Cationic lipids and compositions thereof

Abstract

As used herein, cationic lipids having formula I or Ia: TIFF2024522692000044.tif37128 and pharma- ceutically acceptable salts thereof are provided, wherein R′, R 1 , R 2 , R 3 , R 4 , R 5 , R 6a , R 6b , X, and n are as defined herein. Also provided herein is a lipid nanoparticle (LNP) composition comprising a cationic lipid having formula I or Ia and a capsid-free, non-viral vector (e.g., ceDNA). In one aspect of any of the aspects or embodiments herein, these LNPs can be used to deliver the capsid-free, non-viral DNA vector to a target site of interest (e.g., a cell, tissue, organ, etc.).

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 210,204, filed June 14, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

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

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

[0004] Non-viral gene delivery avoids certain disadvantages associated with viral transduction, in particular the humoral and cellular immune responses against the viral structural proteins that form the vector particles, as well as those resulting from any de novo viral gene expression. Among the advantages of non-viral delivery techniques is the use of lipid nanoparticles (LNPs) as carriers. LNPs offer a unique opportunity to allow the design of cationic lipids as LNP components that can avoid the humoral and cellular immune responses that result in significant toxicity associated with viral gene therapy.

[0005] Cationic lipid generally consists of cationic amine moiety, hydrophobic domain that typically has one or two aliphatic hydrocarbon chains (i.e., aqueous tail that can be saturated or unsaturated), and linker or biodegradable group that connects cationic amine moiety and hydrophobic domain.Cationic amine moiety and polyanionic nucleic acid interact electrostatically to form positively charged liposome or lipid membrane structure.Therefore, cellular uptake is promoted and nucleic acid is delivered to cells.

[0006] Some widely used cationic lipids are CLinDMA, DLinDMA (DODAP) and DOTAP.These lipids have been used for ribonucleic acid (siRNA or mRNA) delivery, but suffer from suboptimal delivery efficiency along with toxicity at higher doses.In view of the shortcomings of current cationic lipids, it is necessary to provide lipid scaffolds that not only show enhanced efficacy along with reduced toxicity, but also improve pharmacokinetics and intracellular dynamics such as cellular uptake and nucleic acid release from lipid carriers. Summary of the Invention

[0007] The cationic lipids provided in the present disclosure include one hydrophobic tail that contains a biodegradable group and a hydrophobic tail that does not contain a biodegradable group.Some of the exemplary lipids provided in the present disclosure include a hydrophobic tail that branches at the end to form two branched aliphatic hydrocarbon chains and an unbranched hydrophobic tail.The inventors have found that the cationic lipids of the present disclosure can be synthesized with satisfactory yield and purity.The inventors have also found that the cationic lipids of the present disclosure, when formulated as lipid nanoparticles (LNPs) for carrying therapeutic nucleic acids, provide sustained, excellent and stable in vivo expression of transgene inserts in nucleic acids and are well tolerated. Furthermore, without wishing to be bound by theory, the inventors believe that the delicate interplay between the length (i.e., number of carbon atoms) of the terminal branched aliphatic hydrocarbon chain in the branched hydrophobic tail, the length of the unbranched hydrophobic tail, and the distance between the degradable group and the branched hydrophobic tail is important, among other things, to achieve superior encapsulation efficiency, expression levels, and in vivo tolerability of the LNP compositions.

[0008] Thus, in one aspect, the present specification provides a cationic lipid represented by formula I or Ia:

[0009] [ka] and pharma- ceutically acceptable salts thereof, wherein R′, R 1 , R 2 , R 3 , R 4 , R 5 , R 6a , R 6b , X, and n are each as defined herein for each of formulas I or Ia.

[0010] Also provided are pharmaceutical compositions comprising a cationic lipid described herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0011] Another aspect of the present disclosure relates to a composition comprising a lipid nanoparticle (LNP) comprising a cationic lipid as described herein, or a pharma- ceutically acceptable salt thereof, and a nucleic acid. In one embodiment of any of the aspects or embodiments herein, the nucleic acid is encapsulated in the LNP. In certain embodiments, the nucleic acid is closed-ended DNA (ceDNA).

