Method for encapsulating polynucleotides in reduced-size lipid nanoparticles and a novel formulation thereof

Abstract

A method for encapsulating polynucleotides in reduced-size lipid nanoparticles and a novel formulation thereof are provided. [Solution] Provided herein are reduced-size lipid formulations comprising lipids and capsid-free, nonviral vectors (e.g., ceDNA), and methods for producing such lipid formulations. The lipid particles (e.g., lipid nanoparticles) of this disclosure include lipid formulations that can be used to deliver capsid-free, nonviral DNA vectors to a target site of interest (e.g., cells, tissues, organs, etc.). Provided herein are novel formulation processes and methods used to produce LNPs with a significantly smaller diameter than those previously described.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 053,274 filed on 17 July 2020 and U.S. Provisional Application No. 63 / 194,620 filed on 28 May 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Lipid nanoparticles (LNPs) are a clinically validated strategy for delivering small interfering RNA (siRNA) cargoes to hepatocytes in the liver. Despite these advances, LNP-mediated delivery of large, rigid polynucleotide cargoes (e.g., double-stranded linear DNA, plasmid DNA, closed-end double-stranded DNA (ceDNA)) presents further challenges compared to small and / or flexible cargoes (e.g., siRNA). One such challenge relates to the size of the resulting LNPs when large, rigid cargoes are encapsulated. For example, LNPs with a diameter of 80-120 nm, encapsulating closed-end linear DNA (ceDNA) of length >3000 bp (base pairs), are routinely observed, with an average diameter of 92 nm (n=28), using a “state-of-the-art” process of high-pressure microfluidic mixing of aqueous ceDNA in H2O or aqueous buffer from one stream and ethanol lipids (100% EtOH from another stream in acidic buffer (pH 3-4) (see, for example, International Application No. PCT / US2020 / 021328)).

[0003] The relatively large size of these LNPs reduces the therapeutic index for hepatic indications by several mechanisms: (1) larger LNPs cannot efficiently bypass the endothelial cell window covering the hepatic sinusoids, hindering access to target cells (hepatocytes); (2) larger LNPs cannot be efficiently taken up by hepatocytes via clathrin-mediated endocytosis with several different receptors (e.g., ethial glycoprotein receptor (ASGPR), low-density lipoprotein (LDL) receptor); and (3) LNPs exceeding a certain threshold size tend to be preferentially taken up by cells of the reticuloendothelial system, potentially triggering a dose-limiting immune response. Therefore, there is an urgent need for manufacturing processes that can encapsulate large, rigid therapeutic nucleic acid molecules in relatively small LNPs (less than 75 nm in diameter). [Overview of the project]

[0004] Provided herein are novel formulation processes and methods used to produce LNPs with significantly smaller diameters than those previously described. The novel formulation process described herein involves reversible compression of TNA in 80% to 100% low molecular weight alcohol (e.g., ethanol, propanol, isopropanol, butanol, or methanol) prior to the aggregation of microfluidic nanoparticles with alcoholic (e.g., enolic) lipids, thereby resulting in LNPs with an average diameter of 75 nm (±3 nm) or less.

[0005] According to several embodiments, the LNPs described herein have average diameters in the range of approximately 20 nm to 75 nm, 20 nm to 70 nm, 20 nm to 60 nm, 30 nm to 75 nm, 30 nm to 70 nm, 30 nm to 60 nm, 40 nm to 75 nm, or 40 nm to 70 nm. Small LNPs offer more efficient tissue diffusion and more efficient uptake and / or targeting. Particularly in the liver, small LNPs are required to pass through the hepatic sinusoidal endothelial cell (LSEC) window (<100 nm) and induce ASGPR-mediated endocytosis (≤70 nm). Such miniaturization is also advantageous for targeting and avoiding undesirable immune responses, as immune cells can easily evade them. The formulation processes and methods described herein can encapsulate considerably more therapeutic nucleic acids (e.g., rigid double-stranded DNA including ceDNA) than those previously reported. The LNPs described herein can encapsulate more than 60% to about 90% of rigid double-stranded DNA such as ceDNA. According to some embodiments, the LNPs described herein can encapsulate more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% of rigid double-stranded DNA such as ceDNA.

[0006] The formulation process described herein utilizes the discovery that ceDNA compression occurs in a solvent containing 80% to 100% low molecular weight (LMW) alcohol. LMW alcohols that can be used for compression include, but are not limited to, methanol, ethanol, propanol, isopropanol, butanol, or other organic solvents such as acetone. Preferably, compression of rigid DNA such as ceDNA can be prepared using an ethanol solution or an ethanol-methanol mixture (e.g., an EtOH-MeOH 1:1 mixture) at a final concentration of about 80% to about 98%. According to several embodiments, the final concentration of low molecular weight alcohols in solution is approximately 80% to 98%, 80% to 95%, 80% to 92%, 80% to 90%, 80% to 85%, 85% to 98%, 85% to 95%, 85% to 92%, 85% to 90%, 90% to 98%, 87% to 97%, and 87% to 95%. The percentages are approximately 87% to 92%, 87% to 90%, 90% to 95%, 90% to 92%, 95% to 98%, or approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%. For example, when ceDNA in a 90% EtOH aqueous solution is added to or mixed with another ethanol solution of lipids (e.g., 90% EtOH) in a ratio such that the resulting solution is, for example, 90-92% ethanol and 8-10% water or aqueous buffer, the ceDNA is observed to exist in a highly compressed or denatured state due to dynamic light scattering. In such solvents (e.g., 90-92% ethanol, 8-10% water), both lipids and ceDNA are solubilized without any detectable precipitates of either component, resulting in more efficient encapsulation of rigid double-stranded DNA like ceDNA into small LNPs.

[0007] Therefore, the formulation process described herein reduces LNP diameter while maintaining equivalent or better encapsulation efficiency of rigid TNAs such as ceDNA compared to standard processes. While we do not wish to be bound by theory, this change is likely due to the compression of rigid TNAs such as ceDNA by preferably 90-92% or up to 95% in an LMW alcohol solution such as ethanol solvent prior to LNP formation. Once LNP formation is initiated by mixing with an acidic aqueous buffer, the lipids can nucleate around a smaller, more compact DNA (e.g., ceDNA) core, in contrast to standard aqueous processes, resulting in significantly smaller particles. Using the process described herein, rigid TNAs such as ceDNA can be efficiently encapsulated in larger numbers, resulting in TNA-LNPs with much smaller diameters, a beneficial attribute of LNPs for targeting various tissues with size constraints.

[0008] In some embodiments, the formulation includes TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of about 75 nm (±3 nm). In some embodiments, the formulation includes TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of about 72 nm (±3 nm). In some embodiments, the formulation includes TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of about 70 nm (±4 nm). In some embodiments, the formulation includes TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of about 68 nm (±4 nm). In some embodiments, the formulation includes TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of about 65 nm (±4 nm). In some embodiments, the formulation includes TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of about 60 nm (±4 nm). In some embodiments, the formulation includes TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of about 55 nm (±4 nm). In some embodiments, the formulation comprises TNA (e.g., ceDNA) encapsulated in LNPs having an average diameter of approximately 50 nm (±4 nm).

[0009] According to a first aspect, the present disclosure provides a pharmaceutical composition comprising lipid nanoparticles (LNPs), the LNPs comprising a lipid and a rigid therapeutic nucleic acid (rTNA), and the average diameter of the LNPs being from about 20 nm to about 75 nm.

[0010] According to some embodiments, the rigid nucleic acid therapeutic is a double-stranded nucleic acid. According to some embodiments, the rigid nucleic acid therapeutic is a closed-ended DNA.

[0011] According to some embodiments, the lipid is selected from an ionizable lipid, a non-cationic lipid, a sterol or its derivative, a conjugated lipid, or any combination thereof. According to some embodiments, the ionizable lipid is a cationic lipid. According to some embodiments, the cationic lipid is a lipid capable of SS cleavage.

[0012] According to some embodiments of the aspects and embodiments disclosed herein, the ionizable lipid has the formula (I):

Chemical formula

[0013] According to some embodiments of the aspects and embodiments disclosed herein, the ionized lipid is of formula (II): [ka] or represented by its pharmaceutically acceptable salt, 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 is absent, or (C2-C 20 ) Alkenyl, -C(O)O(C2-C 20 )alkyl, and (C2-C 20 ) Selected from alkyl-substituted cyclopropyls, R 2 (C2-C 20 It is alkyl.

[0014] According to some embodiments of the aspects and embodiments disclosed herein, the ionized lipid is of formula (V): [ka] or represented by its pharmaceutically acceptable salt, in the formula, R 1 and R 1’ Each of them operates independently, R a (C1-C6) alkylenes optionally substituted with one or more groups selected from the following: R 2 and R 2’ Each of them is independently a (C1-C2) alkylene, R 3 and R 3’ Each of them operates independently, R b It is an alkyl group (C1-C6) optionally substituted with one or more groups selected from the following, Alternatively, R 2 and R 3and / or R 2’ and R 3’ together with the intervening N atom thereof form a 4- to 7-membered heterocyclyl, R 4 and R 4 ’ are each (C2-C6) alkylene interrupted by -C(O)O-, R 5 and R 5 ’ are each independently (C2-C 30 ) alkyl or (C2-C 30 ) alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6) cycloalkyl, R a and R b are each halo or cyano.

[0015] According to some embodiments, the ionizable lipid is of formula (XV):

Chemical formula

Chemical formula

[0016] According to some embodiments, the ionizable lipid is of formula (XX):

Chemical formula

[0017] According to some embodiments, the ionized lipid is selected from any lipid in Table 2, Table 5, Table 6, Table 7, or Table 8.

[0018] According to some embodiments, ionized lipids have the following structure: [ka] Or a lipid having a pharmaceutically acceptable salt thereof.

[0019] According to some embodiments, cationic lipids have the following structure: [ka] It is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA or MC3) which has the following properties.

[0020] According to some embodiments of the aspects and embodiments disclosed herein, the LNP further comprises a sterol. According to some embodiments, the sterol is cholesterol.

[0021] According to some embodiments of the aspects and embodiments disclosed herein, the LNP further comprises polyethylene glycol (PEG). According to some embodiments, the PEG is l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0022] According to some embodiments of the aspects and embodiments disclosed herein, the LNP further comprises a noncationic lipid. According to some embodiments, noncationic lipids include distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and distearoylphosphatidylethanolamine. (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethylPE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethylPE), 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (S M), Dimyristoyl phosphatidylcholine (DMPC), Dimyristoyl phosphatidylglycerol (DMPG), Distearoyl phosphatidylglycerol (DSPG), Diylcoyl phosphatidylcholine (DEPC), Palmitoyl oleoyl phosphatidylglycerol (POPG), Dielydoyl phosphatidylethanolamine (DEPE), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,Selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.

[0023] According to some embodiments, the noncationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

[0024] According to some embodiments, PEG or PEG-lipid conjugates are present in concentrations of approximately 1.5% to 3%.

[0025] According to some embodiments, cholesterol is present in a molar percentage of approximately 20% to 40%, and lipids are present in a molar percentage of approximately 80% to 60%.

[0026] According to some embodiments, cholesterol is present at approximately 40% of the molar percentage, and lipids are present at approximately 50% of the molar percentage.

[0027] According to some embodiments, the composition further comprises cholesterol, PEG or PEG-lipid conjugate, and noncationic lipids.

[0028] According to some embodiments, PEG or PEG-lipid conjugates are present in concentrations of approximately 1.5% to 3%, approximately 1.5% to 2.75%, approximately 1.5% to 2.5%, approximately 1.5% to 2%, approximately 2% to 3%, approximately 2% to 2.75%, approximately 2% to 2.5%, approximately 2.5% to 3%, approximately 2.5% to 2.75%, or approximately 2.5% to 3%.

[0029] According to some embodiments, cholesterol is present in molar percentages of approximately 30% to 50%, 30% to 45%, 30% to 40%, 30% to 35%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to 50%, or 45% to 50%.

[0030] According to some embodiments, lipids are present in molar percentages of approximately 42.5% to 62.5%, 42.5% to 57.5%, 42.5% to 52.5%, 42.5% to 47.5%, 47.5% to 62.5%, 47.5% to 57.5%, 47.5% to 52.5%, 52.5% to 62.5%, 52.5% to 57.5%, or 57.5% to 62.5%.

[0031] According to some embodiments, noncationic lipids are present in molar percentages of approximately 2.5% to 12.5%, 2.5% to 10.5%, 2.5% to 8.5%, 2.5% to 6.5%, 2.5% to 4.5%, 4.5% to 12.5%, 4.5% to 10.5%, 4.5% to 8.5%, 4.5% to 6.5%, 6.5% to 12.5%, 6.5% to 10.5%, 6.5% to 8.5%, 8.5% to 12.5%, 8.5% to 10.5%, or 10.5% to 12.5%.

[0032] According to some embodiments of the aspects and embodiments of this specification, cholesterol is present at about 40% molar percentage, lipids at about 52.5% molar percentage, noncationic lipids at about 7.5% molar percentage, and PEG at about 3%.

[0033] According to some embodiments of the aspects and embodiments disclosed herein, the composition further comprises dexamethasone palmitate.

[0034] According to some embodiments of the aspects and embodiments disclosed herein, the LNP is less than about 75 nm in size. According to some embodiments of the aspects and embodiments disclosed herein, the LNP is less than about 70 nm in size, for example, 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 the aspects and embodiments disclosed herein, the LNP is less than about 70 nm, 69 nm, 68 nm, 67 nm, 66 nm, 65 nm, 64 nm, 63 nm, 62 nm, 61 nm, 60 nm, 59 nm, 58 nm, 57 nm, 56 nm, 55 nm, 54 nm, 53 nm, 52 nm, 51 nm, or 50 nm in size.

[0035] According to some embodiments of the aspects and embodiments disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 15:1.

[0036] According to some embodiments of the aspects and embodiments disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 30:1.

[0037] According to some embodiments of the aspects and embodiments disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 40:1.

[0038] According to some embodiments of the aspects and embodiments disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 50:1.

[0039] According to some embodiments of the embodiments and models disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 15:1 to about 30:1. According to some embodiments of the embodiments and models disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 15:1 to about 40:1. According to some embodiments of the embodiments and models disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 15:1 to about 50:1. According to some embodiments of the embodiments and models disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 30:1 to about 40:1. According to some embodiments of the embodiments and models disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 30:1 to about 50:1. According to some embodiments of the embodiments and models disclosed herein, the composition has a total lipid to rigid therapeutic nucleic acid (rTNA) ratio of about 40:1 to about 50:1. According to some embodiments of the aspects and embodiments disclosed herein, the composition further comprises N-acetylgalactosamine (GalNAc). According to some embodiments, GalNAc is present in LNPs at a molar percentage of 0.5% of total lipids. According to some embodiments, GalNAc is present in LNPs at molar percentages of about 0.3% to about 0.9%, about 0.4% to about 0.8%, and about 0.5% to about 0.6% of total lipids.

[0040] According to some embodiments of the aspects and embodiments disclosed herein, rigid therapeutic nucleic acids (rTNAs) are closed-end DNA (ceDNAs).

[0041] According to some embodiments of the aspects and embodiments disclosed herein, rigid therapeutic nucleic acid (rTNA) comprises an expression cassette containing a promoter sequence and a transgene.

[0042] According to some embodiments, the rigid therapeutic nucleic acid (rTNA) comprises an expression cassette containing a polyadenylated sequence.

[0043] According to some embodiments, rigid therapeutic nucleic acid (rTNA) comprises at least one inverted end repeat (ITR) adjacent to either the 5' or 3' end of the expression cassette.

[0044] According to some embodiments, the expression cassette is adjacent to two ITRs, the two ITRs comprising one 5'ITR and one 3'ITR.

[0045] According to some embodiments, the expression cassette is ligated to an ITR (3'ITR) at its 3' end. According to some embodiments, the expression cassette is ligated to an ITR (5'ITR) at its 5' end.

[0046] According to some embodiments, at least one of the 5'ITR or 3'ITR is a wild-type AAV ITR. According to some embodiments, at least one of the 5'ITR and 3'ITR is a modified ITR.

[0047] According to some embodiments, rigid therapeutic nucleic acids (rTNAs) further include a spacer sequence between the 5'ITR and the expression cassette.

[0048] According to some embodiments, rigid therapeutic nucleic acids (rTNAs) further include a spacer sequence between the 3'ITR and the expression cassette.

[0049] According to some embodiments, the spacer sequence is at least 5 base pairs long. According to some embodiments, the spacer sequence is at least 5 to 100 base pairs long. According to some embodiments, the spacer sequence is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs long. According to some embodiments, the spacer sequence is at least 5 to 500 base pairs long. According to some embodiments, the spacer sequence is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 base pairs long.

[0050] According to some embodiments, rigid therapeutic nucleic acids (rTNAs) have nicks or gaps.

[0051] According to some embodiments, the ITR is selected from ITRs derived from AAV serotypes, ITRs derived from goose virus ITRs, ITRs derived from B19 virus ITRs, or wild-type ITRs derived from parvovirus.

[0052] According to some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0053] According to some embodiments, the ITR is a mutant ITR, and the ceDNA optionally includes additional ITRs different from the first ITR.

[0054] According to some embodiments, the ceDNA contains two mutant ITRs at both the 5' and 3' ends of the expression cassette, and optionally, the two mutant ITRs are symmetric mutants.

[0055] According to some embodiments of the aspects and embodiments disclosed herein, rigid therapeutic nucleic acids (rTNAs) are selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), ribozymes, ceDNAs, ministrings, doggybone™, protelomere-bound DNA, or dumbbell linear DNA, Dicer substrate dsRNAs, small hairpin RNAs (shRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), mRNAs, tRNAs, rRNAs, DNA viral vectors, viral RNA vectors, nonviral vectors, and any combination thereof.

[0056] According to some embodiments of the aspects and embodiments disclosed herein, the nucleic acid for rigid therapeutics is a plasmid.

[0057] According to some embodiments of the aspects and embodiments disclosed herein, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0058] In another aspect, the present disclosure provides a method for producing a lipid nanoparticle (LNP) formulation, wherein the LNP comprises an ionized lipid and closed-end DNA (ceDNA), and the method comprises: adding aqueous ceDNA to a solution of one or more low molecular weight alcohols (e.g., ethanol, methanol, propanol, or isopropanol) containing cationic or ionized lipids to form a ceDNA / lipid solution, wherein the final concentration of alcohol in the solution is about 80% to about 98%; mixing the ceDNA / lipid solution with an acidic aqueous buffer; and exchanging the buffer with a neutral pH aqueous buffer to produce an LNP formulation. According to several embodiments, the final concentration of low molecular weight alcohols in solution is approximately 80% to 98%, 80% to 95%, 80% to 92%, 80% to 90%, 80% to 85%, 85% to 98%, 85% to 95%, 85% to 92%, 85% to 90%, 90% to 98%, 87% to 97%, and 87% to 95%. These are approximately 87% to 92%, 87% to 90%, 90% to 95%, 90% to 92%, 95% to 98%, or approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.

[0059] In another aspect, the Disclosure provides a method for producing lipid nanoparticle (LNP) formulations comprising ionized lipids and closed-end DNA (ceDNA), the method comprising adding ceDNA to a solution of one or more low molecular weight alcohols (e.g., ethanol, methanol, propanol, or isopropanol) such that the alcohol content of the resulting solution exceeds 80%, and adding the ceDNA with an alcohol content of >80% to a cationic or ionized lipid in 80% alcohol. The process includes adding to a substance to form a ceDNA / lipid solution, wherein the concentration of low molecular weight alcohol in the ceDNA-lipid solution is approximately 80% to approximately 95% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%), mixing the ceDNA / lipid solution with an acidic aqueous buffer, and exchanging the buffer with a neutral pH aqueous buffer to produce an LNP formulation. According to several embodiments, the final concentration of low molecular weight alcohols in solution is approximately 80% to 98%, 80% to 95%, 80% to 92%, 80% to 90%, 80% to 85%, 85% to 98%, 85% to 95%, 85% to 92%, 85% to 90%, 90% to 98%, 87% to 97%, and 87% to 95%. These are approximately 87% to 92%, 87% to 90%, 90% to 95%, 90% to 92%, 95% to 98%, or approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.

[0060] According to some embodiments, this method further includes the step of diluting the ceDNA / lipid mixture with an acidic aqueous buffer.

[0061] According to some embodiments, one or more low molecular weight alcohols are selected from the group consisting of methanol, ethanol, propanol, and isopropanol. According to some embodiments, one or more low molecular weight alcohols are ethanol. According to some embodiments, one or more low molecular weight alcohols are propanol. According to some embodiments, one or more low molecular weight alcohols are ethanol. According to some embodiments, one or more low molecular weight alcohols are a mixture of ethanol and methanol.

[0062] According to some embodiments, the acidic aqueous buffer is selected from malic acid / sodium malate or acetic acid / sodium acetate. According to some embodiments, the acidic aqueous buffer has a concentration of about 10 to 40 mmol (mM), for example, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 20 mM to about 30 mM, about 20 mM to about 40 mM, about 30 mM to about 40 mM, or about 10 mM to about 15 mM. According to some embodiments, the acidic aqueous buffer has a pH of about 3 to 5.

[0063] According to some embodiments, the neutral pH aqueous buffer is Dulbecco's phosphate-buffered saline, pH 7.4.

[0064] According to some embodiments, the ceDNA / lipid solution is mixed with an acidic aqueous buffer using microfluidic mixing.

[0065] According to some embodiments, the final alcohol content after the dilution step is about 4% to about 15% (for example, about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%).

[0066] According to some embodiments, the flow rate ratio between the acidic aqueous buffer and the ceDNA / lipid solution is 2:1, 3:2, 3:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1.

[0067] According to some embodiments, the LNP has an average diameter of about 20 nm to about 70 nm, for example, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, or about 70 nm.

[0068] According to some embodiments, cationic lipids have the following structure: [ka] It is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA or MC3) which has the following properties.

[0069] According to some embodiments, the ionized lipid is a disulfide-cleavable lipid containing disulfide bonds and tertiary amines.

[0070] According to some embodiments, SS-cleavable lipids are given by the following formula: [ka] Or it contains ss-OP lipids of a pharmaceutically acceptable salt thereof.

[0071] According to some embodiments, this disclosure provides LNP formulations produced by methods described herein and in the embodiments thereof.

[0072] In another aspect, the present disclosure provides a method for treating a genetic disorder in a subject, the method comprising administering an effective amount of the pharmaceutical composition described in any one of the prior claims to the subject.

[0073] According to some embodiments, the subject is human.

[0074] According to some embodiments, hereditary disorders include sickle cell anemia, melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency) and hemophilia B (coagulation factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary hepatic metabolic disorders, Lesch-Neyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Schayet syndrome (MPS type I S), Hurler-Scheyet syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS III (Types A, B, C, and D), Morquio A and B (MPS IVA and MPS IVB), Malotoramy syndrome (MPS VI), Sly syndrome (MPS VII), Hyaluronidase deficiency (MPS IX type), Niemann-Pick disease A / B, C1 and C2 types, Fabry disease, Schindler's disease, GM2-gangliosidosis type II (Sandhoff disease), Taysachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis I, II / III and IV types, sialidosis I and II types, glycogen storage disorder I and II types (Pompe disease), Gaucher disease I, II and III types, cystinosis, Batten disease, aspartylglucosamineuria, Salah disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosinosis The group is selected from alidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophy-epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, systemic arterial calcification in infancy (GACI), Leber congenital amaurosis, Stargard macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, and cathepsin A deficiency. According to some embodiments, the hereditary disorder is Leber congenital amaurosis (LCA).

[0075] According to some embodiments, the LCA is LCA10.

[0076] According to some embodiments, the hereditary disorder is Niemann-Pick disease. According to some embodiments, the hereditary disorder is Stargard macular dystrophy. According to some embodiments, the hereditary disorder is glucose-6-phosphatase (G6Pase) deficiency (glycogen storage disorder type I) or Pompe disease (glycogen storage disorder type II). According to some embodiments, the hereditary disorder is hemophilia A (factor VIII deficiency). According to some embodiments, the hereditary disorder is hemophilia B (factor IX deficiency). According to some embodiments, the hereditary disorder is Hunter syndrome (mucopolysaccharidosis type II). According to some embodiments, the hereditary disorder is cystic fibrosis. According to some embodiments, the hereditary disorder is dystrophy epidermolysis bullosa (DEB). According to some embodiments, the hereditary disorder is phenylketonuria (PKU). According to some embodiments, the hereditary disorder is hyaluronidase deficiency.

[0077] According to some embodiments of the aspects and embodiments disclosed herein, the method further includes administering an immunosuppressant.

[0078] According to some embodiments, the immunosuppressant is dexamethasone.

[0079] According to some embodiments of the aspects and embodiments disclosed herein, the subject exhibits a reduced immune response level to the pharmaceutical composition compared to the immune response level observed with LNPs containing MC3 as the major cationic lipid, with the immune response level to the pharmaceutical composition being at least 50% lower than the level observed with LNPs containing MC3.

[0080] According to some embodiments, the immune response is measured by detecting levels of pro-inflammatory cytokines or chemokines.

[0081] According to some embodiments, the pro-inflammatory cytokine or chemokine is selected from the group consisting of IL-6, IFNα, IFNγ, IL-18, TNFα, IP-10, MCP-1, MIP1α, MIP1β, and RANTES.

