 mg / kg to 1 mg / kg, it exhibited a higher increase in the expression and / or activity of luciferase compared to LNP 19 tested at the same two dosage levels. These results suggest that the LNP formulated with the ionizable lipid of the present disclosure is more sensitive to different dosage levels, and that the expression level of the transgenic insert in ceDNA encapsulated by the LNP formulated with the ionizable lipid of the present disclosure can be more easily adjusted to the level required to exert a therapeutic effect for a specific disease, thereby demonstrating another desirable technical feature of such ionizable lipids as lipid nanoparticle delivery vehicles.

[0439] Research D

[0440] In Study D, lipids 6, 7, and 8 (respectively, Fig. 4a and do 4b of LNP 24, LNP 25, and LNP 26) and LNPs formulated with ceDNA-luc were evaluated in mice for luciferase expression and / or activity, and also for tolerability, and ionizable lipid A and SS-OP lipids (respectively, Fig. 4a and do 4b LNP 22 and LNP 23) were compared with LNPs formulated with ceDNA-luc. Fig. 4a As shown in [figure], on day 4, the group of mice treated with ceDNA-luc constructs formulated with lipid 6, lipid 7, and lipid 8 showed equivalent or higher luciferase expression and / or activity compared to the group treated with ceDNA-luc formulated with SS-OP lipid (i.e., LNP 23). do 4bThis demonstrates that the ce-DNA-luc constructs formulated with lipid 6, lipid 7, and lipid 8 were also well tolerated in mice, as they did not induce body weight changes in mice on Day 1. In contrast, do 4b As can be seen in [the image], mice treated with ceDNA-luc formulated with ionizable lipid A (i.e., LNP 22) experienced significant body weight loss on day 1, indicating that the lipid was not well tolerated in animals.

[0441] Research E

[0442] In Study E, lipids 9, 10, and 11 (respectively, Fig. 5a and do 5b Evaluated LNPs formulated with ceDNA-luc and LNPs (LNP 28, LNP 29, and LNP 30) and luciferase expression and / or activity, and also tolerability in mice, and SS-OP lipids ( Fig. 5a and do 5b It was compared with LNP 27) and LNP formulated with ceDNA-luc. Fig. 5a As shown in [figure], on day 4, the group of mice treated with ceDNA-luc constructs formulated with lipid 9, lipid 10, and lipid 11 showed equivalent or higher luciferase expression and / or activity compared to the group treated with ceDNA-luc formulated with SS-OP lipid (i.e., LNP 27). do 5b This shows that, excluding outlier data points at LNP 30, the ce-DNA-luc constructs formulated with lipid 9, lipid 10, and lipid 11 did not cause significant body weight changes in mice on day 1, indicating that they were generally well tolerated in mice.

[0443] Accordingly, Studies A through E generally describe that the LNP formulated with the ionizable lipid of the present disclosure (i) excellent of the ceDNA transgene insert In vivo (ii) because it indicates expression levels; and (ii) responds to different dosage levels, the transgene insert of ceDNA In vivo The expression level can be adjusted as needed; (iii) In vivo It demonstrates excellent tolerability.

[0444] References

[0445] All publications and references, including patents and patent applications, cited herein in the embodiments and without limitation, are incorporated herein by reference in their entirety as indicated herein, so that each individual publication or reference is described in its entirety and individually. Any patent application for which this application claims priority is also incorporated herein by reference in the manner described above with respect to publications and references.