[0012] A further aspect of the present disclosure relates to a method of treating a genetic disorder in a subject using the disclosed cationic lipids or compositions described herein. [Brief description of the drawings]

[0013]

[0013] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to the exemplary embodiments of the present disclosure, which are depicted in the accompanying drawings. The accompanying drawings, however, depict only typical embodiments of the present disclosure and therefore should not be considered as limiting in scope, since the present disclosure may admit of other equally effective embodiments. [Figure 1] As observed in preclinical studies, ceDNA-luciferase expression on day 4 achieved by using lipid nanoparticles LNP2, LNP3, and LNP4, each formulated with lipid 6, as a delivery vehicle compared to LNP1 formulated with reference lipid A (positive control) and PBS (negative control) (dose = 0.25 mg / kg). [Figure 2A] 1 is a bar graph showing ceDNA-luciferase expression on day 4, as measured by total flux, achieved by using lipid nanoparticles LNP8, LNP9, and LNP10 formulated with lipid 7, lipid 11, and lipid 1, respectively, as delivery vehicles compared to LNP5 formulated with reference lipid A (positive control), LNP6 formulated with MC3, and LNP7 formulated with reference lipid B (positive control), and PBS (negative control), as observed in preclinical studies (dose=0.5 mg / kg). [Figure 2B]The weight change over time of mice from day 0 to day 4 in the same study is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs), such as non-viral vectors (e.g., closed-end DNA) or synthetic viral vectors, that can be taken up by cells and maintain high levels of expression. For example, immunogenicity associated with viral vector-based gene therapy has limited the number of patients that can be treated due to pre-existing background immunity and has prevented re-dosing of patients to titrate to effective levels for each patient or to maintain efficacy over time. Furthermore, other nucleic acid modalities suffer greatly from immunogenicity due to innate DNA or RNA sensing mechanisms that trigger a cascade of immune responses. Lacking pre-existing immunity, the TNA lipid particles (e.g., lipid nanoparticles) described herein allow for additional doses of TNAs, such as mRNA, siRNA, synthetic viral vectors, or ceDNA, as needed, further expanding patient access, including pediatric populations that may require subsequent doses depending on tissue growth. Furthermore, it is a discovery of the present disclosure that lipid compositions comprising one or more tertiary amino groups, and TNA lipid particles (e.g., lipid nanoparticles) comprising disulfide bonds, in particular, provide more efficient delivery of TNA (e.g., ceDNA), better tolerability, and improved safety profile. The TNA lipid particles (e.g., lipid nanoparticles) described herein are free of packaging constraints imposed by the space within the viral capsid, so theoretically, the only size limitation of the TNA lipid particles (e.g., lipid nanoparticles) is the expression (e.g., DNA replication, or RNA translation) efficiency of the host cell.

[0015] One of the biggest hurdles in developing therapies, especially in rare diseases, is the large number of individual pathologies. Approximately 350 million people on the planet live with a rare disorder, and the National Institutes of Health defines a rare disorder as a disorder or condition with fewer than 200,000 diagnosed humans. Approximately 80 percent of these rare disorders are genetic in origin, and approximately 95 percent of them do not have FDA-approved treatments (rarediseases.info.nih.gov / diseases / pages / 31 / faqs-about-rare-diseases). Among the advantages of the TNA lipid particles (e.g., lipid nanoparticles) described herein is that they provide an approach that can be rapidly adapted to multiple diseases that can be treated with specific modalities of TNA, particularly rare monogenic diseases that can meaningfully change the current state of treatment for many genetic disorders or diseases.