[0082] According to some embodiments, at least one of the pro-inflammatory cytokines is below a detectable level in the serum of the subject 6 hours after administration of the pharmaceutical composition.

[0083] According to several embodiments, LNPs containing SS-cleavable lipids and closed-end DNA (ceDNA) are not phagocytosed or exhibit a phagocytic level at least 50% lower than that of LNPs containing MC3 as the major cationic lipid administered under similar conditions.

[0084] According to some embodiments, SS-cleavable lipids are given by the following formula: [ka] Or it contains ssOP lipids of a pharmaceutically acceptable salt thereof.

[0085] According to some embodiments, the LNP further comprises cholesterol and PEG-lipid conjugates.

[0086] According to some embodiments, the LNP further comprises a noncationic lipid.

[0087] According to some embodiments, the noncationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

[0088] According to some embodiments, the LNP further comprises N-acetylgalactosamine (GalNAc).

[0089] According to some embodiments, GalNAc is present in LNPs at a molar percentage of 0.5% of total lipids.

[0090] In another aspect, the present disclosure provides a method for increasing the targeting of a therapeutic nucleic acid to the liver of a subject in need of treatment, the method comprising administering an effective amount of the pharmaceutical composition described in any one of the prior claims to the subject, wherein the LNP comprises a therapeutic nucleic acid, an ss-cleavable lipid, a sterol, and polyethylene glycol (PEG) and N-acetylgalactosamine (GalNAc).

[0091] According to some embodiments, PEG is l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0092] According to some embodiments, the LNP further comprises a noncationic lipid.

[0093] According to some embodiments, the noncationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

[0094] According to some embodiments, GalNAc is present in LNPs at a molar percentage of 0.5% of total lipids.

[0095] According to some embodiments, the subjects suffer from a genetic disorder.

[0096] According to some embodiments, the hereditary disorder is hemophilia A (factor VIII deficiency). According to some embodiments, the hereditary disorder is hemophilia B (factor IX deficiency). According to some embodiments, the hereditary disorder is phenylketonuria (PKU).

[0097] According to some embodiments, therapeutic nucleic acids are selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), ribozymes, ceDNA, ministrings, doggybone™, protelomere-closed DNA, or dumbbell linear DNA, Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, DNA viral vectors, viral RNA vectors, nonviral vectors, and any combination thereof.

[0098] According to some embodiments, the therapeutic nucleic acid is ceDNA.

[0099] According to some embodiments, the ceDNA includes an expression cassette comprising a promoter sequence and a transgene.

[0100] According to some embodiments, the ceDNA includes at least one inverted end repeat (ITR) adjacent to either the 5' or 3' end of the expression cassette.

[0101] According to some embodiments, the ceDNA is selected from the group consisting of CELiD, MIDGE, ministering DNA, dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITR at the 5' and 3' ends of the expression cassette, or doggybone® DNA, wherein the ceDNA is capsid-free and linear double-stranded DNA.

[0102] In some embodiments, the present disclosure provides a method for reducing the complement response in subjects requiring treatment with therapeutic nucleic acid (TNA), the method comprising administering an effective amount of the pharmaceutical composition described in any one of the prior claims to the subject, wherein the LNP comprises TNA, ss-cleavable lipids, sterols, polyethylene glycol (PEG), and N-acetylgalactosamine (GalNAc).

[0103] According to some embodiments, the subjects suffer from a genetic disorder.

[0104] According to some embodiments, hereditary disorders include sickle cell anemia, melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency) and hemophilia B (coagulation factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary hepatic metabolic disorders, Lesch-Neyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Schayet syndrome (MPS type I S), Hurler-Scheyet syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS III (Types A, B, C, and D), Morquio A and B (MPS IVA and MPS IVB), Malotoramy syndrome (MPS VI), Sly syndrome (MPS VII), Hyaluronidase deficiency (MPS IX type), Niemann-Pick disease A / B, C1 and C2 types, Fabry disease, Schindler's disease, GM2-gangliosidosis type II (Sandhoff disease), Taysachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis I, II / III and IV types, sialidosis I and II types, glycogen storage disorder I and II types (Pompe disease), Gaucher disease I, II and III types, cystinosis, Batten disease, aspartylglucosamineuria, Salah disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosinosis The group is selected from alidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophy-epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, systemic arterial calcification in infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, and cathepsin A deficiency.

[0105] According to some embodiments, the rigid therapeutic nucleic acid is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), ribozymes, ceDNA, ministrings, doggybone™, protelomere-closed DNA, or dumbbell linear DNA, Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, DNA viral vectors, viral vectors, nonviral vectors, and any combination thereof.

[0106] According to some embodiments, the ceDNA is selected from the group consisting of CELiD, MIDGE, ministering DNA, dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITR at the 5' and 3' ends of the expression cassette, or doggybone® DNA, wherein the ceDNA is capsid-free and linear double-stranded DNA.

[0107] According to some embodiments, PEG is l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0108] According to some embodiments, PEG is present in LNPs at a molecular percentage of approximately 2-4%. According to some embodiments, PEG is present in LNPs at a molecular percentage of approximately 3%.

[0109] According to some embodiments, the LNP further comprises a noncationic lipid. According to some embodiments, the noncationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

[0110] According to some embodiments, GalNAc is present in LNPs at a molar percentage of approximately 0.3–1% of total lipids. According to some embodiments, GalNAc is present in LNPs at a molar percentage of approximately 0.5% of total lipids. [Brief explanation of the drawing]

[0111] The embodiments of this disclosure, briefly summarized above and discussed in more detail below, can be understood by referring to exemplary embodiments of this disclosure depicted in the accompanying drawings. However, the accompanying drawings only illustrate typical embodiments of this disclosure and should not be considered limiting in scope, as this disclosure may allow for other equally valid embodiments.

[0112] [Figure 1A] Figure 1A is a graph showing ceDNA condensation determined by dynamic light scattering. The dynamic light scattering correlation function shows ceDNA condensation as the ethanol content increases. [Figure 1B] Figure 1B is a graph showing that compression is reversible through rehydration. [Figure 2] Figure 2 is a graph showing a comparison of the diameters of ceDNA LNPs produced by the standard formulation process and the novel formulation process described herein. [Figure 3A] Figures 3A and 3B are transmission electron microscope (TEM) images of ceDNA and plasmid DNA (pDNA) samples stored in deionized (DI) water, respectively. Figure 3A shows a TEM image of ceDNA stored in deionized water. Figure 3B shows a TEM image of plasmid stored in deionized water. [Figure 3B] Figures 3A and 3B are transmission electron microscope (TEM) images of ceDNA and plasmid DNA (pDNA) samples stored in deionized (DI) water, respectively. Figure 3A shows a TEM image of ceDNA stored in deionized water. Figure 3B shows a TEM image of plasmid stored in deionized water. [Figure 4A] Figures 4A and 4B are TEM images of ceDNA and pDNA samples stored in a 90.9% 1:1 ethanol:methanol low molecular weight alcohol / aqueous solution in deionized water, respectively. Figure 4A shows a TEM image of ceDNA stored in a 90.9% 1:1 ethanol:methanol solution in deionized water. Figure 4B shows a TEM image of plasmid stored in a 90.9% 1:1 ethanol:methanol solution in deionized water. [Figure 4B]Figures 4A and 4B are TEM images of ceDNA and pDNA samples stored in a 90.9% 1:1 ethanol:methanol low molecular weight alcohol / aqueous solution in deionized water, respectively. Figure 4A shows a TEM image of ceDNA stored in a 90.9% 1:1 ethanol:methanol solution in deionized water. Figure 4B shows a TEM image of plasmid stored in a 90.9% 1:1 ethanol:methanol solution in deionized water. [Figure 5] Figure 5 shows a TEM image of a ceDNA sample stored in 100% low molecular weight alcohol (ethanol:methanol = 1:1, no water). [Figure 6A] Figures 6A and 6B are TEM images of ceDNA and pDNA, respectively, that were stored under basic denaturation conditions in a 100 mM sodium hydroxide (NaOH) aqueous solution. [Figure 6B] Figures 6A and 6B are TEM images of ceDNA and pDNA, respectively, that were stored under basic denaturation conditions in a 100 mM sodium hydroxide (NaOH) aqueous solution. [Modes for carrying out the invention]

[0113] The immunogenicity associated with viral vector-based gene therapies has limited the number of patients that can be treated due to existing background immunity and has hindered patient re-administration to titrate each patient to an effective level or to maintain efficacy over the long term. Because of the lack of existing immunity, the therapeutic nucleic acid lipid particles (e.g., lipid nanoparticles) described herein allow for additional doses of therapeutic nucleic acids as needed, further expanding patient access, including pediatric populations that may require subsequent administrations as tissue grows. The therapeutic nucleic acid lipid particles (e.g., lipid nanoparticles) produced by the processes described herein, and in particular those comprising a cationic or ionized lipid composition containing one or more tertiary amino groups, are smaller than LNPs produced from conventional LNP manufacturing processes, thus providing more efficient delivery of therapeutic nucleic acids, better tolerability, and an improved safety profile. Because the therapeutic nucleic acid lipid particles (e.g., lipid nanoparticles) described herein are not subject to the packaging constraints imposed by space within the viral capsid, theoretically, the only size limitation of therapeutic nucleic acid lipid particles (e.g., lipid nanoparticles) lies in the host cell DNA replication efficiency. As described and illustrated herein, according to some embodiments, the therapeutic nucleic acid is a therapeutic nucleic acid (TNA) such as double-stranded DNA (e.g., ceDNA). As described and illustrated herein, according to some embodiments, the therapeutic nucleic acid is ceDNA. As also described herein, according to some embodiments, the therapeutic nucleic acid is mRNA.

[0114] One of the most challenging hurdles in developing treatments for rare diseases, in particular, is the sheer number of individual conditions. Approximately 350 million people worldwide live with rare disorders, and the National Institutes of Health (NIH) defines rare disorders as those diagnosed in fewer than 200,000 people. About 80% of these rare disorders are of genetic origin, and about 95% of them do not have FDA-approved treatments (rarediseases.info.nih.gov / diseases / pages / 31 / faqs-about-rare-diseases). Among the advantages of the ceDNA lipid particles (e.g., lipid nanoparticles) described herein is that they offer an approach that can be rapidly adapted to multiple diseases, particularly rare monogenic disorders, which could meaningfully alter the current state of treatment for many genetic disorders or diseases.

[0115] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art in which this disclosure pertains. This disclosure is not limited to, but may vary, the specific methodologies, protocols, and reagents described herein. The terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of this disclosure as defined by the claims. For definitions of general terms in immunology and molecular biology, see The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), and Robert S. Porter et al. (eds.), Fields Virology, 6th Edition. Edition, published by Lippincott Williams&Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999 - 2012 (ISBN9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN1 - 56081 - 569 - 8), Immunology by Werner Luttmann, published by Elsevier, 2006, Janeway’s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor&Francis Limited, 2014 (ISBN0815345305, 9780815345305), Lewin’s Genes XI, published by Jones&Bartlett Publishers, 2014 (ISBN - 1449659055), Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN1936113414), Davis et al. Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN044460149X),Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN0124199542),Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005, and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John This information can be found in Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), and all of this information is incorporated herein by reference in its entirety.

[0116] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise clearly indicated.

[0117] The abbreviation "e.g." originates from the Latin "exempli gratia" and is used herein to indicate a non-restrictive example. Therefore, the abbreviation "e.g." is synonymous with "for example."

[0118] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0119] As used herein, the term “about” means, when referring to a measurable value such as quantity or temporal duration, to include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, in order to be appropriate for carrying out the disclosed method.

[0120] When used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified.

[0121] As used herein, “comprise,” “comprising,” “comprises,” and “comprised of” are synonymous with “include,” “including,” “includes,” or “contain,” “containing,” and “contains,” and are, for example, comprehensive or free-form terms that specify the presence of a component followed by another component, and do not exclude or preclude the presence of additional, unreferenced components, features, elements, members, or steps that are known or disclosed in the art.

[0122] The term "consisting of" refers to the compositions, methods, processes, and their respective components described herein, excluding any elements not enumerated in the description of the embodiments.

[0123] As used herein, the term “essentially derived from” refers to elements necessary for a given embodiment. The term permits the presence of additional elements that do not substantially affect the basic and novel or functional features of that embodiment of the disclosure.

[0124] As used herein, terms such as “etc.” and “for example” are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Methods and materials similar to or equivalent to those described herein may be used in the conduct of the tests of this disclosure, but preferred materials and methods are described herein.

[0126] As used herein, the terms “administer,” “to administer,” and variations thereof refer to the introduction of a composition or drug (e.g., nucleic acids, particularly ceDNA) into a subject, and include the simultaneous and sequential introduction of one or more compositions or drugs. “Administer” may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administer” also encompasses in vitro and ex vivo treatments. Introduction of a composition or drug into a subject may be by any preferred route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration may include self-administration and administration by another person. Administration may be carried out by any preferred route. A preferred route of administration allows the composition or drug to perform its intended function. For example, if the preferred route is intravenous, the composition is administered by introducing the composition or drug into the vein of the subject.

[0127] As used herein, phrases such as “anti-therapeutic nucleic acid immune response,” “anti-transfer vector immune response,” “immune response to therapeutic nucleic acid,” and “immune response to transfer vector” refer to any undesirable immune response to therapeutic nucleic acid, whether of viral or nonviral origin. In some embodiments, the undesirable immune response is an antigen-specific immune response to the viral transfer vector itself. In some embodiments, the immune response is specific to the transfer vector, which may be double-stranded DNA, single-stranded RNA, or double-stranded RNA. In other embodiments, the immune response is specific to the sequence of the transfer vector. In other embodiments, the immune response is specific to the CpG content of the transfer vector.

[0128] As used herein, the term "aqueous solution" means a composition that contains water in whole or in part.

[0129] As used herein, “bases” include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0130] As used herein, the term “carrier” includes any and all solvents, dispersions, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase “pharmaceutically acceptable” means molecular entities and compositions that, when administered to a host, do not produce a toxic, allergic, or similarly undesirable reaction.

[0131] As used herein, the term “ceDNA” means capsid-free closed-end linear double-stranded (ds) double-stranded DNA for synthetic or other nonviral gene delivery. According to some embodiments, ceDNA is closed-end linear double-stranded (CELiD) CELiD DNA. According to some embodiments, ceDNA is a DNA-based minicircle. According to some embodiments, ceDNA is a minimal immunologically defined gene expression (MIDGE) vector. According to some embodiments, ceDNA is ministering DNA. According to some embodiments, ceDNA is dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITR at the 5' and 3' ends of the expression cassette. According to some embodiments, ceDNA is doggybone® DNA. A detailed description of ceDNA is found in International Patent Application No. PCT / US2017 / 020828, filed on 3 March 2017, which is expressly incorporated herein by reference. Certain methods for producing ceDNA containing various inverted end repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application PCT / US18 / 49996, filed on 7 September 2018, and PCT / US2018 / 064242, filed on 6 December 2018, each of which is incorporated herein by whole reference. Certain methods for producing synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Patent Application PCT / US2019 / 14122, filed on 18 January 2019, which is incorporated herein by whole reference.

[0132] As used herein, the term “closed-end DNA vector” refers to a capsid-free DNA vector having at least one covalent closed end and having at least a portion of the vector having an intramolecular double-strand structure.

[0133] As used herein, the terms “ceDNA vector” and “ceDNA” are interchangeable and refer to a closed-end DNA vector containing at least one terminal palindrom. In some embodiments, the ceDNA contains two covalent closed ends.

[0134] As used herein, the term “ceDNA-bacmid” means an infectious baculovirus genome that contains a ceDNA genome as an intermolecular double helix that can be propagated as a plasmid in E. coli, thereby enabling it to function as a baculovirus shuttle vector.

[0135] As used herein, the term "ceDNA-baculovirus" means a baculovirus that contains the ceDNA genome as an intermolecular double helix within the baculovirus genome.

[0136] As used herein, the terms “ceDNA-baculovirus-infected insect cells” and “ceDNA-BIIC” are interchangeable and mean invertebrate host cells (including, but not limited to, insect cells (e.g., Sf9 cells)) infected with ceDNA-baculovirus.

[0137] As used herein, the term “ceDNA genome” means an expression cassette further incorporating at least one inverted end repeat (ITR) region. The ceDNA genome may further include one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0138] As used herein, the terms “DNA regulatory sequence,” “regulatory element,” and “regulatory element” are interchangeable herein and mean transcriptional and translational regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., which provide and / or regulate the transcription of non-coding sequences (e.g., DNA-targeted RNA) or coding sequences (e.g., site-directed modified polypeptides or Cas9 / Csn1 polypeptides) and / or regulate the translation of encoded polypeptides.

[0139] As used herein, the terms “rigid therapeutic nucleic acid,” “rigid TNA,” or “rTNA” refer to therapeutic nucleic acids as defined herein that have a compact structure or are in a compact state as a result of a process during the preparation of an LNP composition containing rTNA as described herein. In one embodiment, the preparation comprises an LMW alcohol-based process by which rTNA and lipids are mixed in an LMW alcohol solution, the LMW alcohol mixture containing rTNA and lipids is introduced into a microfluidic synthesis system (e.g., NanoAssemblr) via one channel, and an aqueous buffer is introduced into the system via another channel to produce an LNP composition encapsulating rTNA. As used herein, the terms “terminal repeat” or “TR” include any viral or nonviral terminal sequence or synthetic sequence including at least one minimally required replication origin and a region containing a palindromic hairpin structure. Rep-binding sequences (also called "RBS" or Rep-binding elements (RBEs)) and terminal degradation sites ("TRS") together constitute the "minimum necessary replication origin" of AAV, and therefore, a TR contains at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs," respectively. In the context of viruses, ITRs play a crucial role in mediating replication, packaging of viral particles and DNA, incorporation of DNA, and rescue of the genome and provirus. TRs that are not reverse complements (palindromes) over their entire length can still perform the conventional function of an ITR, and therefore, the term ITR is used to refer to TRs in viral or nonviral AAV vectors that can mediate replication within host cells. It will be understood by those skilled in the art that there may be three or more ITRs or asymmetric ITR pairs in a composite AAV vector configuration.

[0140] An "ITR" can be artificially synthesized using a set of oligonucleotides containing one or more desirable functional sequences (e.g., palindromic sequences, RBS). The ITR sequence may be an AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., an ITR fragment removed from a viral genome). For example, an ITR may be derived from the Parvoviridae family, which includes parvoviruses and dependviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or an SV40 hairpin, which serves as the origin of SV40 replication, may be used as an ITR, which can be further modified by cleavage, substitution, deletion, insertion, and / or addition. Parvoviridae viruses consist of two subfamilies: the Parvovirinae, which infects vertebrates, and the Densovirinae, which infects invertebrates. Dependent parvoviruses include a family of adeno-associated viruses (AAVs) capable of replicating in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep. Typically, ITR sequences can originate not only from AAVs but also from parvoviruses, lentiviruses, goose viruses, and B19, in wild-type, "doggy bone," and "dumbbell-shaped," symmetric or asymmetric ITR orientation configurations. While ITRs are typically present at both the 5' and 3' ends of AAV vectors, they can be present at only one end of a linear vector. For example, an ITR may be present only at the 5' end. In some other cases, an ITR may be present only at the 3' end of a synthetic AAV vector. For convenience, in this specification, ITRs located 5' (upstream) of the expression cassette in a synthetic AAV vector are referred to as "5'ITR" or "left ITR," and ITRs located 3' (downstream) of the expression cassette in the vector or synthetic AAV are referred to as "3'ITR" or "right ITR."

[0141] "Wild-type ITR" or "WT-ITR" refers to a sequence of a naturally occurring ITR sequence in the AAV genome or other dependent viruses that maintains, for example, Rep-binding activity and Rep-nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring canonical sequence due to genetic coding or drift degeneracy; therefore, WT-ITR sequences incorporated herein include WT-ITR sequences resulting from naturally occurring variations (e.g., replication errors).

[0142] As used herein, the terms “substantially symmetric WT-ITR” or “substantially symmetric WT-ITR pair” refer to a pair of WT-ITRs in a synthetic AAV vector that are both wild-type ITRs having inversely complementary sequences over their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, as long as the changes do not affect the physical and functional properties of the sequence as well as its overall three-dimensional structure (two-dimensional and three-dimensional structure). In some embodiments, the deviating nucleotides represent a conserved sequence change. As a non-limiting example, a sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the canonical sequence (e.g., measured using BLAST with default settings) and has a three-dimensional spatial configuration that is symmetric with respect to other WT-ITRs such that their three-dimensional structures have the same shape in geometric space. Substantially symmetric WT-ITRs have the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetrical WT-ITR can be functionally confirmed as WT by determining that it possesses a manipulable Rep-binding site (RBE or RBE') and terminal degradation sites (trs) that pair with the appropriate Rep protein. Optionally, other functions, including transgene expression under tolerable conditions, can be tested.

[0143] As used herein, the terms “modified ITR,” “mod-ITR,” or “mutant ITR” are interchangeable and refer to an ITR having mutations in at least one nucleotide compared to a WT-ITR from the same serotype. Mutations may result in changes to one or more of the A, C, C', B, and B' regions of the ITR compared to the three-dimensional spatial configuration of a WT-ITR from the same serotype, and may result in changes to the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space).

[0144] As used herein, the term “asymmetric ITR,” also referred to as “asymmetric ITR pair,” refers to a pair of ITRs in a single-stranded synthetic AAV genome that are not inversely complementary over their entire length. As a non-limiting example, an asymmetric ITR pair does not have a three-dimensional spatial configuration symmetric to its congener ITRs, such that their three-dimensional structures have different shapes in geometric space. In other words, an asymmetric ITR pair has a different overall geometric structure, i.e., a different configuration of their A, C-C', and B-B' loops in three-dimensional space (for example, one ITR may have a shorter C-C' arm and / or a shorter B-B' arm compared to a congener ITR). Sequence differences between two ITRs may result from one or more nucleotide additions, deletions, cleavages, or point mutations. In one embodiment, one ITR in an asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR as defined herein (e.g., a non-wild-type or synthetic ITR sequence). In another embodiment, neither ITR in an asymmetric ITR pair is a wild-type AAV array, and the two ITRs are modified ITRs having different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR in an asymmetric ITR pair may have a short C-C' arm, and the other ITR may have a different modification (e.g., a single arm, or a short B-B' arm) so that they have a different three-dimensional spatial configuration compared to a congeneral asymmetric mod-ITR.

[0145] As used herein, the term “symmetric ITR” refers to a pair of ITRs in a single-stranded AAV genome that are wild-type or mutant (e.g., modified relative to wild-type) dependent virus ITR sequences and are reverse-complementary over their entire length. In one non-limiting example, both ITRs are wild-type ITR sequences from AAV2. In another example, neither ITR is a wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and their sequences may differ from wild-type ITRs due to nucleotide additions, deletions, substitutions, cleavage, or point mutations. For convenience, in this specification, an ITR located 5' (upstream) of the expression cassette in a synthetic AAV vector is referred to as a “5' ITR” or “left ITR,” and an ITR located 3' (downstream) of the expression cassette in a synthetic AAV vector is referred to as a “3' ITR” or “right ITR.”

[0146] As used herein, the terms “substantially symmetric modified ITR” or “substantially symmetric mod-ITR pair” refer to a pair of modified ITRs in a synthetic AAV, both having reverse complementary sequences over their entire length. For example, a modified ITR can be considered substantially symmetric even if there are some nucleotide sequences that deviate from the reverse complementary sequence, as long as the changes do not affect the properties and overall shape. As a non-limiting example, sequences have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) with respect to the canonical sequence, and have a three-dimensional spatial configuration that is symmetric with respect to their congenerated modified ITRs, such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops configured in three-dimensional space. In some embodiments, ITRs from a mod-ITR pair may have different reverse complementary nucleotide sequences, but may still have the same symmetric three-dimensional spatial configuration. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR in a mod-ITR pair (e.g., 5'ITR) may originate from one serotype, and the other ITR (e.g., 3'ITR) may originate from a different serotype, but both may have the same corresponding mutations (e.g., if the 5'ITR has a deletion in the C region, the modified 3'ITR of the same family from the different serotype will have a deletion at the corresponding position in the C' region), thereby the modified ITR pair having the same symmetrical three-dimensional spatial configuration. In such embodiments, each ITR in a modified ITR pair may originate from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and the modification of one ITR is reflected at the corresponding position in the ITR of the same family from the different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) insofar as differences in nucleotide sequences between the ITRs do not affect their properties or overall shape, and they have substantially the same shape in three-dimensional space.As a non-limiting example, mod-ITRs have at least 95%, 96%, 97%, 98%, or 99% alignment identity with a canonical mod-ITR, determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and have a symmetric three-dimensional spatial configuration such that their three-dimensional structures have the same shape in geometric space. Substantially symmetric mod-ITR pairs have the same A, C-C', and B-B' loops in three-dimensional space. For example, if a modified ITR of a substantially symmetric mod-ITR pair has a C-C' arm deletion, then a related mod-ITR has a corresponding C-C' loop deletion and a similar three-dimensional structure of the remaining A and B-B' loops with the same shape in geometric space.