Claims

Claim 1 Chemical formula ( I Lipids having ) or pharmaceutically acceptable salts thereof: ( I )[In the formula, a is an integer in the range of 1 to 20; b is an integer in the range of 2 to 10; R 1 (C2-C 20 )Alkenyl, -C(O)O(C2-C 20 )alkyl, and (C2-C 20 Selected from the group consisting of alkyl-substituted cyclopropyl; R 2 is (C2-C 20 )alkyl is]. Claim 2 In paragraph 1, the chemical formula ( II Lipids or pharmaceutically acceptable salts thereof having ): ( II )[In the expression, c and d are each independently integers in the range of 1 to 8]. Claim 3 A lipid or a pharmaceutically acceptable salt thereof, wherein c and d are each independently integers in the range of 2 to 8, or 4 to 8, or 6 to 8. Claim 4 In paragraph 2, a lipid or a pharmaceutically acceptable salt thereof, wherein c and d are each independently 1, 3, 5 or 7. Claim 5 A lipid or a pharmaceutically acceptable salt thereof, wherein at least one of c and d is 7 in any one of paragraphs 2 to 4. Claim 6 In paragraph 1, the chemical formula ( III Lipids or pharmaceutically acceptable salts thereof having ): ( III ). Claim 7 A lipid or a pharmaceutically acceptable salt thereof, wherein b is an integer in the range of 3 to 9 or 5 to 7 in any one of paragraphs 1 to 4 and 6. Claim 8 In any one of paragraphs 1 through 4 and 6, a lipid or a pharmaceutically acceptable salt thereof in which b is 5 or 7. Claim 9 A lipid or a pharmaceutically acceptable salt thereof, wherein in any one of claims 1 to 4 and 6, a is an integer in the range of 2 to 18, or 3 to 17, or 6 to 18, or 4 to 12, or 2 to 5, or 6 to 8, or 16 to 18, or 9 to 11. Claim 10 In paragraph 9, a lipid or a pharmaceutically acceptable salt thereof in which a is 3, 7, 8, 10, or 17. Claim 11 In any one of paragraphs 1 through 4 and 6, R 1 This (C5-C 15 )Alkenyl, -C(O)O(C4-C 18 )alkyl, and (C4-C 16 A lipid or a pharmaceutically acceptable salt thereof selected from the group consisting of alkyl-substituted cyclopropyl. Claim 12 In Paragraph 11, R 1 This (C5-C 12 )Alkenyl, -C(O)O(C4-C 12 )alkyl, and (C4-C 12 A lipid or a pharmaceutically acceptable salt thereof selected from the group consisting of alkyl-substituted cyclopropyl. Claim 13 In Paragraph 12, R 1 This (C5-C 10 )Alkenyl, -C(O)O(C4-C 10 )alkyl, and (C4-C 10 A lipid or a pharmaceutically acceptable salt thereof selected from the group consisting of alkyl-substituted cyclopropyl. Claim 14 In any one of paragraphs 1 through 4 and 6, R 1 This C 10 Alkenyls, lipids, or pharmaceutically acceptable salts thereof. Claim 15 In paragraph 1, R 1 For -C(O)O(C2-C 20 A lipid or a pharmaceutically acceptable salt thereof in which the alkyl group is an unbranched alkyl. Claim 16 In paragraph 15, R 1 A lipid or a pharmaceutically acceptable salt thereof that is -C(O)O(C9alkyl). Claim 17 In Clause 11, -C(O)O(C4-C 18 A lipid or a pharmaceutically acceptable salt thereof in which the alkyl group is a branched alkyl. Claim 18 In Paragraph 17, R 1 This -C(O)O(C 17 Alkyl) lipids or pharmaceutically acceptable salts thereof. Claim 19 In any one of paragraphs 1 through 4 and 6, R 1 this Selected from a group consisting of; R 2 go A lipid or a pharmaceutically acceptable salt thereof selected from the group consisting of Claim 20 In paragraph 1, the geological structure or selected from the group consisting of pharmaceutically acceptable salts thereof; or a lipid or pharmaceutically acceptable salt thereof in which the lipid is the following lipid 7. 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanyl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl) 9-(tridecane-5-yl)nonandioate. Claim 21 A lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, 6 and 20; and a lipid nanoparticle (LNP) comprising a nucleic acid. Claim 22 Lipid nanoparticles according to claim 21, wherein the nucleic acid is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, ceDNA, ministrings, doggybone™, protelomere closed DNA, dumbbell linear DNA, Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, DNA viral vectors, viral RNA vectors, non-viral vectors, and any combination thereof. Claim 23 Lipid nanoparticles according to claim 21, further comprising at least one lipid selected from the group consisting of sterols, PEG-lipid conjugates, and non-cationic lipids. Claim 24 In