[0016] I. Definition The term "alkyl" refers to a monovalent radical of a saturated, straight-chain (i.e., unbranched) or branched-chain hydrocarbon. 16 As used herein, the term "alkyl" applies to both branched and unbranched alkyl groups, unless specifically stated to be an unbranched alkyl group. Exemplary alkyl groups include C1-C 16 Unbranched alkyl, C7-C 12 Alkyl, C7-C 11 Alkyl, C8-C 10 Alkyl, C2-C 14 Unbranched alkyl, C2-C 12 Unbranched alkyl, C2-C 10 Unbranched alkyl, C2-C7 unbranched alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C7 unbranched alkyl, C8 unbranched alkyl, C9 unbranched alkyl, C 10 Unbranched alkyl, C 11 Unbranched alkyl, C8 alkyl, C 10 Alkyl, C 12Examples of alkyl groups include, but are not limited to, alkyl, methyl, ethyl, propyl, isopropyl, 2-methyl-1-butyl, 3-methyl-2-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decanyl, undecanyl, dodecanyl, tridecanyl, tetradecanyl, pentadecanyl, hexadecanyl, heptadecanyl, octadecanyl, nonadecanyl, eicosanyl, and the like.

[0017] The term "alkylene" refers to a divalent radical of a saturated, straight-chain or branched-chain hydrocarbon. Alkylene can be unbranched, e.g., C3-C 10 As used herein, the term "alkylene" applies to both branched and unbranched alkylene groups, unless specifically stated to be unbranched and C1-C8 alkylene. Exemplary alkylene groups include, but are not limited to, C3-C9 alkylene, C3-C8 alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C2-C8 alkylene, C3-C7 alkylene, C5-C7 alkylene, C7 alkylene, C5 alkylene, and alkylenes corresponding to any of the exemplary alkyl groups above.

[0018] The term "alkenyl" refers to a monovalent radical of a linear or branched hydrocarbon having one or more (e.g., one or two) carbon-carbon double bonds; alkenyl radicals include radicals having "cis" and "trans" orientations, or, alternatively nomenclature, "E" and "Z" orientations. An alkenyl can be unbranched, e.g., C2-C 16 As used herein, the term "alkenyl" applies to both branched and unbranched alkenyl groups, unless specifically stated to be an unbranched alkenyl. Exemplary alkenyl groups include C2-C 16 Unbranched alkenyl, C7-C16 Alkenyl, C8-C 14 Alkenyl, C2-C 14 Unbranched alkenyl, C2-C 12 Unbranched alkenyl, C2-C 10 Unbranched alkenyl, C2-C7 unbranched alkenyl, C2-C6 alkenyl, C2-C4 alkenyl, C2-C3 alkenyl, C8 alkenyl, C 10 Alkenyl, C 12 This includes, but is not limited to, alkenyl, and the alkenyl corresponding to any of the above exemplary alkyl groups containing two or more carbon atoms.

[0019] The term "alkenylene" refers to a straight or branched chain hydrocarbon divalent radical having one or more (e.g., one or two) carbon-carbon double bonds; alkenyl radicals include radicals having "cis" and "trans" orientations, or, alternatively, "E" and "Z" orientations. Alkenylene can be unbranched, e.g., C3-C 10 As used herein, the term "alkenylene" applies to both branched and unbranched alkenylene groups, unless specifically stated to be unbranched alkylene. Exemplary alkenylene groups include, but are not limited to, C3-C9 alkenylene, C3-C8 alkenylene, C2-C8 alkenylene, C2-C6 alkenylene, C3-C7 alkenylene, C5-C7 alkenylene, C2-C4 alkenylene, C1-C8 alkylene, C2-C8 alkylene, C3-C7 alkylene, C5-C7 alkylene, C7 alkylene, C5 alkylene, and alkenyl corresponding to any of the above exemplary alkyl groups containing 2 or more carbon atoms.

[0020] As used herein, the term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable organic or inorganic salts of the cationic lipids of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may include the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counter ion. A counter ion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0021] As used in this specification and the appended claims, the term "about," when referring to a measurable value, such as an amount, temporal duration, and the like, is meant to encompass variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.5%, and even more preferably ±0.1%, from the specified value as appropriate for performing the disclosed methods.