[0147] As used herein, the terms “effective dose” or “therapeutic effective dose” of an activator or therapeutic agent, such as a therapeutic nucleic acid, mean an amount sufficient to produce a desired effect, e.g., inhibition of the expression of a target sequence, compared to the expression level detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include protein or RNA level tests using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0148] As used herein, the term “expression” means cellular processes involved in the production of RNA and proteins, and, as appropriate, secreted proteins, including but not limited to, transcription, transcriptional processing, translation, and protein folding, modification, and processing. As used herein, the term “expression product” includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0149] As used herein, the term “expression vector” means a vector that directs the expression of RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often heterologous to the host cell, but not necessarily. An expression vector may contain additional elements; for example, an expression vector may have two replication systems so that it can be maintained in two organisms, such as human cells in the case of expression and a prokaryotic host in the case of cloning and amplification. An expression vector may also be a recombinant vector.

[0150] As used herein, the term “adjacent” refers to the relative position of one nucleic acid sequence to another nucleic acid sequence. Generally, in sequence ABC, A and C are adjacent to B on both sides. The same is true for arrangement A×B×C. Therefore, an adjacent sequence may precede or follow the adjacent sequence, but it does not need to be continuous with or immediately next to the adjacent sequence.

[0151] As used herein, the term “spacer region” means an intervening sequence that separates functional elements within a vector or genome. In some embodiments, a spacer region holds two functional elements at a desired distance for optimal functionality. In some embodiments, a spacer region provides or increases the genetic stability of the vector or genome. In some embodiments, a spacer region facilitates easy genetic manipulation of the genome by providing a convenient location for cloning sites and gaps in base pair design numbers.

[0152] As used herein, the terms “expression cassette” and “expression unit” mean a heterologous DNA sequence that is interchangeable and manipulably ligated to a promoter or other DNA regulatory sequence sufficient to direct the transcription of a transgene in a DNA vector, such as a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. Promoters may also be of AAV origin.

[0153] As used herein, the terms “hereditary disorder” or “hereditary disorder” mean a disorder caused, directly or indirectly, partially or completely, by one or more abnormalities in the genome, particularly a condition present from birth. The abnormality may be a mutation, insertion, or deletion in a gene. The abnormality may affect the coding sequence or its regulatory sequence of a gene.

[0154] As used herein, the term “lipid” refers to a group of organic compounds including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three classes: (1) “simple lipids” including fats, oils, and waxes; (2) “complex lipids” including phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0155] Typical examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other phosphorus-deficient compounds, such as sphingolipids, the sphingoglycolipid family, diacylglycerols, and β-acyloxy acids, are also included in the group called amphiphilic lipids. In addition, the above amphiphilic lipids can be mixed with other lipids, including triglycerides and sterols.

[0156] In one embodiment, the lipid composition comprises one or more tertiary amino groups, one or more phenyl ester bonds, and a disulfide bond.

[0157] As used herein, the term “lipid conjugate” means a conjugated lipid that inhibits the aggregation of lipid particles (e.g., lipid nanoparticles). Such lipid conjugates include, but are not limited to, PEG-lipid complexes such as PEG conjugated with dialkyloxypropyl (e.g., PEG-DAA complex), PEG conjugated with diacylglycerol (e.g., PEG-DAG complex), PEG conjugated with cholesterol, PEG conjugated with phosphatidylethanolamine, and PEG conjugated with ceramide (see, for example, U.S. Patent No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid complexes (e.g., POZ-DAA complex, e.g., U.S. Provisional Application No. 61 / 294,828 filed January 13, 2010, and U.S. Provisional Application No. 61 / 295,140 filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid complexes), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in International Patent Application Publication No. 2010 / 006282. PEG or POZ can be directly conjugated to lipids or conjugated to lipids via a linker moiety. For example, any linker moiety suitable for conjugating PEG or POZ to lipids can be used, including non-ester-containing and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties such as amides or carbamates are used. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0158] As used herein, the term “lipid-encapsulated” means lipid particles that provide an activator or therapeutic agent, such as a nucleic acid (e.g., ceDNA), in a fully encapsulated, partially encapsulated, or both manner. In preferred embodiments, the nucleic acid is fully encapsulated within the lipid particle (e.g., to form a lipid particle containing the nucleic acid).

[0159] As used herein, the terms “lipid particles” or “lipid nanoparticles” refer to lipid formulations that can be used to deliver therapeutic agents, such as nucleic acid therapeutics, to target sites of interest (e.g., cells, tissues, organs, etc.). In one embodiment, the lipid particles of the Disclosure are nucleic acid-containing lipid particles, which are typically formed from cationic lipids, non-cationic lipids, and optionally conjugated lipids to prevent particle aggregation. In other preferred embodiments, therapeutic agents, such as therapeutic nucleic acids, can be encapsulated in the lipid portion of the particles, thereby protecting them from enzymatic degradation. In one embodiment, the lipid particles comprise nucleic acids (e.g., ceDNA) and lipids containing one or more tertiary amino groups, one or more phenyl ester bonds, and disulfide bonds.

[0160] According to several embodiments, the lipid particles of the Disclosure are typically approximately 20 nm to approximately 75 nm, approximately 20 nm to approximately 70 nm, approximately 25 nm to approximately 75 nm, approximately 25 nm to approximately 70 nm, approximately 30 nm to approximately 75 nm, approximately 30 nm to approximately 70 nm, approximately 35 nm to approximately 75 nm, approximately 35 nm to approximately 70 nm, approximately 35 nm to approximately 70 nm, approximately 40 nm to approximately 75 nm, approximately 40 nm to approximately 70 nm, approximately 45 nm to approximately 75 nm, approximately 50 nm to approximately 75 nm, approximately 50 nm to approximately 70 nm, approximately 60 nm to approximately 75 nm, approximately 60 nm to approximately 70 nm, approximately 65 nm to approximately 75 nm, and approximately 6 It has an average diameter of approximately 5nm to 70nm, or approximately 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm, 60nm, 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm, or approximately 75nm (±3nm).

[0161] Generally, the lipid particles (e.g., lipid nanoparticles) of this disclosure have an average diameter selected to provide the intended therapeutic effect.

[0162] According to some embodiments, the lipid particles of the present disclosure typically have an average diameter of 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, and less than about 20 nm.

[0163] As used herein, the term “cationic lipid” refers to any lipid that is positively charged at physiological pH. Cationic lipids in lipid particles may include one or more cationic lipids such as, for example, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), “SS-cleavable lipids,” or mixtures thereof. In some embodiments, cationic lipids are also ionized lipids, i.e., ionized cationic lipids. All cationic lipids described herein are considered to have corresponding quaternary lipids (i.e., those in which the nitrogen atom of the cationic moiety is protonated and has four substituents) within the scope of this disclosure. Any cationic lipid described herein can be converted to the corresponding quaternary lipid, for example, by treatment with acetonitrile (CH3CN) and chloromethane (CH3Cl) in chloroform (CHCl3).

[0164] As used herein, the term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups bound to neutral lipids.

[0165] As used herein, the term "hydrophobic lipid" refers to a compound having nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Preferred examples include, but are not limited to, diacylglycerol, dialkylglycerol, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0166] As used herein, the term “ionized lipid” means a lipid having at least one protonable or deprotonable group such that the lipid is positively charged below the physiological pH (e.g., pH 7.4) and neutral above a second pH, preferably above the physiological pH, such as a cationic lipid. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that references to charged or neutral lipids refer to the properties of the dominant species, and that not all lipids need to exist in charged or neutral forms. Generally, ionized lipids have a pKa of protonable groups in the range of about 4 to about 7. In some embodiments, ionized lipids may include “cleavable lipids” or “SS-cleavable lipids.”

[0167] As used herein, the term “neutral lipid” means any of the many lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

[0168] As used herein, the term “noncationic lipid” means any amphiphilic lipid and any other neutral or anionic lipid.

[0169] As used herein, the terms “cleavable lipid” or “SS-cleavable lipid” refer to lipids containing cleavable units of disulfide bonds. Cleavable lipids may include cleavable disulfide bonds ("ss") containing lipid-like substances, including pH-sensitive tertiary amines and autodegradable phenyl esters. For example, SS-cleavable lipids may be ss-OP lipids (COATSOME® SS-OP), ss-M lipids (COATSOME® SS-M), ss-E lipids (COATSOME® SS-E), ss-EC lipids (COATSOME® SS-EC), ss-LC lipids (COATSOME® SS-LC), ss-OC lipids (COATSOME® SS-OC), and ss-PalmE lipids (see formulas I-IV, e.g.), or lipids described by Togashi et al., (2018) Journal of Controlled Release, "A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug," 279:262-270. Additional examples of cleavable lipids are described in U.S. Patents 9,708,628 and 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, the cleavable lipid comprises a tertiary amine that responds to disulfide bonds that can be cleaved in reducing environments such as acidic compartments, e.g., endosomes or lysosomes for membrane destabilization, and the cytoplasm. In one embodiment, the cleavable lipid is a cationic lipid. In one embodiment, the cleavable lipid is an ionized cationic lipid. Cleavable lipids are described in more detail herein.

[0170] As used herein, the term "organic lipid solution" means a composition containing an organic solvent that is entirely or partially lipid-containing.

[0171] As used herein, the term “liposome” refers to a lipid molecule assembled in a spherical structure that encapsulates an internal aqueous volume separated from an aqueous external environment. A liposome is a vesicle having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of formulation development. They act by fusing with the cell membrane and repositioning its lipid structure to deliver the drug or active formulation component. Liposome compositions for such delivery are typically composed of phospholipids, particularly compounds having a phosphatidylcholine group, although these compositions may also contain other lipids.

[0172] As used herein, the term “local delivery” means the direct delivery of an active agent, such as interfering RNA (e.g., siRNA), to a target site within a living organism. For example, a drug can be delivered locally by direct injection to a disease site such as a tumor, or another target site such as an inflammatory site, or to a target organ such as the liver, heart, pancreas, or kidney.

[0173] As used herein, the term “nucleic acid” means a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be, for example, in the form of antisense molecules, plasmid DNA, DNA-DNA double helix, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-end linear double-strand DNA (CELiD or ceDNA), doggybone® DNA, dumbbell DNA, minimal immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include those containing known nucleotide analogs or modified backbone residues or ligatures, which are synthetic, naturally occurring, and non-natural, and possess similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholino), phosphoramides, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, locked nucleic acids (LNA®), and peptide nucleic acids (PNA). Unless otherwise limited, this term encompasses nucleic acids containing known analogs of naturally occurring nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise specified, any given nucleic acid sequence implicitly includes its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as any explicitly indicated sequences.

[0174] As used herein, the terms “nucleic acid therapy,” “therapeutic nucleic acid,” and “TNA” are interchangeable and refer to any modality of therapy that uses nucleic acid as the active ingredient of a therapeutic agent for treating a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-exclusive examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-exclusive examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, DOGGYBONE® DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped minimal DNA vectors ("dumbbell DNA").

[0175] As used herein, “nucleotide” comprises a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups.

[0176] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil, and various types of wetting agents. The term also includes drugs approved by U.S. federal regulatory authorities or listed in the United States Pharmacopeia for use in animals, including humans, as well as carriers or diluents that do not cause significant irritation to the subject and do not impair the biological activity and properties of the administered compound.

[0177] As used herein, the term “gap” refers to a broken portion of the synthetic DNA vector of this disclosure, creating a stretch of single-stranded DNA portion in the remaining double-stranded ceDNA. The gap may be a single strand of the double-stranded DNA ranging in length from 1 to 100 base pairs. Typical gaps designed and created by the methods described herein, and synthetic vectors produced by such methods, may have lengths of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 bp. Exemplary gaps in this disclosure may have lengths of 1 bp to 10 bp, 1 to 20 bp, or 1 to 30 bp.

[0178] As used herein, the term “nick” typically refers to a discontinuity in a double-stranded DNA molecule where there is no phosphodiester bond between adjacent single-stranded nucleotides, typically due to damage or enzymatic action. It is understood that one or more nicks allow for the unwinding of strand twists during DNA replication, and nicks are also thought to play a role in facilitating the binding of transcription mechanisms.

[0179] As used herein, the term “Subject” means a human or animal to which a treatment, including prophylactic treatment with therapeutic nucleic acids according to this disclosure, is provided. Typically, animals are vertebrates such as primates, rodents, domesticated animals, or game animals, but are not limited to these. Examples of primates include, but are not limited to, chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, ostriches, and fish such as trout, catfish, and salmon. In certain embodiments of the forms described herein, the subject is a mammal, such as a primate or a human. The subject may be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a neonatal or fetal subject, for example, the subject is in the womb. Preferably, the subject is a mammal. Mammals may be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be advantageously used as subjects representing animal models of diseases and disorders. In addition, the methods and compositions described herein may be used in domesticated animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, for example, Caucasian (white), Asian, African, Black, African American, Afro-European, Latin American, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already receiving treatment. In some embodiments, the subject is an embryo, fetus, neonatal, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonatal, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.

[0180] As used herein, the phrase “Subjects requiring treatment” means, unless the context and usage of the phrase indicate otherwise, (i) subjects who are to be administered ceDNA lipid particles (or a pharmaceutical composition containing ceDNA lipid particles) in accordance with the disclosed herein, (ii) subjects who have received ceDNA lipid particles (or a pharmaceutical composition containing ceDNA lipid particles) in accordance with the disclosed herein, or (iii) subjects who have received ceDNA lipid particles (or a pharmaceutical composition containing ceDNA lipid particles) in accordance with the disclosed herein.

[0181] As used herein, the terms “suppress,” “reduce,” “interfere,” “inhibit,” and / or “reduce” (and similar terms) generally refer to the act of directly or indirectly reducing concentration, level, function, activity, or behavior to natural, expected, or average, or to control conditions.

[0182] As used herein, the term “systemic delivery” refers to the delivery of lipid particles that result in widespread biodistribution of an active agent, such as interfering RNA (e.g., siRNA), within a living organism. Depending on the administration technique, systemic delivery of a particular drug may or may not be achieved. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the drug is exposed to most parts of the body. To achieve widespread biodistribution, the drug generally requires a blood lifetime such that it is not rapidly degraded or eliminated (by first-pass organs (liver, lungs, etc.) or by rapid nonspecific cell binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles (e.g., lipid nanoparticles) can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal delivery. In preferred embodiments, systemic delivery of lipid particles (e.g., lipid nanoparticles) is by intravenous delivery.

[0183] Where used herein, the terms “therapeutic dose,” “therapeutic effective dose,” “effective dose,” or “pharmaceutical effective dose” of an activator (e.g., ceDNA lipid particles as described herein) are interchangeable and refer to an amount sufficient to provide the intended therapeutic benefit. However, dose levels are based on a variety of factors, including the type of injury, age, weight, sex, the patient’s condition, the severity of the condition, the route of administration, and the specific activator used. Therefore, the dosage plan may vary considerably but can be routinely determined by a physician using standard methods. Additionally, the terms “therapeutic dose,” “therapeutic effective dose,” and “pharmaceutical effective dose” include prophylactic or preventative doses of the compositions described herein. In the prophylactic or preventative uses described herein, the pharmaceutical composition or agent is administered in an amount sufficient to eliminate or reduce risk, reduce severity, or delay the onset of the disease, disorder, or condition, including the biochemical, histological and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes that appear during the onset of the disease, disorder, or condition, to patients who are susceptible to the disease, disorder, or condition, or otherwise at risk thereof. According to some medical judgment, it is generally preferable to use the maximum dose, i.e., the safest dose. The terms “dose” and “administered dose” are used interchangeably herein.

[0184] As used herein, the term “therapeutic effect” refers to the outcome of treatment, which is deemed desirable and beneficial. A therapeutic effect may include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. A therapeutic effect may also include, directly or indirectly, the prevention, reduction, or elimination of disease progression.

[0185] For any therapeutic agent described herein, the effective therapeutic dose can first be determined from preliminary in vitro studies and / or animal models. The effective therapeutic dose can also be determined from human data. The dose applied can be adjusted based on the relative bioavailability and potency of the compound administered. Adjusting the dose to achieve maximum potency based on the above methods and other well-known methods is within the capabilities of those skilled in the art. General principles for determining therapeutic efficacy, as can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th Edition, McGraw-Hill (New York) (2001), which are incorporated herein by reference, are summarized below.

[0186] Pharmacokinetic principles provide a basis for modifying dosing regimens to achieve the desired level of therapeutic effect while minimizing unacceptable side effects. Plasma drug concentrations can be measured, and in situations related to therapeutic concentration ranges, additional guidance on dosage adjustments can be obtained.

[0187] As used herein, the terms “to treat,” “to treat,” and / or “treatment” include, to substantially inhibit, delay, or reverse the progression of a condition, substantially improve the clinical symptoms of a condition, or substantially prevent the appearance of the clinical symptoms of a condition, or to obtain a beneficial or desirable clinical outcome. To treat further means to achieve one or more of the following: (a) reducing the severity of the disability; (b) limiting the onset of symptoms characteristic of the disability being treated; (c) limiting the exacerbation of symptoms characteristic of the disability being treated; (d) limiting the recurrence of the disability in patients who previously had the disability; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic with respect to the disability.

[0188] Beneficial or desired clinical outcomes, such as pharmacological and / or physiological effects, include, but are not limited to, preventing the onset of a disease, disorder, or condition (preventive treatment) in subjects who may be predisposed to the disease, disorder, or condition but have not yet experienced or exhibited symptoms of the disease; alleviating symptoms of the disease, disorder, or condition; reducing the severity of the disease, disorder, or condition; stabilizing the disease, disorder, or condition (i.e., preventing exacerbation); preventing the spread of the disease, disorder, or condition; slowing or delaying the progression of the disease, disorder, or condition; improving or reducing the disease, disorder, or condition; and combinations thereof; as well as extending survival compared to the survival expected without treatment.

[0189] Beneficial or desired clinical outcomes, such as pharmacological and / or physiological effects, include, but are not limited to, preventing the onset of a disease, disorder, or condition (preventive treatment) in subjects who may be predisposed to the disease, disorder, or condition but have not yet experienced or exhibited symptoms of the disease; alleviating symptoms of the disease, disorder, or condition; reducing the severity of the disease, disorder, or condition; stabilizing the disease, disorder, or condition (i.e., preventing exacerbation); preventing the spread of the disease, disorder, or condition; slowing or delaying the progression of the disease, disorder, or condition; improving or reducing the disease, disorder, or condition; and combinations thereof; as well as extending survival compared to the survival expected without treatment.

[0190] As used herein, the term "alkyl" refers to a saturated monovalent hydrocarbon radical (i.e., C) with 1 to 20 carbon atoms. 1~20 This refers to alkyl groups. "Monovalent" means that the alkyl group has one bonding site on the rest of the molecule. In one embodiment, alkyl groups have 1 to 12 carbon atoms (i.e., C 1~12 Alkyl) or 1 to 10 carbon atoms (i.e., C 1~10 It has alkyl. In one embodiment, the alkyl has 1 to 8 carbon atoms (i.e., C 1~8 Alkyl), 1 to 7 carbon atoms (i.e., C 1~7 Alkyl), 1 to 6 carbon atoms (i.e., C1~6 Alkyl), 1 to 4 carbon atoms (i.e., C 1~4 Alkyl) or 1 to 3 carbon atoms (i.e., C 1~3 It has alkyl groups. Examples include, but is not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, etc. "Linear or branched chain C 1~6 "Alkyl", "Straight-chain or branched-chain C" 1~4 "Alkyl" or "linear or branched C" 1~3 The terms "alkyl" and other linear or branched alkyl groups indicate that the saturated monovalent hydrocarbon radical is linear or branched.

[0191] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon radical (i.e., C) comprising 1 to 20 carbon atoms. 1~20 This refers to alkylenes, and examples include, but are not limited to, those having the same core structure as the alkyl group exemplified above. "Divalent" means that the alkylene has two bonding sites in the rest of the molecule. In one embodiment, the alkylene has 1 to 12 carbon atoms (i.e., C 1~12 Alkylene), or 1 to 10 carbon atoms (i.e., C 1~10 It has an alkylene. In one embodiment, the alkylene has 1 to 8 carbon atoms (i.e., C 1~8 Alkylene), 1 to 7 carbon atoms (i.e., C 1~7 Alkylene), 1 to 6 carbon atoms (i.e., C 1~6 Alkylene), 1 to 4 carbon atoms (i.e., C 1~4 Alkylene), or 1 to 3 carbon atoms (i.e., C1~3 It contains alkylene, and is ethylene or methylene. "Straight-chain or branched-chain C 1~6 "Alkylene", "Straight-chain or branched-chain C 1~4 "Alkylene," or "straight-chain or branched-chain C 1~3 The terms "alkylene," "linear," and "branched-chain alkylene" refer to saturated divalent hydrocarbon radicals that are linear or branched.

[0192] The term "alkenyl" refers to a linear or branched aliphatic hydrocarbon group having one or more (e.g., one or two) carbon-carbon double bonds, and alkenyl groups include groups having "cis" and "trans" orientations, or, according to other nomenclature, "E" and "Z" orientations.

[0193] As used herein, "alkenylene" refers to an aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms having one or two carbon-carbon double bonds (i.e., C 2~20 Alkenylene refers to an alkenylene radical, where the alkenylene radical includes radicals having "cis" and "trans" orientations or, in other nomenclature, "E" and "Z" orientations. "Divalent" means that the alkylene has two bonding sites in the rest of the molecule. In one embodiment, the alkenylene has 2 to 12 carbon atoms (i.e., C 2~16 Alkenylene), or 2 to 10 carbon atoms (i.e., C 2~10 It has an alkenylene. In one embodiment, the alkenylene has 2 to 4 carbon atoms (C 2~4 It has ) . Examples include ethyleneylene or vinylene (-CH=CH-), allyl (-CH2CH=CH-), etc., but is not limited to these. "Straight-chain or branched-chain C 2~6 Alkenylene, "Straight-chain or branched-chain C 2~4 "Alkenylene" or "Straight-chain or branched-chain C 2~3 The term "alkenylene," as in "alkenylene," refers to a linear or branched alkenylene, meaning that the unsaturated divalent hydrocarbon radical is linear or branched.

[0194] As used herein, "cycloalkylene" refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. "Divalent" means that the cycloalkylene has two bonding sites in the rest of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or a 3- to 6-membered monocyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, and cyclododecylene. In one embodiment, the cycloalkylene is cyclopropylene.

[0195] The terms “heterocyclic,” “heterocyclyl,” “heterocyclic formula,” and “heterocyclic ring” are used interchangeably herein and refer to a non-aromatic (i.e., partially or completely saturated) cyclic group containing at least one N atom, a heteroatom, and optionally 1 to 3 additional heteroatoms selected from N and S. It can be monocyclic or bicyclic (bridged or fused). Examples of heterocyclic rings include, but are not limited to, azilidinyl, diazilidinyl, thiazilidinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, etc. The heterocyclic ring contains 1 to 4 heteroatoms, which are selected from N and S and may be the same or different. In one embodiment, the heterocycle contains 1 to 3 nitrogen atoms. In another embodiment, the heterocycle contains 1 or 2 nitrogen atoms. In yet another embodiment, the heterocycle contains 1 nitrogen atom. "4- to 8-membered heterocyclyl" means a radical having 4 to 8 atoms arranged in a monocyclic ring (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 nitrogen atoms, or 1 or 2 nitrogen atoms, or 1 nitrogen atom). "5- or 6-membered heterocyclyl" means a radical having 5 or 6 atoms arranged in a monocyclic ring (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 nitrogen atoms, or 1 or 2 nitrogen atoms, or 1 nitrogen atom). The term "heterocycle" is intended to include all possible isomer forms.Heterocyclic compounds are described in Leo A., Principles of Modern Heterocyclic Chemistry (WABenjamin, New York, 1968), particularly chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), particularly volumes 13, 14, 16, 19, and 28; and J.Am.Chem.Soc. (1960) 82:5566. The heterocyclyl group may, if possible, be a carbon (carbon bond) or nitrogen (nitrogen bond) bonded to the rest of the molecule.

[0196] If a group is described as "arbitrarily substituted," it may be (1) unsubstituted or (2) substituted. If a carbon atom of a group is described as being arbitrarily substituted with one or more substituents from the list, one or more hydrogen atoms on the carbon (to the extent they exist) may be replaced separately and / or together with any substituents of independent choice.

[0197] Suitable substituents for alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl groups are those that do not significantly adversely affect the biological activity of the bifunctional compound. Unless otherwise specified, exemplary substituents for these groups include linear, branched, or cyclic alkyl, alkenyl, or alkynyl groups having 1 to 10 carbon atoms; aryl; heteroaryl; heterocyclyl; halogen; guanidinium [-NH(C=NH)NH2]; -OR 100 ;NR 101 R 102 ;-NO2;-NR 101 COR 102 ;-SR 100 ;-SOR 101 Sulfoxide represented by -SO2R 101 Sulfones, sulfonates, -SO3M; sulfates, -SO3M; -SO2NR are represented by these terms. 101 R 102Sulfonamide; cyano; azide; -COR 101 ;-OCOR 101 ;-OCONR 101 R 102 and polyethylene glycol units (-OCH2CH2) n R 101 These are examples, where M is H or a cation (Na + or K + etc., R 101 , R 102 and R 103 Each of these independently comprises: H; a linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms; and a polyethylene glycol unit (-OCH2CH2). n -R 104 (where n is an integer from 1 to 24); selected from aryl rings having 6 to 10 carbon atoms; heterocyclic rings having 3 to 10 carbon atoms; and heteroaryl rings having 5 to 10 carbon atoms, R 104 R is a linear or branched alkyl group having H or 1 to 4 carbon atoms, 100 , R 101 , R 102 , R 103 and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl groups represented by are optionally substituted with one or more substituents (e.g., 2, 3, 4, 5, 6 or more) independently selected from halogens, -OH, -CN, -NO2, and unsubstituted linear or branched alkyl groups having 1 to 4 carbon atoms. Preferably, the substituents for the above optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl groups are halogens, -CN, -NR 101 R 102 -CF3, -OR 100 aryl, heteroaryl, heterocyclyl, -SR 101 -SOR 101 , -SO2R 101 Selected from the group consisting of , and -SO3M. Alternatively, preferred substituents are halogen, -OH, -NO2, -CN, C 1~4 Alkyl, -OR 100, NR 101 R 102 , -NR 101 COR 102 , -SR 100 , -SO2R 101 -SO2NR 101 R 102 , -COR 101 , -OCOR 101 , and -OCONR 101 R 102 Selected from the group consisting of R 100 , R 101 and R 102 Each is independently -H or C 1~4 It is alkyl.