claim 21, lipid nanoparticles having a diameter in the range of 50 nm to 110 nm, or a diameter of less than 100 nm, less than 70 nm, or less than 60 nm. Claim 25 In claim 21, lipid nanoparticles further comprising a tissue-specific targeting moiety. Claim 26 In paragraph 25, lipid nanoparticles in which the tissue-specific targeting moiety is N-acetylgalactosamine (GalNAc) or a peptide. Claim 27 In paragraph 26, the tissue-specific targeting moiety is GalNAc, wherein GalNAc is connected to a second PEG-lipid conjugate to form a GalNAc-linked PEG-lipid conjugate, lipid nanoparticle. Claim 28 In claim 27, lipid nanoparticles in which a GalNAc-linked PEG-lipid conjugate is present in the lipid nanoparticles at a molar percentage of 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%. Claim 29 Lipid nanoparticles comprising lipids or pharmaceutically acceptable salts thereof and nucleic acids according to any one of claims 1 to 4, 6 and 20; A composition for use in the treatment of a genetic disorder in subjects requiring treatment for a genetic disorder, comprising a pharmaceutically acceptable excipient, wherein the genetic disorder is sickle cell anemia, melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency) and hemophilia B (coagulation factor IX (FIX) deficiency), cystic fibrosis (CFTR defect), familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary hepatic metabolic disorder, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharide storage disorder, Niemann-Pick disease. Type A / B, Niemann-Pick disease Type C1, Niemann-Pick disease Type C2, Fabry disease, Schindler disease, GM2-gangliosidosis Type II (Sandhoff Disease), Tay-Sachs disease, Metachromatic leukodystrophy, Krabbe disease, Myxolipilysis Type I, Myxolipilysis Type II / III, Myxolipilysis Type IV, Sialic acidosis Type I, Sialic acidosis Type II, Glycogen storage disease Type I, Glycogen storage disease Type II (Pompe disease), Gaucher disease Type I, Gaucher disease Type II, Gaucher disease Type III, Fabry disease, Cystinosis, Batten disease, Aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency),Lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinoses (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialic acidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification in infants (GACI) A composition selected from the group consisting of arterial calcification of infancy, Leber Congenital Amaurosis, Stargardt macular dystrophy (ABCA4 deficiency), ornithine transcarbamylase (OTC) deficiency, age-related macular degeneration, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), progressive familial intrahepatic cholestasis (PFIC) type II (ABCB11), progressive familial intrahepatic cholestasis (PFIC) type III (ABCB4), progressive familial intrahepatic cholestasis (PFIC) type IV (TJP2), and cathepsin A deficiency. Claim 30 In paragraph 29, a composition in which the subject is a human. Claim 31 A composition according to claim 29, wherein the mucopolysaccharide accumulation disorder is selected from the group consisting of Hurler syndrome (MPS I), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I HS), Hunter syndrome (MPS II), Sanfilippo syndrome type A (MPS IIIA), Sanfilippo syndrome type B (MPS IIIB), Sanfilippo syndrome type C (MPS IIIC), Sanfilippo syndrome type D (MPS IIID), Morquio syndrome type A (MPS IVA), Morquio syndrome type B (MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), and hyaluronidase deficiency (MPS IX). Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete Claim 78 delete Claim 79 delete Claim 80 delete Claim 81 delete Claim 82 delete Claim 83 delete Claim 84 delete Claim 85 delete Claim 86 delete Claim 87 delete Claim 88 delete Claim 89 delete Claim 90 delete Claim 91 delete Claim 92 delete Claim 93 delete Claim 94 delete Claim 95 delete Claim 96 delete Claim 97 delete Claim 98 delete Claim 99 delete Claim 100 delete Claim 101 delete Claim 102 delete Claim 103 delete Claim 104 delete Claim 105 delete Claim 106 delete Claim 107 delete Claim 108 delete Claim 109 delete Claim 110 delete Claim 111 delete Claim 112 delete Claim 113 delete Claim 114 delete Claim 115 delete Claim 116 delete Claim 117 delete Claim 118 delete Claim 119 delete

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