[0022] As used herein, "comprise," "comprising," and "comprises," as well as "comprised of," are meant to be synonymous with "include," "including," "includes," or "contain," "containing," "contains," e.g., are inclusive or open-ended terms specifying the presence of what follows the component, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps that are known in the art or disclosed therein.

[0023] The term "consisting of" refers to compositions, methods, processes, and their respective components described herein, excluding any elements not recited in the description of the embodiment.

[0024] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the invention.

[0025] As used herein, the terms "administration", "administering" and variants thereof refer to the introduction of a composition or agent (e.g., a nucleic acid, particularly ceDNA) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. The introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another. Administration can be performed by any suitable route. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of a subject. In one aspect of any of the aspects or embodiments herein, "administration" refers to therapeutic administration.

[0026] As used herein, phrases such as "anti-therapeutic nucleic acid immune response", "anti-transfer vector immune response", "immune response to therapeutic nucleic acid", "immune response to transfer vector" are meant to refer to any undesired immune response to a therapeutic nucleic acid, whether viral or non-viral in origin. In some embodiments of any of the aspects and embodiments herein, the undesired immune response is an antigen-specific immune response to the viral transfer vector itself. In some embodiments of any of the aspects and embodiments herein, the immune response is specific to the transfer vector, which may be double-stranded DNA, single-stranded RNA, or double-stranded RNA. In other embodiments, the immune response is specific to the sequence of the transfer vector. In other embodiments, the immune response is specific to the CpG content of the transfer vector.

[0027] As used herein, the terms "carrier" and "excipient" are used interchangeably and are meant to include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma-ceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0028] As used herein, the term "ceDNA" is meant to refer to capsid-free closed-ended linear double stranded (ds) duplex DNA for synthetic or other non-viral gene transfer. A detailed description of ceDNA is described in International Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for the production of ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Application No. PCT / US2018 / 049996, filed September 7, 2018, and International Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for the production of synthetic ceDNA vectors, including various ITR sequences and configurations, are described, for example, in International Application No. PCT / US2019 / 14122, filed Jan. 18, 2019, the entire contents of which are incorporated herein by reference. As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a closed-ended linear duplex (CELiD) CELiD DNA. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a DNA-based minicircle. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a minimalistic immunologically-defined gene expression (MIDGE) vector. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a ministring DNA.According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a dumbbell-shaped linear double-stranded closed-end DNA that contains two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is doggybone™ DNA.

[0029] As used herein, the term "ceDNA-bacmid" is meant to refer to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can be propagated as a plasmid in E. coli, thereby acting as a shuttle vector for baculovirus.

[0030] As used herein, the term "ceDNA-baculovirus" is meant to refer to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0031] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and are meant to refer to invertebrate host cells (including, but not limited to, insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0032] As used herein, the term "ceDNA genome" is meant to refer to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments of any of the aspects or embodiments herein, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0033] As used herein, the terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and are meant to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0034] As used herein, the term "exogenous" is meant to refer to a substance present in a cell other than its natural source. As used herein, the term "exogenous" may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that is not normally found and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that is found in relatively low amounts and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to cause ectopic expression or levels. In contrast, as used herein, the term "endogenous" refers to a substance that is natural to a biological system or cell.

[0035] As used herein, the term "expression" is meant to refer to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, for example, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0036] As used herein, the term "expression vector" is meant to refer to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the host cell. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, so that it can be maintained in two organisms, for example, in human cells for expression, and in prokaryotic hosts for cloning and amplification. An expression vector can be a recombinant vector.

[0037] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and are meant to refer to a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene in a DNA vector, e.g., a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. The promoter may also be of AAV origin.

[0038] As used herein, the term "flanking" is meant to refer to the relative position of a nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked by A and C. Similarly for the sequence A x B x C. Thus, a flanking sequence precedes or follows the flanked sequence, but need not be contiguous or immediately adjacent to the flanked sequence. In one embodiment of any of the aspects or embodiments herein, the term flanking refers to the terminal repeats at each end of a linear single-stranded synthetic AAV vector.