[0198] As used herein, "halogen" refers to F, Cl, Br, or I. "Cyano" refers to -CN.

[0199] In this specification, "amine" or "amino" as used interchangeably refers to a functional group containing a basic nitrogen atom having a lone pair of electrons.

[0200] As used herein, the term “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt of the ionized lipids of this disclosure. Examples of salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, acidic tartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, sugarates, formates, benzoates, glutamates, methanesulfonates "mesylates", ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may include the inclusion of another molecule, such as an acetate ion, a succinate ion, or another counterion. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. If multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0201] The grouping of alternative elements or embodiments disclosed herein should not be construed as limitation. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of any such inclusion or removal, the specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0202] In some embodiments of the embodiments described herein, the disclosures described herein do not relate to human cloning processes, processes for correcting the genetic identity of human germline cells, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without providing any substantial medical benefit to humans or animals, or processes for correcting the genetic identity of animals resulting from such processes.

[0203] Other terms are defined within the descriptions of various aspects of this disclosure.

[0204] All patents and other publications cited throughout this application, including references to literature, issued patents, published patent applications, and pending patent applications, are expressly incorporated herein by reference for the purpose of explaining and disclosing methodologies described in such publications that may be used, for example, in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an acknowledgment by the inventor that there is no prior right to such disclosure, either because of or for any other reason. All statements regarding dates or expressions regarding the content of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or content of these documents.

[0205] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. While specific embodiments and examples of the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be apparent to those skilled in the art. For example, while the steps or functions of a method are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as needed. The various embodiments described herein may be combined to provide further embodiments. The aspects of the disclosure may be modified as needed to provide further embodiments of the disclosure using the compositions, functions, and concepts of the above-mentioned references and applications. Furthermore, several modifications to the protein structure may be made without affecting the type or amount of biological or chemical action, taking into consideration the equivalence of biological function. These and other modifications may be made in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0206] Certain elements of any of the embodiments described above can be combined with or replaced by elements of other embodiments. Furthermore, while advantages related to certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments necessarily need to demonstrate such advantages in order to be within the scope of the present disclosure.

[0207] The techniques described herein are further illustrated by the following examples and should not be construed as further limiting them. This disclosure is not limited in any way to the specific methodologies, protocols, reagents, etc. described herein, and should be understood to be subject to change. The terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of this disclosure as defined by the claims.

[0208] II. Lipid Nanoparticle Compositions Provided herein are pharmaceutical compositions comprising lipid nanoparticles (LNPs), wherein the LNPs comprise lipids and rigid therapeutic nucleic acids (rTNAs), and the average diameter of the LNPs is approximately 20 nm to approximately 70 nm. The LNPs described herein offer numerous therapeutic advantages, including miniaturization that allows for the encapsulation of large rigid therapeutic nucleic acid molecules. According to some embodiments, the lipids are cationic lipids. According to some embodiments, the rigid therapeutic nucleic acids are closed-end DNA (ceDNA). According to some embodiments, the LNPs further comprise non-cationic lipids. According to some embodiments, the LNPs further comprise sterols or derivatives thereof. According to some embodiments, the LIPs further comprise PEG conjugated to lipids.

[0209] Cationic lipids In some embodiments, lipid nanoparticles having an average diameter of 20–74 nm contain cationic lipids. In some embodiments, the cationic lipids are, for example, non-fusion cationic lipids. "Non-fusion cationic lipids" means cationic lipids that can condense and / or encapsulate nucleic acid cargo such as ceDNA, but have little to no fusion activity.

[0210] In some embodiments, cationic lipids are determined to be non-fusible, as measured by, for example, membrane-impermeable fluorescent dye exclusion assays, such as those described in the Examples section of this specification, as described in the International and U.S. Patent Application Publications listed in Table 1 below. The contents of all patent documents of these International and U.S. Patent Application Publications listed in Table 1 below are incorporated herein by reference in their entirety. [Table 1-1] [Table 1-2]

[0211] In some embodiments, the cationic lipid is N-[1-(2,3-dioleyloxy)propyl lN,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl lN,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC), 1,2- Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)aminolbutylcarboxamide ethyl 11-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamide-glycylpermine (DOGS), 3b-[N-(N',N'-di Selected from the group consisting of methylaminoethyl)carbamoyl cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint-lipids (e.g., SAINT-2, N-methyl-1-4-(dioleyl)methylpyridinium), 1,2-dimyristyloxypropionate-1-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyloxypropionate-1-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropionate-1-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoid-1-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimyristreoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, such as cationic lipids, is, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-1-4-(2-dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA), 1,2-dioleyloxy-3- These are lipids such as dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-di-1-dioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-di-1-dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethicone-4,5-bis(oleoyloxy)pentane-1-aminium chloride (DOTAPen). In some embodiments, the condensed lipid is DOTAP.

[0212] Ionized lipids According to some embodiments, pharmaceutical compositions containing LNPs having an average diameter of 20–70 nm are also provided herein, wherein the LNPs include ionized lipids and rigid therapeutic nucleic acids such as nonviral vectors (e.g., ceDNA). Such LNPs may be used, for example, to deliver capsid-free nonviral DNA vectors to a target site of interest (e.g., cells, tissues, organs, etc.).

[0213] Exemplary ionized lipids are described in International PCT Patent Publications 2015 / 095340, 2015 / 199952, 2018 / 011633, 2017 / 049245, 2015 / 061467, 2012 / 040184, 2012 / 000104, 2015 / 074085, 2016 / 081029, 2017 / 004143, 2017 / 075531, 2017 / 117528, 2011 / 022460, 2013 / 148541, 2013 / 116126, and 20 11 / 153120, 2012 / 044638, 2012 / 054365, 2011 / 090965, 2013 / 016058, 2012 / 162210, 2008 / 042973, 2010 / 129709, 2010 / 1 No. 44740, No. 2012 / 099755, No. 2013 / 049328, No. 2013 / 086322, No. 2013 / 086 No. 373, No. 2011 / 071860, No. 2009 / 132131, No. 2010 / 048536, No. 2010 / 088537 No. 2010 / 054401, No. 2010 / 054406, No. 2010 / 054405, No. 2010 / 054384, Same No. 2012 / 016184, Same No. 2009 / 086558, Same No. 2010 / 042877, Same No. 2011 / 000106, Same No. 2011 / 000107, 2005 / 120152, 2011 / 141705, 2013 / 126803, 20 06 / 007712, 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 / U.S. Patent Publications 127060, 2011 / 141704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, and 2013 / 086354, as well as U.S. Patent Publications 2016 / 0311759, 2015 / 0376115, 2016 / 0151284, 2017 / 0210697, 2015 / 0140070, and 2013 / 0178541,Same No. 2013 / 0303587, No. 2015 / 0141678, No. 2015 / 0239926, No. 2016 / 0376224, No. 2017 / 0119904, No. 2012 / 014989 No. 4, No. 2015 / 0057373, No. 2013 / 0090372, No. 2013 / 0274523, No. 2013 / 0274504, No. 2013 / 0274504, No. 2009 / 002 No. 3673, No. 2012 / 0128760, No. 2010 / 0324120, No. 2014 / 0200257, No. 2015 / 0203446, No. 2018 / 0005363, No. 2014 / 0308304, 2013 / 0338210, 2012 / 0101148, 2012 / 0027796, 2012 / 0058144, 2013 / 0323269, 201 1 / 0117125, 2011 / 0256175, 2012 / 0202871, 2011 / 0076335, 2006 / 0083780, 2013 / 0123338, 2015 / 0064242, 2006 / 0051405, 2013 / 0065939, 2006 / 0008910, 2003 / 0022649, 2010 / 0130588, This is described in issues 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, all of which are incorporated herein by reference in their entirety.

[0214] In some embodiments, the ionized lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA or MC3) having the following structure. [ka]

[0215] The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the contents of which are incorporated herein by reference in their entirety.

[0216] In some embodiments, the ionized lipid is lipid ATX-002, as described in WO2015 / 074085 (the contents of which are incorporated herein by reference in their entirety).

[0217] In some embodiments, the ionized lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (compound 32), as described in WO2012 / 040184 (the contents of which are incorporated herein by reference in their entirety).

[0218] In some embodiments, the ionized lipid is compound 6 or compound 22, as described in WO2015 / 199952 (the contents of which are incorporated herein by reference in their entirety).

[0219] Equations (I) and (I') According to some embodiments, ionized lipids are given by formula (I): [ka] or represented by its pharmaceutically acceptable salt, in the formula, R 1 and R 1’ Each of them is independent of C 1~3 It is alkylene, R 2 and R 2’ Each of these is independently a linear or branched chain C 1~6 Alkylene, or C 3~6 It is a cycloalkylene, R 3 and R 3’ Each of these is an arbitrarily substituted C, independently of the others. 1~6 Alkyl or optionally substituted C 3~6Is it cycloalkyl? Alternatively, R 2 ' is branched chain C 1~6 When it is alkylene, and R 3 C 1~6 If it is alkyl, R 2 'and R 3 ', together with the intervening N atoms, form 4- to 8-membered heterocyclines. Alternatively, R 2 ' is branched chain C 1~6 When it is alkylene, and R 3 C 1~6 If it is alkyl, R 2 'and R 3 ', together with the intervening N atoms, form 4- to 8-membered heterocyclines. R 4 and R 4’ These are, independently, -CH, -CH2CH, or -(CH2)2CH, R 5 and R 5’ Each of them is independent of C 1~20 Alkylene or C 2~20 It is alkenylene, R 6 and R 6’ For each occurrence, independently, C 1~20 Alkilen, C 3~20 Cycloalkylene, or C 2~20 It is alkenylene, m and n are each independently selected integers from 1, 2, 3, 4, and 5.

[0220] Alternatively, according to some embodiments, the ionized lipid is given by formula (I'): [ka] or represented by its pharmaceutically acceptable salt, in the formula, R 1 and R 1’ Each of them is independent of C 1~3 It is alkylene, R 2 and R 2’Each of these is independently a linear or branched chain C 1~6 Alkylene, or C 3~6 It is a cycloalkylene, R 3 and R 3’ Each of these is an arbitrarily substituted C, independently of the others. 1~6 Alkyl or optionally substituted C 3~6 Is it cycloalkyl? Alternatively, R 2 ' is branched chain C 1~6 When it is alkylene, and R 3 C 1~6 If it is alkyl, R 2 'and R 3 ', together with the intervening N atoms, form 4- to 8-membered heterocyclines. Alternatively, R 2 ' is branched chain C 1~6 When it is alkylene, and R 3 C 1~6 If it is alkyl, R 2 'and R 3 ', together with the intervening N atoms, form 4- to 8-membered heterocyclines. R 4 and R 4’ These are, independently, -CH, -CH2CH, or -(CH2)2CH, R 5 and R 5’ These are, independently, hydrogen and C 1~20 Alkylene or C 2~20 It is alkenylene, R 6 and R 6’ For each occurrence, independently, C 1~20 Alkilen, C 3~20 Cycloalkylene, or C 2~20 It is alkenylene, m and n are each independently selected integers from 1, 2, 3, 4, and 5.

[0221] According to any of the embodiments or aspects of this specification, R 2 and R 2’ Each is independently C1~3 It is alkylene.

[0222] According to any of the embodiments or aspects of this specification, R 1 or R 1’ A linear or branched chain C represented by 1~3 Alkilen, R 2 or R 2’ A linear or branched chain C represented by 1~6 Alkylenes and optionally substituted linear or branched C 1~6 Each alkyl group is optionally substituted with one or more halo and cyano groups.

[0223] According to some embodiments of any aspect or embodiment of this specification, the combined R 1 and R 2 C 1~3 It is alkylene, and together with R 1’ and R 2’ C 1~3 Alkylenes, such as ethylene, are examples of this.

[0224] According to any of the embodiments or aspects of this specification, R 3 and R 3’ Each of these is an arbitrarily substituted C, independently of the others. 1~3 Alkyl compounds, such as methyl compounds.

[0225] According to any of the embodiments or aspects of this specification, R 4 and R 4’ Each of these is -CH.

[0226] According to any of the embodiments or aspects of this specification, R 2 This is an arbitrarily substituted branched chain C 1~6 It is alkylene, R 2 and R 3 These, together with the intervening N atoms, form a 5-membered or 6-membered heterocycline. According to some embodiments of any of the aspects or embodiments of this specification, R2 This is an arbitrarily substituted branched chain C 1~6 It is alkylene, R 2 and R 3 These, together with the intervening N atoms, form five- or six-membered heterocyclines such as pyrrolidinyl or piperidinyl.

[0227] According to any of the embodiments or aspects of this specification, R 4 -C(R a )2CR a , or -[C(R a )2]2CR a And R a C 1~3 It is alkyl, R 3 and R 4 These, together with the intervening N atoms, form a 5-membered or 6-membered heterocycline. According to some embodiments of any of the aspects or embodiments of this specification, R 4 -C(R a )2CR a , or -[C(R a )2]2CR a And R a C 1~3 It is alkyl, R 3 and R 4 These, together with the intervening N atoms, form five- or six-membered heterocyclines such as pyrrolidinyl or piperidinyl.

[0228] According to any of the embodiments or aspects of this specification, R 5 and R 5’ Each of them is independent of C 1~10 Alkylene or C 2~10 It is an alkenylene. In one embodiment, R 5 and R 5’ Each of them is independent of C 1~8 Alkylene or C 1~6 It is alkylene.

[0229] According to any of the embodiments or aspects of this specification, R6 and R 6’ For each occurrence, independently, C 1~10 Alkilen, C 3~10 Cycloalkylene, or C 2~10 It is an alkenylene. 1~6 Alkilen, C 3~6 Cycloalkylene, or C 2~6 It is an alkenylene. 3~10 Cycloalkylene or C 3~6 The cycloalkylene is cyclopropylene. According to some embodiments of any aspect or embodiment of this specification, m and n are each 3.

[0230] According to some embodiments of any aspect or embodiment of this specification, the ionized lipid is selected from any one of the lipids in Table 2 or a pharmaceutically acceptable salt thereof. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12]

[0231] Formula (II) In some embodiments, ionized lipids are given by formula (II): [ka] or represented by its pharmaceutically acceptable salt, in the formula, a is an integer in the range of 1 to 20 (for example, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), b is an integer in the range of 2 to 10 (for example, b is 2, 3, 4, 5, 6, 7, 8, 9, or 10), R 1 is absent, or (C2-C 20 ) Alkenyl, -C(O)O(C2-C 20 )alkyl, and (C2-C 20 ) Selected from alkyl-substituted cyclopropyls, R 2 (C2-C 20 It is alkyl.

[0232] In the second chemical embodiment, the ionized lipid of formula (II) is formula (XIII): [ka] or a pharmaceutically acceptable salt thereof, where 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 as described for formula (XII).

[0233] In the third chemical embodiment, c and d in the ionized lipid of formula (II) or (III) are, independently, integers in the range of 2-8, 3-8, 3-7, 3-6, 3-5, 4-8, 4-7, 4-6, 5-8, 5-7, or 6-8, and the remaining variables are as described for formula (XII).

[0234] In the fourth chemical embodiment, c in the ionized lipid of formula (II) or (III) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for formula (XII) or the second or third chemical embodiment. Alternatively, as part of the fourth chemical embodiment, c and d in the ionized lipid of formula (XII) or (XIII), or a pharmaceutically acceptable salt thereof, are independently 1, 3, 5, or 7, and the remaining variables are as described for formula (XII) or the second or third chemical embodiment.

[0235] In the fifth chemical embodiment, d in the ionized lipid of formula (II) or (III) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for formula (II) or the second, third, or fourth chemical embodiment. Alternatively, as part of the fourth chemical embodiment, at least one of c and d in the ionized lipid of formula (II) or (III), or a pharmaceutically acceptable salt thereof, is 7, and the remaining variables are as described for formula (II) or the second, third, or fourth chemical embodiment.

[0236] In the sixth chemical embodiment, the ionized lipid of formula (II) or (III) is formula (IV): [ka] or a pharmaceutically acceptable salt thereof, where the remaining variables are as described for formula (I).

[0237] In the seventh chemical embodiment, b in the ionized lipid of formula (II), (III), or (IV) is an integer in the range of 3 to 9, and the remaining variables are as described for formula (II) or the second, third, fourth, or fifth chemical embodiment. Alternatively, as part of the seventh chemical embodiment, b in the ionized lipid of formula (II), (III), or (IV) 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 as described for formula (II) or the second, third, fourth, or fifth chemical embodiment. Alternatively, as part of the seventh chemical embodiment, b in the ionized lipid of formula (II), (III), or (IV) is 3, 4, 5, 6, 7, 8, or 9, and the remaining variables are as described for formula (XII), or the second, third, fourth, or fifth chemical embodiment.

[0238] In the eighth chemical embodiment, a in the ionized lipid of formula (II), (III), or (IV) is an integer in the range of 2 to 18, and the remaining variables are as described for formula (II), or for the second, third, fourth, fifth, or seventh chemical embodiments. Alternatively, as part of the eighth embodiment, a in the ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof, 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, 3-8, 3-7, 3-6, 3-5, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 25-8, 5-7, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 11-18, 11-1 The integers are in the range of 7, 11-16, 11-15, 11-14, 11-13, 12-18, 12-17, 12-16, 12-15, 12-14, 13-18, 13-17, 13-16, 13-15, 14-18, 14-17, 14-16, 15-18, 15-17, or 16-18, and the remaining variables are as described in formula (II) or for the second, third, fourth, fifth, or seventh chemical embodiment.In another alternative, as part of the eighth embodiment, a in the ionized lipid of formula (II), (III), or (IV) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, with the remaining variables as described for formula (II) or the second, third, fourth, fifth, or seventh chemical embodiment.

[0239] In the ninth chemical embodiment, R in an ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof 1 is absent, or (C5-C 15 ) Alkenyl, -C(O)O(C4-C 18 )alkyl, and (C4-C 16 ) Selected from alkyl-substituted cyclopropyl, where the remaining variables are as described for formula (II), (III), or (IV), or for the second, third, fourth, fifth, seventh, or eighth chemical embodiment. Alternatively, as part of the ninth chemical embodiment, R in the ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof. 1 is absent, or (C5-C 15 ) Alkenyl, -C(O)O(C4-C 16 )alkyl, and (C4-C 16 ) Selected from alkyl-substituted cyclopropyl, where the remaining variables are as described for formula (II), (III), or (IV), or for the second, third, fourth, fifth, seventh, or eighth chemical embodiment. Alternatively, as part of the ninth chemical embodiment, R in the ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof. 1 is absent, or (C5-C 12 ) Alkenyl, -C(O)O(C4-C 12 )alkyl, and (C4-C 12) Selected from alkyl-substituted cyclopropyls, where the remaining variables are as described for formula (II), (III), or (IV), or for the second, third, fourth, fifth, seventh, or eighth chemical embodiment. Alternatively, as part of the ninth chemical embodiment, R in the ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof. 1 is absent, or (C5-C 10 ) Alkenyl, -C(O)O(C4-C 10 )alkyl, and (C4-C 10 ) Selected from alkyl-substituted cyclopropyl, where the remaining variables are as described for formula (II), (III), or (IV), or for the second, third, fourth, fifth, seventh, or eighth chemical embodiment.

[0240] In the tenth chemical embodiment, R 1 C 10 It is an alkenyl, and the remaining variables in the formula are as described in one of the embodiments described above.

[0241] In the eleventh chemical embodiment, R in an ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof 1 C(O)O(C2-C 20 )alkyl, -C(O)O(C4-C 18 )alkyl, -C(O)O(C4-C 12 )alkyl, or -C(O)O(C4-C 10 )The alkyl in alkyl is an unbranched alkyl, and the remaining variables are as described in any one of the embodiments described above. In one chemical embodiment, R 1 R is -C(O)O(C9 alkyl). Alternatively, in the 11th chemical embodiment, ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof, R 1 -C(O)O(C4-C 18 )alkyl, -C(O)O(C4-C 12 )alkyl, or -C(O)O(C4-C10 )The alkyl in alkyl is a branched alkyl, and the remaining variables are as described in any one of the embodiments described above. In one chemical embodiment, R 1 is -C(O)O(C 17 The formula is alkyl, and the remaining variables are as described in one of the chemical embodiments described above.

[0242] In the twelfth chemical embodiment, R in an ionized lipid of formula (II), (III), or (IV), or a pharmaceutically acceptable salt thereof. 1 The group is selected from any group listed in Table 3 below, where the wavy bond in each group indicates the bond site of the group to the remainder of the lipid molecule, and the remaining variables are as described for formula (II), (III), or (IV), or for the second, third, fourth, fifth, seventh, or eighth chemical embodiment. This disclosure further relates to the R in Table 4. 1 One of the bases and R in Table 5 2 The combination is intended with any one of the bases, and the remaining variables are as described in formula (II), (III), or (IV), or in the second, third, fourth, fifth, seventh, or eighth chemical embodiment. [Table 3]

[0243] In the 13th chemical embodiment, R in the ionized lipid of formula (II) or a pharmaceutically acceptable salt thereof 2 The group is selected from any of the groups listed in Table 4 below, where the wavy bond in each group indicates the bond point of the group to the remainder of the lipid molecule, and the remaining variables are as described for formula (II), or for the 7th, 8th, 9th, 10th, or 11th chemical embodiments. [Table 4]

[0244] Specific examples are provided in the illustrative section following Table 5 and are included as part of the 14th chemical embodiment of the ionized lipid of formula (II) herein. This also includes pharmaceutically acceptable salts as well as ionized and neutral forms. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0245] Formula (V) In some embodiments, the ionized lipid is given by formula (V): [ka] A substance thereof, or a pharmaceutically acceptable salt thereof, in the formula, R 1 and R 1’ Each of them operates independently, R a (C1-C6) alkylenes optionally substituted with one or more groups selected from the following: R 2 and R 2’ Each of them is independently a (C1-C2) alkylene, R 3 and R 3’ Each of them operates independently, R b It is an alkyl group (C1-C6) optionally substituted with one or more groups selected from the following, Alternatively, R 2 and R 3 And / or R 2’ and R 3’These, together with the intervening N atoms, form 4- to 7-membered heterocyclines. R 4 and R 4 Each of these is a (C2-C6) alkylene interrupted by -C(O)O-, R 5 and R 5 'Each is independent of the others, (C2-C 30 ) Alkyl or (C2-C 30 ) are alkenyls, each of which may be optionally interrupted with -C(O)O- or (C3-C6)cycloalkyl groups. R a and R b These are either halo or cyano, respectively.

[0246] In the second chemical embodiment, R in the ionized lipid of formula (V) 1 and R 1’ Each of these is independently a (C1-C6) alkylene, and the remaining variables in the formula are as described above for formula (V). Alternatively, as part of a second chemical embodiment, R in the ionized lipid of formula (V) 1 and R 1’ These are (C1-C3)alkylenes, each independently, and the remaining variables in the formula are as described above for formula (V).

[0247] In the third chemical embodiment, the ionized lipid of formula (V) is Equation (VI): [ka] The substance thereof, or a pharmaceutically acceptable salt thereof, wherein the remaining variables in the formula are as described for formula (V).

[0248] In the fourth chemical aspect, the ionized lipid of formula (V) is formula (VII) or (VIII): [ka] The substance thereof, or a pharmaceutically acceptable salt thereof, wherein the remaining variables in the formula are as described for formula (V).

[0249] In the fifth chemical aspect, the ionized lipid of formula (V) is formula (IX) or (VI): [ka] The substance thereof, or a pharmaceutically acceptable salt thereof, wherein the remaining variables in the formula are as described for formula (V).

[0250] In the sixth chemical aspect, the ionized lipid of formula (V) is formula (XI), (XII), (XIII), or (XIV): [ka] The substance or a pharmaceutically acceptable salt thereof, where the remaining variables are as described for formula (XV).

[0251] In the seventh chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 and R 5´ At least one of them is a branched alkyl or branched alkenyl (the number of carbon atoms described above for formulas (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV)). In another alternative, as part of the seventh chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV). 5 and R 5´ One of these is a branched alkyl or branched alkenyl. Another alternative is R in ionized lipids of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) as part of the seventh chemical aspect. 5is a branched alkyl or branched alkenyl. Alternatively, as part of the seventh chemical aspect, R in ionized lipids of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV). 5’ These are branched alkyl or branched alkenyl compounds.

[0252] In the eighth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C6-C 26 ) Alkyl or (C6-C 26 ) are alkenyls, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl, where the remaining variables are as described above for formula (I). Alternatively, as part of the seventh chemical aspect, R in ionized lipids of formulas (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C6-C 26 ) Alkyl or (C6-C 26 ) are alkenyls, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, where the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in ionized lipids of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C7-C 26 ) Alkyl or (C7-C 26 ) are alkenyls, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, where the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in ionized lipids of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is, (C8-C 26 ) alkyl or (C8-C26 ) are alkenyls, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, where the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in ionized lipids of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C6-C 24 ) Alkyl or (C6-C 24 ) are alkenyls, each of which can optionally be interrupted with -C(O)O- or cyclopropyl, and in the formula, the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is, (C8-C 24 ) alkyl or (C8-C 24 ) is an alkenyl, and the (C8-C 24 The alkyl group is optionally interrupted with -C(O)O- or cyclopropyl, and in the formula, the remaining variables are as described above for formula (V). Alternatively, as part of the eighth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 is, (C8-C 10 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV). 5 It was interrupted with cyclopropyl (C 14 -C 16 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV).5 (C 10 -C 24 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV). 5 is, (C 16 -C 18 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V). In another alternative, as part of the eighth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV). 5 is -(CH2)3C(O)O(CH2)8CH3, -(CH2)5C(O)O(CH2)8CH3, -(CH2)7C(O)O(CH2)8CH3, -(CH2 )7C(O)OCH[(CH2)7CH3]2, -(CH2)7-C3H6-(CH2)7CH3, -(CH2)7CH3, -(CH2)9CH3, -(CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3, or -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remaining variables in the equation are as described above for equation (XV).

[0253] In the ninth chemical aspect, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C 15 -C 28 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V) or the eighth chemical embodiment. Alternatively, as part of the ninth chemical embodiment, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV). 5 (C 17 -C 28) is alkyl, where the remaining variables are as described above for formula (V) or the eighth chemical embodiment. Alternatively, as part of the ninth embodiment, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C 19 -C 28 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V) or the eighth chemical embodiment. Alternatively, as part of the ninth chemical embodiment, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C 17 -C 26 ) is alkyl, where the remaining variables are as described above for formula (V) or the eighth chemical embodiment. Alternatively, as part of the ninth embodiment, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C 19 -C 26 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V) or the eighth chemical embodiment. Alternatively, as part of the ninth chemical embodiment, R in the ionized lipid of formula (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV) 5 (C 20 -C 26 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V) or the eighth chemical embodiment. In another alternative, as part of the ninth embodiment, R 5 (C 22 -C 24 ) is alkyl, and in the formula, the remaining variables are as described above for formula (V) or the eighth chemical embodiment. In another alternative, as part of the ninth embodiment, R5’ is -(CH2)5C(O)OCH[(CH2)7CH3]2, -(CH2)7C(O)OCH[(CH2)7CH3]2, -(CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or -(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, where the remaining variables are as described above for formula (V) or the eighth chemical embodiment.

[0254] In another embodiment, the ionized lipid of formula (V), (VI), (VIII), (VIII), (IX), (X), (XII), (XIII), or (XIV) may be selected from any of the following lipids or pharmaceutically acceptable salts thereof listed in Table 6. [Table 6-1] [Table 6-2]

[0255] Formula (XV) In some embodiments, ionized lipids are given by formula (XV): [ka] A substance thereof, or a pharmaceutically acceptable salt thereof, in the formula, R' is absent, hydrogen, or a C1-C6 alkyl group, however, if R' is hydrogen or a C1-C6 alkyl group, then R', R 1 , and R 2 The nitrogen atom to which all of these atoms are bonded is protonated. R 1 and R 2 Each of these is independently hydrogen, a C1-C6 alkyl group, or a C2-C6 alkenyl group. R 3 C1-C 12 Alkylene or C2-C 12 It is alkenylene, R 4 C1-C 16 Unbranched alkyl, C2-C 16It is either an unbranched alkenyl or [ka] And in the formula, R 4a and R 4b Each is independent of C1-C 16 Unbranched alkyl or C2-C 16 It is an unbranched alkenyl, R 5 It is absent, C1-C8 alkylene, or C2-C8 alkenylene. R 6a and R 6b Each of them operates independently, C7-C 16 Alkyl or C7-C 16 It is alkenil, however R 6a and R 6b The total number of carbon atoms inside is greater than 15. X 1 and X 2 These are, independently of each other, -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-,-C(R a )=NO-, -ON=C(R a )-,-C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O-, or OC(=O)(CR a 2) C(=O)-, and in the formula, R a For each occurrence, it is independently hydrogen or a C1-C6 alkyl group. n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0256] In the second embodiment, the ionized lipid or a pharmaceutically acceptable salt thereof according to the first embodiment is X 1 and X2 The same, and all other remaining variables are as described for formula (V) or the first embodiment.

[0257] In the third embodiment, the ionized lipid or a pharmaceutically acceptable salt thereof according to the first or second embodiment is X 1 and X 2 Each of these is independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-, or X 1 and X 2 Each of these is independently -C(=O)O-, -C(=O)S-, or -SS-, or X 1 and X 2 These are, independently, -C(=O)O- or -SS-, and all other remaining variables are as described for formula V or for either of the embodiments described above.

[0258] In the fourth embodiment, the ionized lipid of the present disclosure is of formula (XVI): [ka] or represented by its pharmaceutically acceptable salt, where n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for formula (XV) or one of the embodiments described above.

[0259] In the fifth embodiment, the ionized lipid of the present disclosure is of formula (XVII): [ka] or represented by a pharmaceutically acceptable salt thereof, where n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for formula (XV), formula (XVI), or any one of the embodiments described above.

[0260] In the sixth embodiment, the ionized lipid of the present disclosure is of formula (XVIII): [ka] Or represented by a pharmaceutically acceptable salt thereof, all other remaining variables as described for formula (XV), formula (XVI), formula (XVII), or any one of the embodiments described above.

[0261] In the seventh embodiment, an ionized lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 1 and R 2 Each of these is independently hydrogen, a C1-C6 alkyl or C2-C6 alkenyl, or a C1-C5 alkyl or C2-C5 alkenyl, or a C1-C4 alkyl or C2-C4 alkenyl, or a C6 alkyl, or a C5 alkyl, or a C4 alkyl, or a C3 alkyl, or a C2 alkyl, or a C1 alkyl, or a C6 alkenyl, or a C5 alkenyl, or a C4 alkenyl, or a C3 alkenyl, or a C2 alkenyl, and all other remaining variables are as described for formula (XV), formula (XVI), formula (XVII), formula (XVIII), or any one of the embodiments described above.

[0262] In the eighth embodiment, the ionized lipid of the present disclosure is of formula (XIX): [ka] Or represented by a pharmaceutically acceptable salt thereof, all other remaining variables as described for formula (XV), formula (XVI), formula (XVII), (XVIII), or any one of the embodiments described above.

[0263] In the ninth embodiment, an ionized lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 3is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2-C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 C 12 Alkilen, C 11 Alkilen, C 10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 Alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above. Alternatively, in the ninth embodiment, a cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 3 is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2-C7 alkenylene, C1-C6 alkylene or C2-C6 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 C 12 Alkilen, C 11 Alkilen, C 10Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 The variables are alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above.

[0264] In the tenth embodiment, a cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene, or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene, or R 5 is absent, or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above.

[0265] In the eleventh embodiment, a cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 4C1-C 14 Unbranched alkyl, C2-C 14 Unbranched alkenyls, or [ka] And R 4a and R 4b Each is independent of C1-C 12 Unbranched alkyl or C2-C 12 It is either an unbranched alkenyl or R 4 C2-C 12 Unbranched alkyl or C2-C 12 It is either an unbranched alkenyl or R 4 is either a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 It is either an unbranched alkenyl, a C9 unbranched alkenyl, a C8 unbranched alkenyl, a C7 unbranched alkenyl, a C6 unbranched alkenyl, a C5 unbranched alkenyl, a C4 unbranched alkenyl, a C3 unbranched alkenyl, or a C2 alkenyl, or R 4 teeth, [ka] And R4a and R 4b Each is independently C2-C 10 Unbranched alkyl or C2-C 10 It is either an unbranched alkenyl or R 4 teeth, [ka] And R 4a and R 4b Each of them is independent of C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 The variables are unbranched alkenyls, C9 unbranched alkenyls, C8 unbranched alkenyls, C7 unbranched alkenyls, C6 unbranched alkenyls, C5 unbranched alkenyls, C4 unbranched alkenyls, C3 unbranched alkenyls, or C2 alkenyls, and all other remaining variables are as described for formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above.

[0266] In the twelfth embodiment, a cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 6a and R 6b Each of them operates independently, C6-C14 Alkyl or C6-C 14 It is an alkenyl, or R 6a and R 6b Each of them operates independently, C8-C 12 Alkyl or C8-C 12 It is an alkenyl, or R 6a and R 6b Each of them is independent of C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenil, C 15 Alkenil, C 14 Alkenil, C 13 Alkenil, C 12 Alkenil, C 11 Alkenil, C 10 Alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, however R 6a and R 6b The total number of carbon atoms in the compound is greater than 15, and all other remaining variables are as described for formulas (XV), (XVI), (XVII), (XVIII), (XIX), or any one of the embodiments described above.

[0267] In the 13th embodiment, a cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 6a and R 6b These contain an equal number of carbon atoms, or R 6a and R 6b are the same, or R 6a and R 6b Both are C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenil, C 15 Alkenil, C 14 Alkenil, C 13 Alkenil, C 12 Alkenil, C 11 Alkenil, C 10 Alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, however R 6a and R 6b The total number of carbon atoms in the compound is greater than 15, and all other remaining variables are as described for formulas (XV), (XVI), (XVII), (XVIII), (XIX), or any one of the embodiments described above.

[0268] In the fourteenth embodiment, a cationic lipid according to formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, is defined as R as defined in any one of the embodiments described above. 6a and R 6b Each contains a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms differs by only one or two carbon atoms, or R 6a and R 6b The number of carbon atoms differs by only one carbon atom, or R 6a It is a C7 alkyl, and R 6a It is a C8 alkyl, and R 6a It is a C8 alkyl, and R 6a It is a C7 alkyl, and R 6a It is a C8 alkyl, and R 6a It is a C9 alkyl, and R 6a It is a C9 alkyl, and R 6a It is a C8 alkyl, and R 6a It is a C9 alkyl, and R 6a C 10 It is alkyl, R 6a C 10 It is alkyl, R6a It is a C9 alkyl, and R 6a C 10 It is alkyl, R 6a C 11 It is alkyl, R 6a C 11 It is alkyl, R 6a C 10 It is alkyl, R 6a C 11 It is alkyl, R 6a C 12 It is alkyl, R 6a C 12 It is alkyl, R 6a C 11 It is alkyl, R 6a It is a C7 alkyl, and R 6a It is a C9 alkyl, and R 6a It is a C9 alkyl, and R 6a It is a C7 alkyl, and R 6a It is a C8 alkyl, and R 6a C 10 It is alkyl, R 6a C 10 It is alkyl, R 6a It is a C8 alkyl, and R 6a It is a C9 alkyl, and R 6a C 11 It is alkyl, R 6a C 11 It is alkyl, R 6a It is a C9 alkyl, and R 6a C 10 It is alkyl, R 6a C 12 It is alkyl, R 6a C 12 It is alkyl, R 6a C 10 It is alkyl, R 6a C 11 It is alkyl, R 6a C 13 Alkyl or R 6a C 13 It is alkyl, R 6a C 11The elements are alkyl, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the embodiments described above.

[0269] In one embodiment, the cationic lipid of the present disclosure or the cationic lipid of formula (XV), formula (XVI), formula (XVII), formula (XVIII), or formula (XIX) is any one lipid selected from the lipids in Table 7 or pharmaceutically acceptable salts thereof: [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0270] Formula (XX) In some embodiments, ionized lipids are given by formula (XX): [ka] A substance thereof, or a pharmaceutically acceptable salt thereof, in the formula, R' is absent, hydrogen, or a C1-C3 alkyl group, however, if R' is hydrogen or a C1-C3 alkyl group, then R', R 1 , and R 2 The nitrogen atom to which all of these atoms are bonded is protonated. R 1 and R 2 Each of these is independently hydrogen or a C1-C3 alkyl group. R 3 C3-C 10 Alkylene or C3-C 10 It is alkenylene, R 4 C1-C 16 Unbranched alkyl, C2-C 16It is either an unbranched alkenyl or [ka] And in the formula, R 4a and R 4b Each is independent of C1-C 16 Unbranched alkyl or C2-C 16 It is an unbranched alkenyl, R 5 It is absent, C1-C6 alkylene, or C2-C6 alkenylene. R 6a and R 6b Each of them operates independently, C7-C 14 Alkyl or C7-C 14 It is alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-,-C(R a )=NO-, -ON=C(R a )-,-C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O-, or OC(=O)(CR a 2) C(=O)-, and in the formula, R a For each occurrence, it is independently hydrogen or a C1-C6 alkyl group. n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0271] In the second embodiment, in the ionized lipid or pharmaceutically acceptable salt thereof according to the first embodiment, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS, and all other remaining variables are as described for formula (XX) or the first embodiment.

[0272] In a third embodiment, the ionized lipid of the present disclosure is of formula (XXI): [ka] or represented by a pharmaceutically acceptable salt thereof, where n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for formula (XX) or any one of the embodiments described above. In another third embodiment, n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for formula (XX) or any one of the embodiments described above.

[0273] In the fourth embodiment, the ionized lipid of the present disclosure is of formula (XXII): [ka] Or represented by a pharmaceutically acceptable salt thereof, all other remaining variables as described for formula (XX), formula (XXI), or any one of the embodiments described above.

[0274] In the fifth embodiment, in the ionized lipid or pharmaceutically acceptable salt thereof according to the first embodiment, R 1 and R 2 Each of these is independently hydrogen, a C1-C2 alkyl group, or a C2-C3 alkenyl group, or R', R 1 , and R 2 These are, independently, hydrogen and a C1-C2 alkyl group, and all other remaining variables are as described for formula (XX), (XXI), or any one of the embodiments described above.

[0275] In the sixth embodiment, the ionized lipid of the present disclosure is of formula (XXII): [ka] Or represented by a pharmaceutically acceptable salt thereof, all other remaining variables as described for formula (XX), formula (XXI), formula (XXII), or any one of the embodiments described above.

[0276] In the seventh embodiment, an ionized lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 5 is either absent or C1-C8 alkylene, or R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene, or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene, or R 5 is absent, or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), or any one of the embodiments described above.

[0277] In the eighth embodiment, the ionized lipid of the present disclosure is of formula (XXIV): [ka] Or represented by a pharmaceutically acceptable salt thereof, all other remaining variables as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII) or any one of the embodiments described above.

[0278] In the ninth embodiment, an ionized lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 4 C1-C 14Unbranched alkyl, C2-C 14 Unbranched alkenyls, or [ka] And R 4a and R 4b Each is independent of C1-C 12 Unbranched alkyl or C2-C 12 It is either an unbranched alkenyl or R 4 C2-C 12 Unbranched alkyl or C2-C 12 It is either an unbranched alkenyl or R 4 C5-C 12 Unbranched alkyl or C5-C 12 It is either an unbranched alkenyl or R 4 C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 It is either an unbranched alkenyl, a C9 unbranched alkenyl, a C8 unbranched alkenyl, a C7 unbranched alkenyl, a C6 unbranched alkenyl, a C5 unbranched alkenyl, a C4 unbranched alkenyl, a C3 unbranched alkenyl, or a C2 alkenyl, or R 4 teeth, [ka] And R 4a and R 4b Each is independently C2-C 10 Unbranched alkyl or C2-C 10 It is either an unbranched alkenyl or R 4 teeth, [ka] And R 4a and R 4b Each of them is independent of C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 The variables are unbranched alkenyls, C9 unbranched alkenyls, C8 unbranched alkenyls, C7 unbranched alkenyls, C6 unbranched alkenyls, C5 unbranched alkenyls, C4 unbranched alkenyls, C3 unbranched alkenyls, or C2 alkenyls, and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above.

[0279] In the tenth embodiment, an ionized lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 3is either C3-C8 alkylene or C3-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C3-C5 alkylene or C3-C5 alkenylene, or R 3 is C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C1 alkylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above.

[0280] In the eleventh embodiment, an ionized lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 6a and R 6b Each of them operates independently, C7-C 12 Alkyl or C7-C 12 It is an alkenyl, or R 6a and R 6b Each of them operates independently, C8-C 10 Alkyl or C8-C 10 It is an alkenyl, or R 6a and R 6b Each of them is independent of C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenil, C 11 Alkenil, C 10 The variables are an alkenyl, a C9 alkenyl, a C8 alkenyl, or a C7 alkenyl, and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above.

[0281] In the twelfth embodiment, an ionized lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R 6a and R 6b These contain an equal number of carbon atoms, or R 6a and R 6b are the same, or R 6a and R 6b Both are C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenil, C 11 Alkenil, C 10 The variables are an alkenyl, a C9 alkenyl, a C8 alkenyl, or a C7 alkenyl, and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above.

[0282] In the 13th embodiment, an ionized lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, is defined as R as defined in any one of the embodiments described above. 6a and R 6b Each contains a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms differs by only one or two carbon atoms, or R 6a and R 6b The number of carbon atoms differs by only one carbon atom, or R 6a It is a C7 alkyl, and R 6a It is a C8 alkyl, and R 6a It is a C8 alkyl, and R 6a It is a C7 alkyl, and R 6a It is a C8 alkyl, and R 6a It is a C9 alkyl, and R 6a It is a C9 alkyl, and R 6aIt is a C8 alkyl, and R 6a It is a C9 alkyl, and R 6a C 10 It is alkyl, R 6a C 10 It is alkyl, R 6a It is a C9 alkyl, and R 6a C 10 It is alkyl, R 6a C 11 It is alkyl, R 6a C 11 It is alkyl, R 6a C 10 It is alkyl, R 6a C 11 It is alkyl, R 6a C 12 It is alkyl, R 6a C 12 It is alkyl, R 6a C 11 It is alkyl, R 6a It is a C7 alkyl, and R 6a It is a C9 alkyl, and R 6a It is a C9 alkyl, and R 6a It is a C7 alkyl, and R 6a It is a C8 alkyl, and R 6a C 10 It is alkyl, R 6a C 10 It is alkyl, R 6a It is a C8 alkyl, and R 6a It is a C9 alkyl, and R 6a C 11 It is alkyl, R 6a C 11 It is alkyl, R 6a It is a C9 alkyl, and R 6a C 10 It is alkyl, R 6a C 12 It is alkyl, R 6a C 12 It is alkyl, R 6a C 10Alkyl, etc., and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV) or any one of the embodiments described above.

[0283] In the fourteenth embodiment, in the ionized lipid according to formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above, or a pharmaceutically acceptable salt thereof, R' is absent, and all other remaining variables are as described for formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV), or any one of the embodiments described above.

[0284] In one embodiment, the ionized lipid of the present disclosure or the ionized lipid of formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV) is any one lipid selected from the lipids in Table 8 or pharmaceutically acceptable salts thereof: [Table 8-1] [Table 8-2]

[0285] Specific examples are provided in the following illustrative section and are included as part of the ionized lipids described herein. Pharmaceutically acceptable salts and neutral forms are also included.

[0286] Cuttable lipids In some embodiments, provided herein are pharmaceutical compositions comprising cleavable lipids and capsid-free nonviral vectors (e.g., ceDNA) that can be used to deliver the capsid-free nonviral DNA vector to a target site of interest (e.g., cells, tissues, organs, etc.). As used herein, the term “cleavable lipid” refers to a cationic lipid containing disulfide bond ("SS") cleavable units. In one embodiment, the SS-cleavable lipid comprises a tertiary amine that responds to disulfide bonds that can be cleaved in an acidic compartment, for membrane destabilization (e.g., endosomes or lysosomes), and in a reducing environment (e.g., cytoplasm). Examples of SS-cleavable lipids include SS-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids, ss-OC lipids, and other SS-cleavable and pH-activated lipid-like substances.

[0287] According to some embodiments, SS-cleavable lipids are described in International Patent Application Publication No. 2019 / 188867, which is incorporated herein by reference in whole.

[0288] As described herein, ceDNA lipid particles (e.g., lipid nanoparticles) containing cleavable lipids provide more efficient delivery of ceDNA to target cells (e.g., hepatocytes). This disclosure provides novel formulation processes and methods for producing significantly smaller LNPs than those previously described. According to some embodiments, the LNPs produced by the formulation processes and methods described herein have sizes ranging from about 20 to about 70 nm in average diameter, for example, about 20 nm to about 70 nm, about 25 nm to about 70 nm, about 30 nm to about 70 nm, about 35 nm to about 70 nm, about 40 nm to about 70 nm, about 45 nm to about 80 nm, about 50 nm to about 70 nm, about 60 nm to about 70 nm, about 65 nm to about 70 nm, or average diameters of about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, and about 70 nm. According to some embodiments, the average diameter of LNPs is approximately 50 nm to approximately 70 nm. This is very small, which is advantageous for targeting and evading immune responses. Furthermore, the LNPs described herein can encapsulate more than 60% to about 90% of rigid double-stranded DNA such as ceDNA. According to some embodiments, the LNPs described herein can encapsulate more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% of rigid double-stranded DNA such as ceDNA.

[0289] The lipid particles (e.g., nanoparticles) (e.g., ceDNA lipid particles, mRNA lipid particles) described herein can be advantageously used to increase the delivery of nucleic acids (e.g., ceDNA, mRNA) to target cells / tissues compared to LNPs produced by other processes and compared to other lipids, e.g., ionized cationic lipids. Thus, the lipid particles (e.g., nanoparticles) (e.g., ceDNA lipid particles, mRNA lipid particles) described herein provided maximum nucleic acid delivery compared to lipid particles prepared by processes and methods known in the art. Although the mechanism has not yet been determined and is not bound by theory, it is thought that lipid particles (e.g., nanoparticles) (e.g., ceDNA lipid particles or mRNA lipid particles) containing cleavable lipids prepared by the processes described herein improve delivery to hepatocytes, avoid phagocytosis, and transport more efficiently to the nucleus. Another advantage of the lipid particles (e.g., lipid nanoparticles) (e.g., ceDNA lipid particles, mRNA lipid particles) prepared by the processes described herein and containing cleavable lipids is better tolerability compared to other lipids (e.g., ionized cationic lipids, e.g., MC3).

[0290] In one embodiment, the cleavable lipid may comprise three components: an amine tip group, a linker group, and a hydrophobic tail. In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one or more tertiary amino groups, and a disulfide bond. The tertiary amine group provides pH responsiveness and induces endosome escape, the phenyl ester bond enhances structural degradability (autodegradability), and the disulfide bond cleaves in a reducing environment.

[0291] In one embodiment, the cleavable lipid is an ss-OP lipid. In one embodiment, the ss-OP lipid has a structure represented by the following formula A. Lipid A [ka]

[0292] In one embodiment, the SS-cleavable lipid is an SS-cleavable and pH-activated lipid-like substance (ssPalm). ssPalm lipids are well known in the art. See, for example, Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, ssPalm is an ssPalmM lipid containing the structure of lipid B. lipid B [ka]

[0293] In one embodiment, the ssPalmE lipid is an ssPalmE-P4-C2 lipid containing the structure of lipid C. lipid C [ka]

[0294] In one embodiment, the ssPalmE lipid is an ssPalmE-Paz4-C2 lipid containing the structure of lipid D. lipid D [ka]

[0295] In one embodiment, the cleavable lipid is an ss-M lipid. In one embodiment, the ss-M lipid has the structure shown in lipid E ​​below. Lipid E [ka]

[0296] In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, the ss-E lipid has the structure shown in lipid F below. lipid F [ka]

[0297] In one embodiment, the cleavable lipid is an ss-EC lipid. In one embodiment, the ss-EC lipid has the structure shown in lipid G below. lipid G [ka]

[0298] In one embodiment, the cleavable lipid is an ss-LC lipid. In one embodiment, the ss-LC lipid has the structure shown in lipid H below. lipid H [ka]

[0299] In one embodiment, the cleavable lipid is an ss-OC lipid. In one embodiment, the ss-OC lipid has the structure shown in lipid J below. lipid J [ka]

[0300] In one embodiment, lipid particle (lipid nanoparticle) formulations are prepared and loaded with ceDNA obtained by a process disclosed in International Patent Application PCT / US2018 / 050042, filed September 7, 2018, which is incorporated in whole herein by reference. This can be achieved by high-energy mixing of ethanol lipids and aqueous ceDNA at a low pH, which protonates the lipids and provides a favorable energy for ceDNA / lipid association and nucleation of the particles. The particles can be further stabilized by aqueous dilution and removal of the organic solvent. The particles can be concentrated to a desired level. In one embodiment, the present disclosure provides ceDNA lipid particles containing a lipid of formula I prepared by the process described in Example 2.

[0301] Generally, lipid particles (e.g., lipid nanoparticles) are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 60:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) may be in the range of about 1:1 to about 60:1, about 1:1 to about 55:1, about 1:1 to about 50:1, about 1:1 to about 45:1, about 1:1 to about 40:1, about 1:1 to about 35:1, about 1:1 to about 30:1, about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, about 6:1 to about 9:1, and about 30:1 to about 60:1. According to some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with ceDNA (mass or weight) relative to a total lipid ratio of about 60:1. According to some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with ceDNA (mass or weight) relative to a total lipid ratio of about 30:1. By adjusting the amounts of lipids and ceDNA, a desired N / P ratio, such as 3, 4, 5, 6, 7, 8, 9, 10 or higher, can be provided. Generally, the total lipid content of the lipid particle formulation may range from about 5 mg / ml to about 30 mg / mL.

[0302] In some embodiments, the lipid nanoparticles include agents for condensing and / or encapsulating nucleic acid cargo, such as ceDNA. Such agents are also referred to herein as condensants or encapsulants. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acids can be used, as long as it is non-fusionable. In other words, an agent can condense and / or encapsulate nucleic acid cargo, such as ceDNA, but has little or no fusion activity. While we do not wish to be bound by theory, a condensant may have some fusion activity if it does not condense / encapsulate nucleic acids such as ceDNA, but the nucleic acid encapsulating the lipid nanoparticles formed with the condensant may be non-fusionable. The formulation processes described herein take advantage of the discovery that ceDNA compression occurs in solvents with a high ethanol content. When aqueous ceDNA (90% EtOH) is added to an ethanol solution of lipids (e.g., 90% EtOH) in a ratio such that the resulting solution is 90-92% ethanol and 8-10% water, the ceDNA is observed to exist in a compressed state due to dynamic light scattering. In such solvents (90-92% ethanol and 8-10% water), both lipids and ceDNA are solubilized, and no precipitation of either component is detected.

[0303] According to some embodiments, the formulation processes and methods described herein can encapsulate significantly more double-stranded DNA (e.g., ceDNA) than previously reported. According to some embodiments, the LNPs described herein can encapsulate more than about 60% of rigid double-stranded DNA such as ceDNA, more than about 65% of rigid double-stranded DNA such as ceDNA, more than about 70% of rigid double-stranded DNA such as ceDNA, more than about 75% of rigid double-stranded DNA such as ceDNA, more than about 80% of rigid double-stranded DNA such as ceDNA, more than about 85% of rigid double-stranded DNA such as ceDNA, or more than about 90% of rigid double-stranded DNA such as ceDNA.

[0304] According to some embodiments, the solvent contains about 80% ethanol and about 20% water. According to some embodiments, the solvent contains about 81% ethanol and about 19% water. According to some embodiments, the solvent contains about 82% ethanol and about 18% water. According to some embodiments, the solvent contains about 83% ethanol and about 17% water. According to some embodiments, the solvent contains about 84% ethanol and about 16% water. According to some embodiments, the solvent contains about 85% ethanol and about 15% water. According to some embodiments, the solvent contains about 86% ethanol and about 14% water. According to some embodiments, the solvent contains about 87% ethanol and about 13% water. According to some embodiments, the solvent contains about 88% ethanol and about 12% water. According to some embodiments, the solvent contains about 89% ethanol and about 11% water. According to some embodiments, the solvent contains about 90% ethanol and about 10% water. According to some embodiments, the solvent comprises about 91% ethanol and about 9% water. According to some embodiments, the solvent comprises about 92% ethanol and about 8% water. According to some embodiments, the solvent comprises about 93% ethanol and about 7% water. According to some embodiments, the solvent comprises about 94% ethanol and about 6% water. According to some embodiments, the solvent comprises about 95% ethanol and about 5% water.

[0305] Cationic lipids are typically used to condense nucleic acid cargoes, such as ceDNA, at low pH and to drive membrane association and fusion. Generally, cationic lipids are lipids containing at least one amino group that is positively charged or protonated under acidic conditions, for example, pH 6.5 or lower. Cationic lipids can also be ionized lipids, such as ionized cationic lipids. "Non-fusion cationic lipids" refer to cationic lipids that can condense and / or encapsulate nucleic acid cargoes such as ceDNA, but have little to no fusion activity.

[0306] In one embodiment, noncationic lipids may constitute 20-90% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). For example, the cationic lipid molar content may be 20-70% (mol), 30-60% (mol), 40-60% (mol), 40-55% (mol), or 45-55% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, cationic lipids constitute about 50 mol% to about 90 mol% of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0307] In one embodiment, the SS-cleavable lipid is not MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3). DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the contents of which are incorporated herein by reference in their entirety. The structure of D-Lin-MC3-DMA (MC3) is shown below as lipid K. lipid K [ka]

[0308] In one embodiment, the cleavable lipid is not lipid ATX-002. Lipid ATX-002 is described in W02015 / 074085 (the contents thereof are incorporated herein by reference in their entirety). In one embodiment, the cleavable lipid is not (13Z,16Z)- / V, / V-dimethyl-3-nonyldocosa-13,16-diene-l-amine (compound 32). Compound 32 is described in WO2012 / 040184, the contents thereof are incorporated herein by reference in their entirety. In one embodiment, the cleavable lipid is not compound 6 or compound 22. Compounds 6 and 22 are described in WO2015 / 199952, the contents thereof are incorporated herein by reference in their entirety.

[0309] Non-limiting examples of cationic lipids include SS-cleavable and pH-activated lipid-like substances-OP (ss-OP, formula I), SS-cleavable and pH-activated lipid-like substances-M (SS-M, formula V), SS-cleavable and pH-activated lipid-like substances-E (SS-E, formula VI), SS-cleavable and pH-activated lipid-like substances-EC (SS-EC, formula VII), SS-cleavable and pH-activated lipid-like substances-LC (SS-LC, formula VIII), SS-cleavable and pH-activated lipid-like substances-OC (SS-OC, formula IX), polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimers, lipofectin (combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE® (e.g., LIPOFECTAMINE® 2000), DOPE, Cytofectin (Gilead Examples include Sciences (Foster City, Calif.) and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be prepared from N-[l-(2,3-dioloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioloxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3b-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propaneaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide (DDAB). Nucleic acids (e.g., ceDNA or CELiD) may also be compounded with, for example, poly(L-lysine) or avidin, and lipids may or may not be present in this mixture, for example, steryl-poly(L-lysine).

[0310] In one embodiment, the cationic lipid is ss-OP of formula I. In another embodiment, the cationic lipid is SS-PAZ of formula II.

[0311] In one embodiment, the ceDNA vector disclosed herein is delivered using a cationic lipid as described in U.S. Patent No. 8,158,601 or a lipid as described in U.S. Patent No. 8,034,376.

[0312] Noncationic lipids In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further contain noncationic lipids. Noncationic lipids can help enhance fusion and improve the stability of LNPs during formation. Examples of noncationic lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Therefore, noncationic lipids can be neutral, uncharged, zwitterionic, or anionic lipids. Noncationic lipids are typically used to enhance membrane fusion.

[0313] Examples of noncationic lipids include distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and distearoylphosphatidylethanolamine (DSP E) Monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethylPE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethylPE), 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dieluoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dieluoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,Examples include, but are not limited to, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphaticaside, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholines and diacylphosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably C, 10 -C 24 The acyl group is derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0314] Other examples of noncationic lipids suitable for use in lipid particles (e.g., lipid nanoparticles) include, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and nonphosphorus lipids such as sphingomyelin.

[0315] In one embodiment, the noncationic lipid is a phospholipid. In one embodiment, the noncationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the noncationic lipid is DSPC. In other embodiments, the noncationic lipid is DOPC. In other embodiments, the noncationic lipid is DOPE.

[0316] In some embodiments, non-cationic lipids may constitute 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the non-cationic lipid content is 0.5-15% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the non-cationic lipid content is 5-12% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the non-cationic lipid content is 5-10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 6% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 7.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 7.5% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the noncationic lipid content is approximately 8.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In another embodiment, the noncationic lipid content is approximately 9.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In several embodiments, the noncationic lipid content is approximately 10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In yet another embodiment, the noncationic lipid content is approximately 11% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0317] Exemplary noncationic lipids are described in International Patent Application Publication 2017 / 099823 and U.S. Patent Application Publication 2018 / 0028664, both of which are incorporated herein by reference in their entirety.

[0318] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further contain components such as sterols to provide integrity and stability to the lipid particle membrane. In one embodiment, exemplary sterols that can be used in the lipid particles are cholesterol or its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestanol, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholesterol hemysuccinate (CHEMS).

[0319] Exemplary cholesterol derivatives are described in International Patent Application Publication No. 2009 / 127060 and U.S. Patent Application Publication No. 2010 / 0130588, both of which are incorporated herein by reference in their entirety.

[0320] In one embodiment, the membrane-integrating component, such as sterols, may constitute 0-50% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 20-50% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 30-40% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 35-45% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such component constitutes 38-42% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles).

[0321] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. Generally, these are used to inhibit aggregation of the lipid particles (e.g., lipid nanoparticles) and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEGylated lipid, e.g., a (methoxypolyethylene glycol)-conjugated lipid. In some other embodiments, the PEGylated lipid is PEG 2000 -DMG (Dimyristoyl Glycerol).

[0322] Examples of PEGylated lipids include PEG-diacylglycerol (DAG) (e.g., l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG)), PEG-dialkoxypropylcarbam, N-(carbonyl-methoxypoly This includes, but is not limited to, ethylene glycol (2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5,885,613, US6,287,591, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904, all of which are incorporated herein by reference in their entirety.

[0323] In one embodiment, the PEG-DAA PEGylated lipid can be, for example, PEG-dilauroxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be PEG-DMG, PEG-dilauryl glycerol, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauryl glycamide, PEG-dimyristyl glycamide, PEG-dipalmitoyl glycamide, PEG-distearyl glycamide, PEG-cholesterol (l-[8’-(cholesta-5-en-3[beta]-oxy)carboxamido-3’,6’-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[omega]-methyl-poly(ethylene glycol) ether), and one or more of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG lipid is PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] [Chemical formula] can be selected from the group consisting of.

[0324] In some embodiments, the PEGylated lipid is N-(carbonyl-methoxypolyethylene glycoln)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG n , where n is 350, 500, 750, 1000 or 2000), N-(carbonyl-methoxypolyethylene glycol n)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG nSelected from the group consisting of DSPE-polyglycerin-cyclohexyl-carboxylic acid, DSPE-polyglycerin-2-methylglutar-carboxylic acid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG-OH), polyethylene glycol-dimilystoglycerol (PEG-DMG), polyethylene glycol-distearoylglycerol (PEG-DSG), or N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)200011 (C8PEG2000 ceramide) where n is 350, 500, 750, 1000 or 2000. n In some examples, the PEG-lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000). DSPE-PEG is where n is 350, 500, 750, 1000, or 2000. n In some examples, the PEG-lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000). In some embodiments, the PEG-lipid is DSPE-PEG-OH. In some preferred embodiments, the PEG-lipid is PEG-DMG.

[0325] In some embodiments, the bound lipid, such as a PEGylated lipid, includes a tissue-specific targeting ligand, such as a first or second targeting ligand. For example, PEG-DMG conjugated with a GalNAc ligand.

[0326] In one embodiment, lipids conjugated with molecules other than PEG can be used instead of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used instead of or in addition to PEG-lipids. Exemplary conjugated lipids, namely PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in International Patent Application Publications 1996 / 010392, 1998 / 051278, W02002 / 087541, W02005 / 026372, and 2008 / 1 Nos. 47438, W02009 / 086558, W02012 / 000104, 2017 / 117528, 2017 / 099823, 2015 / 199952, W02017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 U.S. Patent Application Publication Nos. / 006282, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0376224, 2016 / 0317 These are described in U.S. Patent Nos. 458, 2013 / 0303587, 2013 / 0303587, and 2011 / 0123453, as well as U.S. Patents Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, all of which are incorporated herein by reference in their entirety.

[0327] In some embodiments, PEGylated lipids may constitute 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the PEGylated lipid content is 0.5-10% (mol). In some embodiments, the PEGylated lipid content is 1-5% (mol). In some embodiments, the PEGylated lipid content is 2-4% (mol). In some embodiments, the PEGylated lipid content is 2-3% (mol). In one embodiment, the PEGylated lipid content is approximately 2% (mol). In one embodiment, the PEGylated lipid content is approximately 2.5% (mol). In some embodiments, the PEGylated lipid content is approximately 3% (mol). In one embodiment, the PEGylated lipid content is approximately 3.5% (mol). In one embodiment, the PEGylated lipid content is approximately 4% (mol).

[0328] It is understood that the molar ratio of cationic lipids, e.g., ionized cationic lipids, to non-cationic lipids, sterols, and PEGylated lipids can be varied as needed. For example, lipid particles (e.g., lipid nanoparticles) may contain 30-70% cationic lipids by mole or total weight of the composition, 0-60% cholesterol by mole or total weight of the composition, 0-30% non-cationic lipids by mole or total weight of the composition, and 2-5% PEGylated lipids by mole or total weight of the composition. In one embodiment, the composition contains lipid particles (e.g., lipid nanoparticles) that comprise 40-60% cationic lipids by mole or total weight of the composition, 30-50% cholesterol by mole or total weight of the composition, 5-15% non-cationic lipids by mole or total weight of the composition, and 2-5% PEGylated or conjugated lipids by mole or total weight of the composition. In one embodiment, the composition comprises 40-60% ionized lipids by mole or total weight, 30-40% cholesterol by mole or total weight, 5-10% noncationic lipids by mole or total weight, and 2-5% PEGylated lipids by mole or total weight. The composition may contain lipid particles (e.g., lipid nanoparticles) comprising 60-70% cationic lipids by mole or total weight, 25-35% cholesterol by mole or total weight, 5-10% noncationic lipids by mole or total weight, and 2-5% PEGylated lipids by mole or total weight. The composition may also contain up to 45-55% ionized lipids by mole or total weight, 35-45% cholesterol by mole or total weight, 2-15% noncationic lipids by mole or total weight, and 2-5% PEGylated lipids by mole or total weight.The formulation may also contain, for example, 8-30% cationic lipids by mole or total weight of the composition, 5-15% non-cationic lipids by mole or total weight of the composition, and 0-40% cholesterol by mole or total weight of the composition; 4-25% cationic lipids by mole or total weight of the composition, 4-25% non-cationic lipids by mole or total weight of the composition, 2-25% cholesterol by mole or total weight of the composition, 10-35% complex lipids by mole or total weight of the composition, and 5% cholesterol by mole or total weight of the composition; or by mole or The composition may also consist of lipid nanoparticles comprising 2-30% cationic lipids by total weight, 2-30% non-cationic lipids by mole or total weight of the composition, 1-15% cholesterol by mole or total weight of the composition, 2-35% PEGylated lipids by mole or total weight of the composition, and 1-20% cholesterol by mole or total weight of the composition; or further comprising up to 90% cationic lipids by mole or total weight of the composition, and 2-10% non-cationic lipids by mole or total weight of the composition; or further comprising 100% cationic lipids by mole or total weight of the composition. In some embodiments, the lipid particle formulation comprises cationic lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of 50:9:38.5:2.5.

[0329] In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation contains cationic lipids, non-cationic phospholipids, cholesterol, and PEGylated lipids (conjugated lipids) in a molar ratio of approximately 50:7:40:3.

[0330] In other embodiments, the Disclosure provides lipid nanoparticle formulations comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0331] In one embodiment, the lipid particles (e.g., lipid nanoparticles) include cationic lipids, non-cationic lipids (e.g., phospholipids), sterols (e.g., cholesterol), and PEGylated lipids (conjugated lipids), with the molar ratio of lipids ranging from 20 to 70 mole percent for cationic lipids (target 30 to 60), from 0 to 30 mole percent for non-cationic lipids (target 0 to 15), from 20 to 70 mole percent for sterols (target 30 to 50), and from 1 to 6 mole percent for PEGylated lipids (conjugated lipids) (target 2 to 5).

[0332] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Patent Application No. PCT / US2018 / 050042, filed on 7 September 2018, which are incorporated herein in their entirety and are intended for use in the methods and compositions disclosed herein.

[0333] The size of lipid particles (e.g., lipid nanoparticles) can be determined by quasi-elastic light scattering using Malvern Zetasizer Nano ZS (Malvern, UK). According to some embodiments, the average diameter of LNPs determined by light scattering is less than about 75 nm or less than about 70 nm. According to some embodiments, the average diameter of LNPs determined by light scattering is about 50 nm to about 75 nm or about 50 nm to about 70 nm.

[0334] The pKa of formulated cationic lipids may correlate with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated herein in their entirety by reference). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles consisting of cationic lipids / DSPC / cholesterol / PEG-lipids (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipids can be prepared using in-line processes described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. The vesicles can be diluted to 24 mM lipids in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH in the range of 2.5 to 11. Aliquots of the TNS solution can be added to a final concentration of 1 mM, and the eddy-mixed emission intensity is subsequently measured at room temperature using an SLM Aminco Series 2 emission spectrophotometer with an excitation wavelength of 321 nm and an oscillation wavelength of 445 nm. S-curve best fit analysis can be applied to the fluorescence data, and the pKa is measured as the pH that produces the semi-optimal fluorescence intensity.

[0335] In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity is compared at doses of 0.3 and 1.0 mg ceDNA / kg and expressed as luciferase ng per g of liver measured 4 hours after administration.

[0336] Without limitation, the lipid particles (e.g., lipid nanoparticles) of this disclosure include lipid formulations 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) include a capsid-free, nonviral DNA vector and a cationic lipid or a salt thereof.

[0337] In one embodiment, the lipid particles (e.g., lipid nanoparticles) contain cationic lipids / non-cationic lipids / sterols / conjugated lipids in a molar ratio of 50:10:38.5:1.5. In one embodiment, the present disclosure provides a lipid particle formulation (e.g., lipid nanoparticles) comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0338] III. Rigid therapeutic nucleic acids Aspects of this disclosure generally provide rigid therapeutic nucleic acids (TNAs), such as closed-end DNA (ceDNA), and lipid particles (e.g., lipid nanoparticles) containing lipids.

[0339] Closed-end DNA (ceDNA) vectors Embodiments of this disclosure are based on methods and compositions comprising closed-end linear double-stranded (ceDNA) vectors capable of expressing transgenes (e.g., therapeutic nucleic acids). ceDNA vectors as described herein are not subject to the packaging constraints imposed by the limited space within a viral capsid. The ceDNA vector represents a variable eukaryotically produced alternative to a prokaryotically produced plasmid DNA vector, in contrast to an encapsulated AAV genome. This allows for the insertion of regulatory elements, such as regulatory switches, large transgenes, or multiple transgenes, as disclosed herein.

[0340] ceDNA vectors preferably have a linear and continuous structure rather than a discontinuous structure. A linear and continuous structure is considered to be more stable against attack by cellular endonucleases and less likely to be recombinated and cause mutagenesis. Therefore, linear and continuous ceDNA vectors are a preferred embodiment. Continuous linear single-stranded intramolecular double-stranded ceDNA vectors may have a sequence covalently bonded to the terminal without encoding the AAV capsid protein. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are cyclic double-stranded nucleic acid molecules of bacterial origin. While the complementary strand of a plasmid can separate following denaturation to produce two nucleic acid molecules, conversely, a ceDNA vector may have a complementary strand but be a single DNA molecule and therefore remain a single molecule even when denatured. In some embodiments, ceDNA vectors may be produced without prokaryotic cell-type DNA base methylation, unlike plasmids. Therefore, ceDNA vectors and ceDNA plasmids differ in both their structure (particularly linear-to-cyclic) and the methods used to produce and purify these different objects, as well as in their DNA methylation, with ceDNA plasmids being prokaryotic and ceDNA vectors being eukaryotic.

[0341] Provided herein are nonviral capsid-free ceDNA molecules (ceDNA) having covalent closed ends. These nonviral capsid-free ceDNA molecules can be produced in permissible host cells from expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrated cell lines) containing heterologous genes (e.g., transgenes, particularly therapeutic transgenes) positioned between two different inverted end repeat (ITR) sequences, the ITRs being different with respect to each other. In some embodiments, one of the ITRs is modified by deletions, insertions, and / or substitutions compared to a wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs includes functional end degradation sites (trs) and Rep-binding sites. The ceDNA vector is preferably double-stranded over at least a portion of the molecule, such as an expression cassette, e.g., self-complementary (e.g., ceDNA is not a double-stranded cyclic molecule). The ceDNA vector has covalent closed ends and is therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37°C for more than 1 hour.

[0342] In one embodiment, the ceDNA vector comprises a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, oriented from 5' to 3'. In one embodiment, the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric with respect to each other; that is, they have different three-dimensional spatial configurations. In exemplary embodiments, the first ITR may be a wild-type ITR and the second ITR may be a mutant or modified ITR, or vice versa; or the first ITR may be a mutant or modified ITR and the second ITR may be a wild-type ITR. In one embodiment, both the first and second ITRs are modified but have different sequences, different modifications, or are not identical modified ITRs, and have different three-dimensional spatial configurations. In other words, a ceDNA vector using asymmetric ITRs may have ITRs in which any modification of one ITR relative to the WT-ITR is not reflected in the other ITRs, or, if the asymmetric ITRs are modified asymmetric ITR pairs, they may have different sequences and different three-dimensional shapes relative to each other.

[0343] In one embodiment, the ceDNA vector comprises a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, in the 5' to 3' direction, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are symmetric or substantially symmetric with respect to each other, i.e., the ceDNA vector may contain ITR sequences having a symmetric three-dimensional spatial configuration, so that their structures are the same shape in geometric space or have the same A, C-C', B-B' loops in three-dimensional space. In such embodiments, the symmetric ITR pair, or substantially symmetric ITR pair, may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair may have one or more modifications from the wild-type ITR and have the same sequence that is inversely complementary (inverted) to each other. In one embodiment, the modified ITR pair is substantially symmetric as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetric three-dimensional shape. In some embodiments, the symmetric ITR, or substantially symmetric ITR, may be wild-type (WT-ITR) as described herein, i.e., both ITRs have wild-type sequences but do not necessarily have to be WT-ITRs of the same AAV serotype. In one embodiment, one WT-ITR may originate from one AAV serotype, and the other WT-ITR may originate from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they may have one or more conserved nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration.

[0344] The wild-type, mutant, or otherwise modified ITR sequences provided herein represent the DNA sequences contained in expression constructs (e.g., ceDNA plasmids, ceDNA bacmids, ceDNA baculoviruses) for the production of ceDNA vectors. Therefore, the ITR sequences actually contained in ceDNA vectors produced from ceDNA plasmids or other expression constructs may be identical or different to the ITR sequences provided herein as a result of naturally occurring changes (e.g., replication errors) that occur during the production process.

[0345] In one embodiment, a ceDNA vector described herein, comprising an expression cassette having a transgene which is a therapeutic nucleic acid sequence, can be operably ligated to one or more regulatory sequences that enable or control the expression of the transgene. In one embodiment, a polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is adjacent to the first and second ITR sequences, and the first and second ITR sequences are asymmetrical or symmetrical with respect to each other.

[0346] In one embodiment, the expression cassette includes, in this order, a promoter located between two ITRs and operably linked to the transgene, a post-transcriptional regulatory element, and one or more polyadenylation and termination signals. In one embodiment, the promoter is regulated-inducible or repressible. The promoter may be any sequence that promotes transcription of the transgene. In one embodiment, the promoter is a CAG promoter or a variation thereof. The post-transcriptional regulatory element is a sequence that modulates the expression of the transgene and, as a non-limiting example, is any sequence that creates a tertiary structure that enhances the expression of the transgene, which is a therapeutic nucleic acid sequence.

[0347] In one embodiment, the post-transcriptional regulatory element comprises WPRE. In one embodiment, the polyadenylation and termination signal comprises BGH polyA. Any cis-regulatory element known in the art, or a combination thereof, such as the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements (including, but not limited to, thymidine kinase genes of herpes simplex virus or hepatitis B virus (HBV)) may be used in addition. In one embodiment, the expression cassette length in the 5'-3' direction exceeds the maximum length known to be capsidized in AAV virion. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0348] In one embodiment, the expression cassette may include more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range of about 4,000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette may include a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 50,000 nucleotides in length. In one embodiment, the expression cassette may include a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 75,000 nucleotides in length. In one embodiment, the expression cassette may include a transgene which is a therapeutic nucleic acid sequence in the range of 500 to 10,000 nucleotides in length. In one embodiment, the expression cassette may include a transgene which is a therapeutic nucleic acid sequence in the range of 1,000 to 10,000 nucleotides in length. In one embodiment, the expression cassette may contain a transgene, which is a therapeutic nucleic acid sequence in the range of 500 to 5,000 nucleotides in length. Because ceDNA vectors do not have the size limitations of capsidized AAV vectors, it is possible to deliver large-sized expression cassettes to the host. In one embodiment, the ceDNA vector lacks prokaryotic cell-specific methylation.

[0349] In one embodiment, the nucleic acid for rigidity therapy may be a plasmid.

[0350] In one embodiment, the ceDNA vector disclosed herein is used for therapeutic purposes (e.g., medical, diagnostic, or veterinary use) or for immunogenic polypeptides.

[0351] The expression cassette may contain any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector contains any gene of interest in a subject, comprising one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0352] In one embodiment, the ceDNA expression cassette may include, for example, an expressible exogenous sequence (e.g., an open reading frame) encoding a protein that is absent, inactive, or insufficiently active in the recipient, or a gene encoding a protein having a desired biological or therapeutic effect. In one embodiment, the exogenous sequence, such as a donor sequence, may encode a gene product that can function to correct the expression of a defective gene or transcript. In one embodiment, the expression cassette may also encode a correction DNA strand and encode a polypeptide, sense or antisense oligonucleotide, or RNA (coding or non-coding; e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). In one embodiment, the expression cassette may include an exogenous sequence encoding a reporter protein used for experimental or diagnostic purposes, e.g., β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0353] Therefore, the expression cassette may contain any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to mutation, or any gene that would produce a therapeutic effect if its overexpression is considered to be within the scope of this disclosure. The ceDNA vector may contain a template or donor nucleotide sequence used as a correction DNA strand to be inserted after a double-strand break (or nick) provided by a nuclease. The ceDNA vector may contain a template nucleotide sequence used as a correction DNA strand to be inserted after a double-strand break (or nick) provided by an inducible RNA nuclease, meganuclease, or zinc finger nuclease.

[0354] therapeutic nucleic acids Examples of therapeutic nucleic acids in this disclosure include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), ribozymes, closed-end double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministrings"), doggybone®, protelomere-closed DNA, or dumbbell linear DNA), Dicer substrate dsRNAs, small hairpin RNAs (shRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), mRNAs, tRNAs, rRNAs, and DNA viral vectors, viral RNA vectors, and any combination thereof.

[0355] siRNA or miRNAs that can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi) are also intended to be nucleic acid therapeutics in this disclosure. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of the siRNA or miRNA is removed by the RISC complex. Upon binding to complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strand. RNAi is achieved by inducing specific disruption of the mRNA, which results in the downregulation of the corresponding protein.

[0356] Antisense oligonucleotides (ASOs) and ribozymes, which inhibit mRNA translation into proteins, can be used as nucleic acid therapies. In the case of antisense constructs, these single-stranded deoxynucleic acids have a sequence complementary to the target protein mRNA sequence, and Watson can bind to the mRNA via click base pairing. This binding prevents translation of the target mRNA and / or induces RNaseH degradation of the mRNA transcript. As a result, antisense oligonucleotides have increased specificity of action (i.e., downregulation of specific disease-related proteins).

[0357] In any of the methods provided herein, the therapeutic nucleic acid may be therapeutic RNA. Such therapeutic RNA may be an mRNA translation inhibitor, an RNA interference (RNAi) agent, a catalytically active RNA molecule (ribozyme), transfer RNA (tRNA), or RNA that binds to mRNA transcripts (ASOs), proteins, or other molecular ligands (aptamers). In any of the methods provided herein, the RNAi agent may be double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, small hairpin RNA, or triple-helix-forming oligonucleotides.

[0358] According to some embodiments, the formulation processes and methods described herein can encapsulate significantly more double-stranded DNA (e.g., ceDNA) than previously reported. According to some embodiments, the LNPs described herein can encapsulate more than about 60% of rigid double-stranded DNA such as ceDNA, more than about 65% of rigid double-stranded DNA such as ceDNA, more than about 70% of rigid double-stranded DNA such as ceDNA, more than about 75% of rigid double-stranded DNA such as ceDNA, more than about 80% of rigid double-stranded DNA such as ceDNA, more than about 85% of rigid double-stranded DNA such as ceDNA, or more than about 90% of rigid double-stranded DNA such as ceDNA.

[0359] IV. Nucleic Acids for Degeneration Therapy Aspects of this disclosure further provide pharmaceutical compositions comprising lipid particles (e.g., lipid nanoparticles) and denatured therapeutic nucleic acids (TNAs), where TNA is as defined above. In one embodiment, the denatured TNA is closed-end DNA (ceDNA). The term “denatured therapeutic nucleic acid” refers to partially or completely TNA whose conformation has been altered from a standard type B structure. Conformational alterations may include changes in secondary structure (i.e., base-pair interactions within a single nucleic acid molecule) and / or tertiary structure (i.e., double helix structure). While not bound by theory, the inventors have believed that TNA treated with alcohol / aqueous solution or a pure alcohol solvent results in denaturation of the nucleic acid to a conformation that enhances encapsulation efficiency by LNPs, producing LNP formulations with smaller diameter sizes (i.e., less than 75 nm, e.g., an average size of approximately 68–74 nm in diameter). All LNP average diameter sizes and size ranges described herein apply to LNPs containing denatured TNAs.

[0360] When DNA is in an aqueous environment, it has a type B structure with 10.4 base pairs per complete helical turn. Gradually changing this aqueous environment by adding a moderately low-polarity alcohol such as methanol loosens the helical twist, and the DNA smoothly changes to a form with only 10.2 base pairs per helical turn, as can be visualized by circular dichroism (CD) spectroscopy. In one embodiment, the denatured TNA in the pharmaceutical composition provided herein has a type 10.2 structure.

[0361] In contrast to this behavior, replacing water with a slightly less polar alcohol such as ethanol results in the same type of structural change occurring until approximately 65% ​​of the water is replaced with ethanol. At this point, the DNA rapidly transforms into a type A structure with a tighter twisted helix containing 11 base pairs per turn of the helix, as visualized in CD. In one embodiment, the denatured TNA in the pharmaceutical composition provided herein has a type A structure.

[0362] According to some embodiments, the modified TNA in the pharmaceutical compositions provided herein has a rod-like structure when visualized by transmission electron microscopy (TEM). According to some embodiments, the modified TNA in the pharmaceutical compositions provided herein has a circular-like structure when visualized by transmission electron microscopy (TEM). In contrast, unmodified TNA has a chain-like structure.

[0363] According to some embodiments, the modified TNA in the pharmaceutical compositions provided herein has little or no hydrogen bonding, no base stacking, and a P-type structure having a condensed tertiary structure.

[0364] V.ceDNA vector production Embodiments of this disclosure are based on methods and compositions comprising closed-end linear double-stranded (ceDNA) vectors capable of expressing transgenes (e.g., TNAs). ceDNA vectors as described herein are not subject to the packaging constraints imposed by the limited space within a viral capsid. The ceDNA vector represents a variable eukaryotically produced alternative to a prokaryotically produced plasmid DNA vector, in contrast to an encapsulated AAV genome. This allows for the insertion of regulatory elements, such as regulatory switches, large transgenes, or multiple transgenes, as disclosed herein.

[0365] A method for producing the ceDNA vector described herein, including asymmetric ITR pairs or symmetric ITR pairs as defined herein, is described in Section IV of PCT / US18 / 49996, filed on 7 September 2018, which is incorporated herein by reference in its entirety. As described herein, a ceDNA vector may be obtained by a process comprising, for example, a) incubating a population of host cells (e.g., insect cells) containing a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), wherein the host cells lack a viral capsid coding sequence for a sufficient period of time in the presence of a Rep protein, and b) harvesting and isolating the ceDNA vector from the host cells. The presence of the Rep protein induces replication of the vector polynucleotide having a modified ITR, thereby producing the ceDNA vector in the host cells.

[0366] The following are provided as non-exclusive examples.

[0367] According to some embodiments, synthetic ceDNA is produced by excision from a double-stranded DNA molecule. The synthetic production of ceDNA vectors is described in Examples 2–6 of International Patent Application PCT / US19 / 14122, filed January 18, 2019, which is incorporated herein by reference in its entirety. One exemplary method for producing a ceDNA vector using a synthetic method involving excision of a double-stranded DNA molecule. Briefly, a ceDNA vector can be produced using a double-stranded DNA construct. See, for example, Figures 7A–8E of PCT / US19 / 14122. In some embodiments, the double-stranded DNA construct is a ceDNA plasmid (see, for example, Figure 6 of International Patent Application PCT / US2018 / 064242, filed December 6, 2018).

[0368] In some embodiments, the construct for creating a ceDNA vector includes additional components for regulating the expression of the transgene, such as a regulatory switch for regulating the expression of the transgene, or a kill switch that can kill cells containing the vector.

[0369] Molecular regulatory switches are those that, in response to a signal, produce a measurable change in state. Such regulatory switches can be usefully combined with the ceDNA vectors described herein to control the output of transgene expression. In some embodiments, the ceDNA vector includes a regulatory switch that helps fine-tune the expression of the transgene. For example, it may serve as a biological containment function for the ceDNA vector. In some embodiments, the switch is an "on / off" switch designed to initiate or stop (i.e., shut down) the controllable and regulated expression of the gene of interest in the ceDNA vector. In some embodiments, the switch may include a "kill switch" that, once activated, can instruct a cell containing the synthetic ceDNA vector to undergo programmed cell death. Exemplary regulatory switches incorporated into use with ceDNA vectors can be used to regulate the expression of transgenes and are discussed more fully in International Application PCT / US18 / 49996, which is incorporated herein by reference in whole.

[0370] Another exemplary method for producing a ceDNA vector using a synthetic method involving the assembly of various oligonucleotides is provided in Example 3 of PCT / US19 / 14122, in which the ceDNA vector is produced by synthesizing 5' oligonucleotides and 3' ITR oligonucleotides and ligating the ITR oligonucleotides to a double-stranded polynucleotide containing an expression cassette. Figure 11B of PCT / US19 / 14122, which is incorporated entirely herein by reference, shows an exemplary method for ligating 5' ITR oligonucleotides and 3' ITR oligonucleotides to a double-stranded polynucleotide containing an expression cassette.

[0371] An exemplary method for producing a ceDNA vector using a synthetic method is provided in Example 4 of PCT / US19 / 14122, which is incorporated entirely herein by reference, and involves using a single-stranded linear DNA containing two sense ITRs covalently bound to two antisense ITRs adjacent to a sense expression cassette sequence and adjacent to an antisense expression cassette, and then ligating the ends of this single-stranded linear DNA to form a closed-end single-stranded molecule. A non-limiting example involves synthesizing and / or producing a single-stranded DNA molecule, annealing a portion of the molecule to form a single linear DNA molecule having one or more base-pairing regions of a secondary structure, and then ligating the free 5' and 3' ends to form a closed-end single-stranded molecule.

[0372] In yet another embodiment, the disclosure provides host cell lines that stably incorporate the DNA vector polynucleotide expression templates (ceDNA templates) described herein into their own genomes for use in the production of nonviral DNA vectors. A method for producing such cell lines is described in Lee, L. et al. (2013) Plos One 8(8):e69879, which is incorporated herein by reference in its entirety. For example, the Rep protein is added to host cells at an MOI of 3. In one embodiment, the host cell line is an invertebrate cell line, preferably an insect Sf9 cell. If the host cell line is a mammalian cell line, preferably a 293 cell, the cell line may have a stably incorporated polynucleotide vector template, and the Rep protein can be introduced into the cells using a second vector, such as a herpesvirus, enabling the excision and amplification of ceDNA in the presence of Rep.

[0373] Any promoter can be manipulatively ligated to a heterologous nucleic acid (e.g., a reporter nucleic acid or therapeutic transgene) of the vector polynucleotide. The expression cassette may contain synthetic regulatory elements such as a CAG promoter. The CAG promoter comprises (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and first intron of the chicken beta-actin gene, and (ii) a splice acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette may contain an alpha-1-antitrypsin (AAT) promoter, a liver-specific (LP1) promoter, or a human elongation factor-1-alpha (EF1-α) promoter. In some embodiments, the expression cassette includes one or more constitutive promoters, e.g., a retroviral Roussarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), a cytomegalovirus (CMV) early promoter (optionally having a CMV enhancer). Alternatively, inducible or repressive promoters, unvariable promoters for transgenes, tissue-specific promoters, or various promoters known in the art may be used. Suitable transgenes for gene therapy are well known to those skilled in the art.

[0374] Capsid-free ceDNA vectors may also be produced from vector polynucleotide expression constructs further comprising cis-regulatory elements, or combinations of cis-regulatory elements. Non-limiting examples include the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and BGH polyA, or, for example, beta-globin polyA. Other post-transcriptional elements include, for example, thymidine kinase genes of herpes simplex virus or hepatitis B virus (HBV). Expression cassettes may include any polyadenylated sequence or modification thereof known in the art, such as natural or synthetic sequences isolated from, for example, bovine BGHpA or viral SV40pA. Some expression cassettes may also include SV40 late polyA signal upstream enhancer (USE) sequences. USE may be used in combination with SV40pA or heterologous polyA signals.

[0375] The time for harvesting and collecting the DNA vectors described herein from cells may be selected and optimized to achieve high yield production of ceDNA vectors. For example, the harvesting time may be selected considering cell viability, cell morphology, cell proliferation, etc. In one embodiment, cells are harvested after they have grown under sufficient conditions and sufficient time has elapsed since baculovirus infection to produce the DNA vector, but before the majority of cells begin to die due to viral toxicity. The DNA vector may be isolated using a plasmid purification kit such as the Qiagen Endo-Free plasmid kit. Other methods developed for plasmid isolation may also be adapted for DNA vectors. In general, any nucleic acid purification method may be employed.

[0376] DNA vectors can be purified by any means known to those skilled in the art for the purification of DNA. In one embodiment, the ceDNA vector is purified as a DNA molecule. In another embodiment, the ceDNA vector is purified as an exosome or microparticle.

[0377] In one embodiment, the capsid-free nonviral DNA vector comprises or is obtained from a plasmid containing a polynucleotide template comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an exogenous DNA expression cassette), and a modified AAV ITR in that order, wherein the template nucleic acid molecule lacks the AAV capsid protein coding. In further embodiments, the nucleic acid template of the Disclosure lacks a viral capsid protein coding sequence (i.e., lacks not only the AAV capsid gene but also the capsid genes of other viruses). In addition, in certain embodiments, the template nucleic acid molecule also lacks the AAV Rep protein coding sequence. Thus, in preferred embodiments, the nucleic acid molecule of the Disclosure lacks both a functional AAV cap and an AAV rep gene.

[0378] In one embodiment, the ceDNA vector may include an ITR structure mutated with respect to the wild-type AAV2 ITR disclosed herein, but still retaining the manipulable RBE, TRS, and RBE' portions.

[0379] ceDNA plasmid A ceDNA plasmid is a plasmid used for late-stage production of a ceDNA vector. In one embodiment, a ceDNA plasmid can be constructed using known techniques that provide, as operably linked components in the transcription direction, at least (1) a modified 5'ITR sequence, (2) an expression cassette containing a cis-regulatory element, e.g., a promoter, inducible promoter, regulatory switch, enhancer, etc., and (3) a modified 3'ITR sequence (the 3'ITR sequence is asymmetric with respect to the 5'ITR sequence). In some embodiments, the expression cassette flanked by the ITRs includes a cloning site for introducing an exogenous sequence. The expression cassette replaces the rep and cap coding regions of the AAV genome.

[0380] In one embodiment, the ceDNA vector is obtained from a plasmid referred to herein as a “ceDNA-plasmid” encoding a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette containing a transgene, and a mutant or modified AAV ITR in that order, the ceDNA-plasmid lacking an AAV capsid protein coding sequence. In an alternative embodiment, the ceDNA-plasmid encodes a first (or 5') modified or mutant AAV ITR, an expression cassette containing a transgene, and a second (or 3') modified AAV ITR in that order, the ceDNA-plasmid lacking an AAV capsid protein coding sequence, and the 5' and 3' ITRs being symmetrical with respect to each other. In an alternative embodiment, the ceDNA plasmid encodes a first (or 5') modified or mutant AAV ITR, an expression cassette containing the transgene, and a second (or 3') mutant or modified AAV ITR in this order, wherein the ceDNA plasmid lacks an AAV capsid protein coding sequence, and the 5' and 3' modified ITRs have the same modifications (i.e., they are inversely complementary or symmetrical to each other).

[0381] In one embodiment, the ceDNA-plasmid system lacks a viral capsid protein-coding sequence (i.e., it lacks not only the AAV capsid gene but also the capsid genes of other viruses). In one embodiment, the ceDNA plasmid also lacks an AAV Rep protein-coding sequence. In one embodiment, the ceDNA-plasmid lacks a variable palindromic sequence that enables hairpin formation, in addition to the functional AAV cap and AAV rep genes (GG-3' in the case of AAV2). In one embodiment, the ceDNA-plasmid of this disclosure can be generated using the native nucleotide sequence of the genome of any AAV serotype known in the art. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8 genomes, and is available, for example, at NCBI:NC 002077, NC 001401, NC001729, NC001829, NC006152, NC 006260, NC 006261, Kotin and Smith, The Springer Index of Viruses, and URLs managed by Springer. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV2 genome. In one embodiment, the ceDNA-plasmid backbone is a synthetic backbone genetically engineered to be contained in the 5' and 3' ITRs derived from one of these AAV genomes. In one embodiment, the ceDNA plasmid may optionally contain selectable or selectable markers for use in establishing ceDNA vector-producing cell lines. In one embodiment, the selectable marker may be inserted downstream of the 3' ITR sequence (i.e., 3'). In another embodiment, the selectable marker may be inserted upstream of the 5' ITR sequence (i.e., 5'). Suitable selectable markers include, for example, those that confer drug resistance. Selectable markers may include, for example, the blasticidin S resistance gene, kanamycin, geneticin, etc.

[0382] VI. Preparation of Lipid Particles Lipid particles (e.g., lipid nanoparticles) can be spontaneously formed when ceDNA and lipids are mixed. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane (e.g., 100 nrn cutoff) using a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. In one embodiment, lipid nanoparticles are formed as described in Example 3 of this specification.

[0383] In general, lipid particles (e.g., lipid nanoparticles) can be formed by any method known in the art. For example, lipid particles (e.g., lipid nanoparticles) can be prepared by methods described, for example, US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129 and US2010 / 0130588, the contents of each of these are incorporated herein by reference in their entirety. In some embodiments, lipid particles (e.g., lipid nanoparticles) can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. Processes and apparatus for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007 / 0042031, the contents of which are incorporated herein by reference in their entirety. A process and apparatus for preparing lipid nanoparticles using a serial dilution process is described in US2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0384] According to several embodiments, this disclosure provides LNPs comprising rigid DNA vectors and ionized lipid-containing ceDNA vectors as described herein. For example, lipid nanoparticle formulations prepared and filled with rigid therapeutic nucleic acids such as ceDNA obtained by a process disclosed in International Patent Application No. PCT / US2018 / 050042 filed on September 7, 2018, are incorporated herein by reference in their entirety. This disclosure includes a process of pre-compressing rigid therapeutic nucleic acids (TNAs) such as ceDNA in an 80% to 100% low molecular weight alcohol solution (e.g., ethanol, methanol, propanol, and isopropanol) before mixing the TNAs with lipids. Subsequently, loading and encapsulation of pre-compressed therapeutic nucleic acids can be achieved by conventional high-energy mixing by using a microfluidic device such as an ethanol-lipid Nanoassemblr™ with aqueous ceDNA (e.g., 80-100% ethanol, methanol, propanol, isopropanol, or a mixture) at a low pH that protonates ionized lipids and provides energy favorable for ceDNA / lipid association and particle nucleation. The particles can be further stabilized by aqueous dilution and removal of the organic solvent. The particles can be concentrated to a desired level. While we do not wish to be bound by theory, a pre-compression step of rigid DNA before mixing with lipids for DNA encapsulation is thought to have a beneficial effect in reducing the size of the resulting LNPs by compressing the DNA molecules in a low molecular weight alcohol solution before encapsulation.

[0385] According to some embodiments, the present disclosure provides a method for producing an LNP formulation, wherein the LNP comprises a cationic lipid and a TNA such as ceDNA, and the method includes adding aqueous TNA (e.g., ceDNA) to a low molecular weight alcohol such as an ethanol solution, where the alcohol concentration in the solution is about 80% to about 95% and the water concentration in the solution is about 20% to about 5%, adding the TNA (e.g., ceDNA) to a lipid solution (e.g., 80% to 100% EtOH) and an acidic aqueous buffer (e.g., malic acid), and optionally exchanging the buffer with a neutral pH aqueous buffer to produce an LNP formulation.

[0386] According to some embodiments, the concentration of low molecular weight alcohol (e.g., ethanol, methanol, propanol, or isopropanol) in the solution is about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 95%, about 85% to about 90%, about 90% to about 95%, or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%.

[0387] According to some embodiments, the concentration of water in the solution is approximately 20% to 5%, approximately 15% to 5%, approximately 10% to 5%, approximately 20% to 10%, approximately 20% to 15%, approximately 15% to 5%, approximately 15% to 10%, or approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 164%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, or approximately 5%.

[0388] According to some embodiments, the low molecular weight alcohol is selected from the group consisting of ethanol, methanol, propanol, and isopropanol. In some embodiments, aqueous TNA (e.g., ceDNA) is in a solution containing a mixture of two or three low molecular weight alcohols. In one embodiment, the low molecular weight alcohol solution is a mixture of ethanol and methanol. In another embodiment, the low molecular weight alcohol solution is a mixture of any combination of ethanol, methanol, propanol, and isopropanol. In yet another embodiment, the low molecular weight alcohol solution is a mixture of ethanol and propanol. In yet another embodiment, the low molecular weight alcohol solution contains 45% ethanol, 45% methanol, and 10% water.

[0389] According to some embodiments, the method further includes the step of diluting the ceDNA / lipid mixture solution with an acidic aqueous buffer. According to some embodiments, the acidic aqueous buffer is selected from malic acid / sodium malate or acetic acid / sodium acetate. According to some embodiments, the acidic aqueous buffer is available in concentrations of about 10–40 mM, about 10 mM–about 35 mM, about 10 mM–about 30 mM, about 10 mM–about 25 mM, about 10 mM–about 20 mM, about 10 mM–about 15 mM, about 15–40 mM, about 15 mM–about 35 mM, about 15 mM–about 30 mM, about 15 mM–about 25 mM, about 15 mM–about 20 mM, about 20–40 mM, about 20 mM–about 35 mM, about 210 mM–about 30 mM, about 20 mM–about 25 mM, about 25 The concentrations are 40 mM, approximately 25 mM to 35 mM, approximately 25 mM to 30 mM, approximately 310 to 40 mM, approximately 30 mM to 35 mM, approximately 35 mM to 40 mM, or approximately 10 mM to 25 mM, approximately 10 mM to 20 mM, approximately 10 mM to 15 mM, or approximately 10 mM, approximately 12 mM, approximately 14 mM, approximately 16 mM, approximately 18 mM, approximately 20 mM, approximately 22 mM, approximately 24 mM, approximately 26 mM, approximately 28 mM, approximately 30 mM, approximately 32 mM, approximately 34 mM, approximately 36 mM, approximately 38 mM, or approximately 40 mM.

[0390] According to some embodiments, the pH of the acidic aqueous buffer is approximately 3 to approximately 5, approximately 3 to approximately 4.5, approximately 3 to approximately 4, approximately 3 to approximately 3.5, approximately 3.5 to approximately 5, approximately 3.5 to approximately 4.5, approximately 3.5 to approximately 4, approximately 4 to approximately 5, approximately 4 to approximately 4.5, approximately 4.5 to approximately 5, or approximately 3, approximately 3.25, approximately 3.5, approximately 3.75, approximately 4, approximately 4.25, approximately 4.5, approximately 4.75, or approximately 5.

[0391] According to some embodiments, the neutral pH aqueous buffer is Dulbecco's phosphate-buffered saline, pH 7.4.

[0392] According to some embodiments, the process for preparing LNPs utilizes the discovery that rigid TNA compression, such as ceDNA, occurs in a solvent having a high alcohol content (>80%) (ethanol, methanol, propanol, and / or isopropanol). According to some embodiments, the formulation process described herein produces LNPs with a size in the range of about 50 to about 70 nm. According to some embodiments, the lipid particles of the present disclosure generally have an average diameter of about 20 nm to about 70 nm, about 25 nm to about 70 nm, about 30 nm to about 70 nm, about 35 nm to about 70 nm, about 40 nm to about 70 nm, about 45 nm to about 80 nm, about 50 nm to about 70 nm, about 60 nm to about 70 nm, about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, or about 70 nm. According to some embodiments, the formulation process described herein produces LNPs that encapsulate more than about 80% of a rigid TNA such as double-stranded ceDNA. According to some embodiments, the LNPs described herein can encapsulate more than about 60% of a rigid TNA such as double-stranded ceDNA, more than about 65% of a rigid TNA such as double-stranded ceDNA, more than about 70% of a rigid TNA such as double-stranded ceDNA, more than about 75% of a rigid TNA such as double-stranded ceDNA, more than about 80% of a rigid TNA such as double-stranded ceDNA, more than about 85% of a rigid TNA such as double-stranded ceDNA, or more than about 90% of a rigid TNA such as double-stranded ceDNA.

[0393] According to some embodiments, when compressed ceDNA-like TNAs in low molecular weight alcohols are mixed with an ethanol solution of lipids (80%-100% EtOH) in a ratio such that the resulting solution is 85-95% ethanol and 15-5% water, the ceDNA-like TNAs are observed to exist in a compressed state by dynamic light scattering. In such solvents, both lipids and ceDNA are solubilized, and no precipitation of either component is detected. Formulation of LNPs resulting in the encapsulation of compressed TNAs results in a much smaller diameter.

[0394] According to some embodiments, when aqueous TNA such as ceDNA is mixed with an ethanol solution of lipids in a ratio such that the resulting solution is 90-92% ethanol and 8-10% water in an acidic state (malic acid), the TNA such as ceDNA is observed to exist in a compact state by dynamic light scattering, and the resulting encapsulation yields LNPs with a much smaller diameter.

[0395] LNP formation is driven by mixing an ethanol solution of ceDNA / lipid solution with an acidic aqueous buffer using microfluidic mixing. According to some embodiments, the flow rate ratio between the acidic aqueous buffer and the ceDNA / lipid ethanol mixture is 2:1, 3:2, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1. According to some embodiments, after mixing is complete, the final solution is diluted with the acidic aqueous buffer so that the final ethanol content is approximately 4% to approximately 15%. According to some embodiments, after mixing is complete, the final solution is diluted with the acidic aqueous buffer so that the final ethanol content is approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, or approximately 15%. According to some embodiments, the final ethanol content is 4%. According to some embodiments, the final ethanol content is 12%. This solution containing LNP is buffered with a neutral pH aqueous buffer.

[0396] In one embodiment, lipid particles (e.g., lipid nanoparticles) can be prepared by an impact jet process. Generally, the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, such as citrate buffer, sodium acetate buffer, sodium acetate and magnesium chloride buffer, malic acid buffer, malic acid and sodium chloride buffer, or sodium citrate and sodium chloride buffer. The lipid-to-ceDNA mixing ratio can be about 45-55% lipid and about 65-45% ceDNA.

[0397] Lipid solutions may contain cationic lipids (e.g., ionized cationic lipids), non-cationic lipids (e.g., phospholipids such as DSPC, DOPE, and DOPC), PEG or PEG-conjugated molecules (e.g., PEG-lipids), and sterols (e.g., cholesterol) in an alcohol, such as ethanol, at a total lipid concentration of 5 to 30 mg / mL, more preferably 5 to 15 mg / mL, and most likely 9 to 12 mg / mL. In lipid solutions, the molar ratio of lipids may range from about 25 to 98%, preferably about 35 to 65%, for cationic lipids; about 0 to 15%, preferably about 0 to 12%, for non-ionizable lipids; about 0 to 15%, preferably about 1 to 6%, for PEG or PEG-conjugated lipid molecules; and about 0 to 75%, preferably about 30 to 50%, for sterols.

[0398] The ceDNA solution may contain ceDNA in a buffer solution having a pH in the range of 3.5 to 5 at a concentration range of 0.3 to 1.0 mg / mL, preferably 0.3 to 0.9 mg / mL.

[0399] To form LNPs, in one exemplary but non-limiting embodiment, two liquids are heated to a temperature in the range of about 15–40°C, preferably about 30–40°C, and then mixed in, for example, an impact jet mixer to immediately form LNPs. The mixing flow rate may be in the range of 10–600 mL / min. The tube ID range may be 0.25–1.0 mm, and the total flow rate may be 10–600 mL / min. The combination of flow rate and tube ID can have the effect of controlling the particle size of the LNPs to 30–200 nm. The solution can then be mixed with a buffer solution at a higher pH in a vol:vol ratio in the range of 1:1–1:3, preferably about 1:2 vol:vol. If necessary, this buffer solution may be at a temperature in the range of 15–40°C or 30–40°C. The mixed LNPs can then undergo an anion exchange filtration step. Before anion exchange, the mixed LNPs can be incubated for a certain period, for example, 30 minutes to 2 hours. The incubation temperature can be in the range of 15–40°C or 30–40°C. After incubation, the solution is filtered through a filter, such as a 0.8 μm filter, which includes an anion exchange separation step. This process can use tube IDs ranging from 1 mm to 5 mm and flow rates of 10–2000 mL / min.

[0400] After formation, the LNPs can be concentrated and filtered through an ultrafiltration process, in which alcohol is removed and the buffer is replaced with a final buffer solution, such as phosphate-buffered saline (PBS) at approximately pH 7, approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4.

[0401] In the ultrafiltration process, a tangential flow filtration format (TFF) with a nominal molecular weight cutoff range of 30–500 kD for the membrane can be used. The membrane format is hollow fiber or flat sheet cassette. In a TFF process with an appropriate molecular weight cutoff, LNP can be retained in the holding solution, and the filtrate or permeate contains waste from alcohol, citrate buffer, and final buffer. The TFF process is a multi-step process to an initial ceDNA concentration of 1–3 mg / mL. After concentration, the LNP solution is ultrafiltered against the final buffer at a volume of 10–20 to remove alcohol and perform buffer exchange. The material can then be further concentrated 1–3 times. The concentrated LNP solution can be sterile filtered.

[0402] VII. Pharmaceutical Compositions and Formulations Also provided herein are pharmaceutical compositions comprising TNA such as ceDNA lipid particles and pharmaceutically acceptable carriers or excipients.

[0403] In one embodiment, TNA (e.g., ceDNA) lipid particles (e.g., lipid nanoparticles) are provided with complete and partial encapsulation of therapeutic nucleic acids. In one embodiment, the nucleic acid therapeutic agent is fully encapsulated in lipid particles (e.g., lipid nanoparticles) to form nucleic acids containing lipid particles. In one embodiment, the nucleic acid may be encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0404] Depending on the intended use of lipid particles (e.g., lipid nanoparticles), the proportion of constituent components may vary, and the delivery efficiency of a particular formulation can be measured, for example, using an endosomal release parameter (ERP) assay.

[0405] In one embodiment, lipid particles (e.g., lipid nanoparticles) may be conjugated with other parts to prevent aggregation. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as PEG conjugated with dialkyloxypropyl (e.g., PEG-DAA complex), PEG conjugated with diacylglycerol (e.g., PEG-DAG complex), PEG conjugated with cholesterol, PEG conjugated with phosphatidylethanolamine, and PEG conjugated with ceramide (see, for example, U.S. Patent No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid complexes (e.g., POZ-DAA complex, e.g., U.S. Provisional Application No. 61 / 294,828 filed January 13, 2010, and U.S. Provisional Application No. 61 / 295,140 filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid complexes), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in PCT Publication 2010 / 006282. PEG or POZ can be conjugated directly to lipids or conjugated to lipids via a linker moiety. For example, any linker moiety suitable for conjugating PEG or POZ to lipids can be used, including non-ester-containing and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties such as amides or carbamates are used. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0406] In one embodiment, TNA (e.g., ceDNA) can be compounded with the lipid portion of a particle or encapsulated at the lipid position of a lipid particle (e.g., lipid nanoparticle). In one embodiment, TNA can be completely encapsulated at the lipid position of a lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by nucleases, for example, in aqueous solutions. In one embodiment, TNA in lipid particles (e.g., lipid nanoparticles) is substantially undegraded after exposure of the lipid particles (e.g., lipid nanoparticles) to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes. In some embodiments, TNA in lipid particles (e.g., lipid nanoparticles) is substantially undegraded after incubation of the particles in serum at 37°C for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0407] In one embodiment, the lipid particles (e.g., lipid nanoparticles) are substantially nontoxic to the target, such as mammals like humans.

[0408] In one embodiment, a pharmaceutical composition comprising a therapeutic nucleic acid of the present disclosure may be formulated into lipid particles (e.g., lipid nanoparticles). In some embodiments, the lipid particles comprising the therapeutic nucleic acid may be formed from cationic lipids. In some other embodiments, the lipid particles comprising the therapeutic nucleic acid may be formed from non-cationic lipids. In preferred embodiments, the lipid particles of the present disclosure are nucleic acids containing lipid particles, which are formed from cationic lipids comprising therapeutic nucleic acids selected from the group consisting of mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or nonviral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone® DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (linear covalent closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("Dumbbell DNA").

[0409] In another preferred embodiment, the lipid particles of the present disclosure are nucleic acid-containing lipid particles, which are formed from noncationic lipids and optionally conjugated lipids that prevent particle aggregation.

[0410] In one embodiment, the lipid particle formulation is an aqueous solution. In another embodiment, the lipid particle (e.g., lipid nanoparticles) formulation is a freeze-dried powder.

[0411] In some embodiments, the present disclosure provides lipid particle formulations further comprising one or more pharmaceutical excipients. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation further comprises sucrose, tris, trehalose, and / or glycine.

[0412] In one embodiment, the lipid particles (e.g., lipid nanoparticles) disclosed herein may be incorporated into a pharmaceutical composition suitable for administration to a target for in vivo delivery to a target cell, tissue, or organ. Typically, the pharmaceutical composition comprises the TNA (e.g., ceDNA) lipid particles (e.g., lipid nanoparticles) disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the TNA (e.g., ceDNA) lipid particles (e.g., lipid nanoparticles) of this disclosure may be incorporated into a pharmaceutical composition suitable for a desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via hyperbaric intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also intended. Pharmaceutical compositions for therapeutic purposes may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high TNA (e.g., ceDNA) vector concentrations. A sterile, injectable solution can be prepared by filtration sterilization, incorporating the required amount of TNA (e.g., ceDNA) vector compound in a suitable buffer, along with one or a combination of the components listed above, as needed.

[0413] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for topical, systemic, intraamniotic, subarachnoid, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), subarachnoid, intrabladder, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, interstitial, intraacular, and intravitreous), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via hyperbaric intravenous or intraarterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also intended.

[0414] A pharmaceutically active composition containing TNA (e.g., ceDNA) lipid particles (e.g., lipid nanoparticles) can be formulated to deliver a transgene in nucleic acid to recipient cells, resulting in the therapeutic expression of the transgene. This composition may also contain a pharmaceutically acceptable carrier.

[0415] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high TNA (e.g., ceDNA) vector concentrations. Sterile, injectable solutions can be prepared by filtration sterilization, incorporating the required amount of ceDNA vector compound in a suitable buffer, along with one or a combination of the components listed above, as needed.

[0416] In one embodiment, the lipid particles (e.g., lipid nanoparticles) are solid core particles having at least one lipid bilayer. In one embodiment, the lipid particles (e.g., lipid nanoparticles) have a non-double-layer structure, i.e., a non-lamellar (i.e., non-double-layer) morphology. Examples of non-double-layer morphologies include, without limitation, three-dimensional tubes, rods, cubic symmetry, and the like. The non-lamellar morphology (i.e., non-double-layer structure) of lipid particles (e.g., lipid nanoparticles) is known to those skilled in the art and can be determined using analytical techniques employed by those skilled in the art. Such techniques include, but are not limited to, cryo-transmission electron microscopy ("Cryo-TEM"), differential scanning calorimeter ("DSC"), and X-ray diffraction. For example, the morphology of lipid particles (lamellar vs. non-lamellar) can be readily evaluated and characterized using, for example, Cryo-TEM analysis as described in US2010 / 0130588 (the contents of which are incorporated herein by reference in their entirety).

[0417] In one embodiment, lipid particles having a non-lamellar morphology (e.g., lipid nanoparticles) have a high electron density.

[0418] In one embodiment, the Disclosure provides lipid particles (e.g., lipid nanoparticles) having either a single-lamellar or multi-lamellar structure. In some embodiments, the Disclosure provides lipid particle (e.g., lipid nanoparticle) formulations comprising multicellular particles and / or foaming base particles. By controlling the composition and concentration of the lipid components, the rate at which lipid conjugates exchange outside the lipid particles (lipid nanoparticles), and consequently the rate at which the lipid nanoparticles become membrane-fusing, can be controlled. In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which the lipid particles (e.g., lipid nanoparticles) become membrane-fusing. Other methods that may be used to control the rate at which the lipid particles (e.g., lipid nanoparticles) become membrane-fusing will become apparent to those skilled in the art based on the Disclosure. It will also become apparent that the lipid particle size can be controlled by controlling the composition and concentration of the lipid conjugate.

[0419] In one embodiment, the pKa of a formulated cationic lipid may correlate with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010). Both of these are incorporated herein by reference in their entirety). In one embodiment, a preferred range of pKa is about 5 to about 7. In one embodiment, the pKa of a cationic lipid may be determined in lipid particles (e.g., lipid nanoparticles) using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0420] In one embodiment, encapsulation of TNA (e.g., ceDNA) in lipid particles (e.g., lipid nanoparticles) can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or PicoGreen® assay, using a dye whose fluorescence is enhanced when associated with nucleic acids. Generally, encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed when a small amount of nonionic surfactant is added. Surfactant-mediated breakdown of the lipid bilayer releases the encapsulated TNA (e.g., ceDNA), allowing it to interact with the membrane-impermeable dye. CeDNA encapsulation can be calculated as E = (Io - I) / Io, where I and Io refer to the fluorescence intensity before and after the addition of the surfactant.

[0421] Unit dose In one embodiment, the pharmaceutical composition may be presented in unit dosage forms. These unit dosage forms will typically be adapted to one or more routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by inhaler. In some embodiments, the unit dosage form is adapted for administration by sprayer. In some embodiments, the unit dosage form is adapted for administration by aerosolizer. In some embodiments, the unit dosage form is adapted for oral administration, buccal administration, or sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intraventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect.

[0422] VIII. Treatment Methods The TNAs (e.g., ceDNA vector lipid particles) and compositions described herein can be used to introduce nucleic acid sequences (e.g., therapeutic nucleic acid sequences) into host cells. In one embodiment, the introduction of nucleic acid sequences into host cells using TNA (e.g., ceDNA vector) lipid particles can be monitored with appropriate biomarkers from the treated patient to evaluate gene expression.

[0423] The pharmaceutical compositions provided herein can be used to deliver transgenes (nucleic acid sequences) for a variety of purposes. In one embodiment, a ceDNA vector (e.g., ceDNA vector lipid nanoparticles) can be used in a variety of ways, including, for example, excitation, in vitro and in vivo application, methodologies, diagnostic procedures, and / or gene therapy regimens.

[0424] Provided herein is a method for treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of TNA (e.g., ceDNA) lipid nanoparticles described herein, along with optionally a pharmaceutically acceptable carrier, into target cells of a subject requiring treatment (e.g., muscle cells or tissues, or other affected cell types). While the TNA lipid nanoparticles may be introduced in the presence of a carrier, such a carrier is not required. The implemented TNA (e.g., ceDNA) lipid nanoparticles contain a target nucleotide sequence useful for treating a disease. In particular, the ceDNA vector may contain a desired exogenous DNA sequence operably linked to a regulatory element that, when introduced into a subject, can direct the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence. The TNA (e.g., ceDNA) lipid nanoparticles described herein can be administered via any preferred route known in the art. In one embodiment, the target cells are in a human subject.

[0425] Provided herein is a method for providing a diagnostically or therapeutically effective amount of a ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) to a subject in need, the method comprising providing a certain amount of the ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) to cells, tissues, or organs of a subject in need for a time sufficient to enable the expression of a transgene from the ceDNA vector, thereby providing the subject with a diagnostically or therapeutically effective amount of a protein, peptide, nucleic acid expressed by the ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)). In one embodiment, the subject is a human.

[0426] Provided herein are methods for diagnosing, preventing, treating, or improving at least one symptom of a disease, disorder, dysfunction, injury, abnormal condition, or trauma in a subject. Generally, these methods include at least the step of administering one or more TNA (e.g., ceDNA) lipid nanoparticles described herein to a subject in need, in an amount and for a duration sufficient to diagnose, prevent, treat, or improve one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the subject. In one embodiment, the subject is a human.

[0427] Provided herein are methods comprising the use of TNA (e.g., ceDNA) lipid nanoparticles as tools for treating or reducing one or more symptoms of a disease or disease state. Several genetic diseases exist in which defective genes are known, and they typically fall into two classes: deficiency states of enzymes that are usually generally inherited recessively, and imbalance states of enzymes that may be involved in regulatory or structural proteins but are not typically always inherited dominantly. In the case of deficiency states, TNAs such as ceDNA lipid nanoparticles can be used to deliver transgenes to introduce normal genes into affected tissue for replacement therapy, and in some embodiments, antisense mutations can be used to create animal models of the disease. In the case of imbalance states, TNA (e.g., ceDNA) lipid nanoparticles can be used to create a disease state in a model system, which can then be used to attempt to counteract that disease state. Thus, the TNA (e.g., ceDNA) lipid nanoparticles and methods disclosed herein enable the treatment of genetic diseases. As used herein, a disease condition is treated by partially or completely repairing the deficiency or imbalance that causes or exacerbates the disease.

[0428] In general, as described above, any transgene can be delivered using TNA such as ceDNA lipid nanoparticles described herein to treat, prevent, or improve any symptoms associated with any disorder of gene expression. Examples of conditions include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes mellitus, muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy), Hurler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondriopathy (e.g., Leber's hereditary optic neuropathy (LHON), Leigh syndrome, and subacute sclerosing encephalitis), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathy), and diseases of solid organs (e.g., brain, liver, kidneys, heart). In some embodiments, ceDNA vectors such as those disclosed herein may be advantageously used in the treatment of individuals with metabolic disorders (e.g., ornithine transcarbamylase deficiency).

[0429] In one embodiment, TNAs such as ceDNA lipid nanoparticles described herein can be used to treat, improve, and / or prevent diseases or disorders caused by mutations in genes or gene products. Exemplary diseases or disorders that can be treated with ceDNA vectors (e.g., ceDNA vector lipid particles 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 disorders); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII, Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC)); hematological diseases or disorders (e.g., hemophilia (A and B), thalassemia, and anemia); cancers and tumors; and genetic diseases or disorders (e.g., cystic fibrosis).

[0430] In one embodiment, a heterologous nucleotide sequence can be delivered using TNA such as ceDNA lipid nanoparticles described herein in situations where it is desirable to regulate the expression level of a transgene (for example, a transgene encoding a hormone or growth factor as described herein).

[0431] In one embodiment, TNAs such as ceDNA lipid nanoparticles can be used to correct abnormal levels and / or functions (e.g., absence or deletion in a protein) of gene products that cause disease or impairment. The lipid nanoparticle ceDNA vectors described herein can produce functional proteins and / or modify protein levels to alleviate, reduce, or confuse symptoms resulting from specific diseases or impairments caused by absence or deletion in proteins. For example, treatment of OTC deficiency can be achieved by producing functional OTC enzymes. Treatment of hemophilia A and B can be achieved by modifying the levels of factor VIII, factor IX, and factor X. Treatment of PKU can be achieved by modifying the levels of phenylalanine hydroxylase enzymes. Treatment of Fabry disease or Gaucher disease can be achieved by producing functional alpha-galactosidase or beta-glucocerebrosidase, respectively. Treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or isuronate-2-sulfatase, respectively. Treatment of cystic fibrosis may be achieved by producing functional transmembrane conductance regulators of cystic fibrosis. Treatment of glycogen storage disease may be achieved by restoring functional G6Pase enzyme function. Treatment of PFIC may be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

[0432] In one embodiment, RNA-based therapeutics can be delivered to cells in vitro or in vivo using TNA (e.g., ceDNA) lipid nanoparticles described herein. 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, in one embodiment, antisense nucleic acids can be delivered to cells in vitro or in vivo using a ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein). For example, if the transgene is an RNAi molecule, the expression of antisense nucleic acid or RNAi in target cells reduces the expression of a particular protein by the cell. Therefore, a transgene that is an RNAi molecule or antisense nucleic acid can be administered to reduce the expression of a particular protein in a target that requires it. Antisense nucleic acids can also be administered to cells in vitro to modulate cell physiology, for example, to optimize a cell or tissue culture system.

[0433] In one embodiment, TNA lipid nanoparticles as described herein can be used to deliver DNA-based therapeutics to cells in vitro or in vivo. Examples of DNA-based therapeutics include, but are not limited to, minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone® DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped minimal DNA vectors ("dumbbell DNA"). For example, in one embodiment, a ceDNA vector (e.g., ceDNA vector lipid particles as described herein (e.g., lipid nanoparticles)) can be used to deliver minicircles to cells in vitro or in vivo. For example, if the transgene is minicircle DNA, the expression of minicircle DNA in target cells reduces the expression of a particular protein by the cell. Therefore, a transgene that is minicircle DNA can be administered to reduce the expression of a particular protein in a target that requires it. Minicircle DNA can also be administered to cells in vitro to modulate cell physiology, for example, to optimize cell or tissue culture systems.

[0434] In one embodiment, exemplary transgenes encoded by TNAs such as ceDNA vectors include lysosomal enzymes (e.g., hexosaminidase A associated with Thai-Sachs disease, or Hunter syndrome / MPS) Examples include, but are not limited to, isuronate sulfatases related to II, erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., interferon, β-interferon, interferon-g, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factor 1 and 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factors 3 and 4, brain-derived neurotrophic factor (BDNF), glial-derived growth factor (GDNF), transforming growth factors α and β, etc.), and receptors (e.g., tumor necrosis factor receptor). In some exemplary embodiments, the transgene encodes a monoclonal antibody specific to one or more desired targets. In some exemplary embodiments, two or more transgenes are encoded by a ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments, the transgene encodes an antibody comprising a full-length antibody or an antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as defined herein. Other exemplary transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, and tumor suppressor gene products.

[0435] Administration In one embodiment, the TNA lipid nanoparticles of the present disclosure can be administered to an organism for in vivo cell transduction. In one embodiment, TNA can be administered to an organism for ex vivo cell transduction.

[0436] Generally, administration is by one of the routes typically used to bring the molecule into final contact with blood or tissue cells. Preferred methods for administering such nucleic acids are available and well known to those skilled in the art, and two or more routes may be used to administer a particular composition, although a particular route may often be more immediate and provide a more effective response than another. Exemplary forms of administration of TNAs, such as ceDNA vectors (e.g., ceDNA lipid nanoparticles) described herein, include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, intrauterine (or intraocular), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to the skeleton, diaphragm, and / or myocardium), intrapleural, intracerebral, and intraarterial), topical (e.g., to skin and mucous membrane surfaces, including the airway surface, and transdermal administration), intralymphatic, and direct tissue or organ injection (e.g., to the liver, eye, skeletal muscle, myocardium, diaphragm, muscle, or brain).

[0437] The administration of TNA lipid particles described herein may be performed on any site of target, including but not limited to sites selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. In one embodiment, the administration of TNA lipid nanoparticles described herein may also be directed at tumors (e.g., within or near tumors or lymph nodes). The most preferred route in any given case will depend on the nature and severity of the condition being treated, improved, and / or prevented, as well as the properties of the specific ceDNA (e.g., ceDNA lipid nanoparticles) used herein. Additionally, ceDNA allows for the administration of two or more transgenes in a single vector or multiple ceDNA vectors (e.g., a ceDNA cocktail).

[0438] In one embodiment, administration of a ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) as described herein) to skeletal muscle includes, but is not limited to, administration to the skeletal muscles of the limbs (e.g., upper arm, lower arm, upper limb, and / or lower limb), waist, neck, head (e.g., tongue), pharynx, abdomen, pelvis / perineum, and / or fingers. The ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) as described herein) may be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion (optionally, isolated limb perfusion of the legs and / or arms, see, e.g., Arruda et al., (2005) Blood 105:3458-3464) and / or direct intramuscular injection. In certain embodiments, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) is administered to the limbs (arms and / or legs) of a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally by isolated limb perfusion (e.g., by intravenous or intra-arterial administration). In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be administered without using "hydrodynamic" techniques.

[0439] Administration of ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) as described herein) to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. CeDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) as described herein) may be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., to the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion. Administration to the diaphragmatic muscle may be carried out by any preferred method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to smooth muscle may be carried out by any preferred method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment, administration may be carried out to endothelial cells present in, near, and / or on the smooth muscle.

[0440] In one embodiment, a ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) as described herein) is administered to skeletal muscle, diaphragmatic muscle and / or myocardium (e.g., to treat, improve, and / or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure)).

[0441] ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be administered to the CNS (e.g., the brain or the eye). ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (cerebrum including striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can also be administered to different areas of the eye, such as the retina, cornea, and / or optic nerve. ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be delivered to the cerebrospinal fluid (e.g., by lumbar puncture). ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0442] In one embodiment, a ceDNA vector (ceDNA vector lipid particles (e.g., lipid nanoparticles) as described herein) may be administered to a desired region of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of sugars such as mannitol), intranasal, intraocular, intraocular (e.g., intravitreous, subretinal, anterior chamber), and periocular (e.g., subtenon region) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[0443] In some embodiments, the ceDNA vector (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) is administered in a liquid formulation by direct injection (e.g., stereotactic injection) into a desired region or compartment in the CNS. According to other embodiments, the ceDNA vector (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) may be provided by topical application to a desired region or by intranasal administration of an aerosol formulation. Administration to the eye may be performed by topical application of a droplet. As a further alternative, the ceDNA vector may be administered as a solid sustained-release formulation (see, for example, U.S. Patent No. 7,201,898, which is incorporated herein in whole by reference). In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be used for retrograde transport to treat, improve, and / or prevent motor neuron-related diseases and disorders (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, a ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) as described herein) can be delivered to muscle tissue and from there migrate into neurons.

[0444] In one embodiment, the therapeutic product can be repeatedly administered until an appropriate level of expression is achieved. Therefore, in one embodiment, the therapeutic nucleic acid can be administered and re-administered multiple times. For example, the therapeutic nucleic acid can be administered on day 0. Following the initial treatment on day 0, the therapeutic nucleic acid can be administered for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or approximately 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or approximately 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years. A second dose of medication (re-administration) can be given approximately 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 years later.

[0445] In one embodiment, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be contained within lipid particles (e.g., lipid nanoparticles) of the Disclosure. In other words, the lipid particles (e.g., lipid nanoparticles) may contain other compounds in addition to ceDNA, or at least a second ceDNA different from the first. Without limitation, the other additional compounds may be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptide mimes, nucleic acids, nucleic acid analogs and derivatives, extracts made from biomolecules, or any combination thereof.

[0446] In one embodiment, one or more additional compounds may be therapeutic agents. The therapeutic agent may be selected from any class suitable for the therapeutic purpose. Therefore, the therapeutic agent may be selected from any class suitable for the therapeutic purpose. The therapeutic agent may be selected according to the desired therapeutic purpose and biological action. For example, in one embodiment, if the ceDNA within the LNP is useful for treating cancer, the additional compound may be an anticancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates)). In one embodiment, if the LNP containing ceDNA is useful for treating an infection, the additional compound may be an antimicrobial agent (e.g., an antibiotic or antiviral compound). In one embodiment, if the LNP containing ceDNA is useful for treating an immune disease or disorder, the additional compound may be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulant, or a compound that modulates one or more specific immune pathways). In one e...

Claims

[Claim 1] The invention described in the specification.

Images