[0039] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid involved in the expression of a given RNA or protein in vitro or in vivo. Thus, a gene includes a region that codes for an expressed RNA (usually including a polypeptide coding sequence) and often regulatory sequences required for their expression. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and may include sequences specifically designed to have desired parameters.

[0040] As used herein, the phrase "genetic disease" or "genetic disorder" is meant to refer to a disease or deficiency caused, in part or in whole, directly or indirectly, by one or more abnormalities in the genome, including conditions that are present from birth. The abnormality may be a mutation, insertion, or deletion in a gene. The abnormality may affect the coding sequence of the gene or its regulatory sequence.

[0041] As used herein, the term "heterologous" is meant to refer to a nucleotide or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein, respectively. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence that encodes a chimeric polypeptide. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleotide sequence that encodes a fusion variant polypeptide.

[0042] As used herein, the term "host cell" refers to any cell type that is amenable to transformation, transfection, transduction, etc., with a nucleic acid therapeutic of the present disclosure. By way of non-limiting example, host cells can include isolated primary cells, pluripotent stem cells, CD34 +The host cell may be either a human cell, an induced pluripotent stem cell, or an immortalized cell line (e.g., HepG2 cell). Alternatively, the host cell may be a cell in situ or in vivo in a tissue, organ, or organism. Furthermore, the host cell may be, for example, a target cell in a mammalian subject (e.g., a human patient in need of gene therapy).

[0043] As described herein, "inducible promoter" is meant to refer to one that is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or agent. As used herein, an "inducer" or "inducing agent" can be an endogenous or usually exogenous compound or protein that is administered in such a way that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments of any of the aspects and embodiments herein, the inducer or agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control or an inducible promoter. In some embodiments of any of the aspects or embodiments herein, the inducible promoter is induced in the absence of a certain agent, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0044] As used herein, the term "in vitro" is meant to refer to assays and methods that do not require the presence of cells with intact membranes, such as a cell extract, and can refer to introducing a programmable synthetic biological circuit into a non-cellular system, e.g., a medium that does not contain cells or cell systems, such as a cell extract.

[0045] As used herein, the term "in vivo" is meant to refer to an assay or process that occurs in or within an organism, such as a multicellular animal. In some of the aspects described herein, the method or use may be said to occur "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses that are carried out using live cells with intact membranes outside the body of a multicellular animal or plant, such as explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells), among others.

[0046] As used herein, the term "lipid" is meant to refer to a group of organic compounds, including but not limited to esters of fatty acids, characterized by poor solubility in water but generally soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids", including fats and oils and waxes; (2) "complex lipids", including phospholipids and glycolipids; and (3) "derived lipids", such as steroids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also included in the group called amphipathic lipids. Additionally, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0047] As used herein, the term "encapsulated" is meant to refer to a lipid particle that provides complete encapsulation, partial encapsulation, or both, of an active or therapeutic agent, such as a nucleic acid (e.g., ASO, mRNA, siRNA, ceDNA, viral vector). In preferred embodiments, the nucleic acid is completely encapsulated within the lipid particle (e.g., to form a lipid particle containing the nucleic acid).

[0048] As used herein, the term "lipid particle" or "lipid nanoparticle" is meant to refer to a lipid formulation that can be used to deliver a therapeutic agent, such as a nucleic acid therapeutic (TNA), to a desired target site (e.g., a cell, tissue, organ, etc.) (referred to as "TNA lipid particle", "TNA lipid nanoparticle" or "TNA LNP"). In one embodiment of any of the aspects or embodiments herein, the lipid particle of the present invention is a LNP containing one or more therapeutic nucleic acids, and the LNP is typically composed of a cationic lipid, a sterol, a non-cationic lipid, and optionally a PEGylated lipid to prevent particle aggregation, and optionally a tissue-specific targeting ligand to deliver the LNP to a desired target site. In other preferred embodiments, the therapeutic agent, such as a therapeutic nucleic acid, can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment of any of the aspects or embodiments herein, the LNP comprises a nucleic acid (e.g., ceDNA) and the LNP formulated with a cationic lipid as described herein.

[0049] As used herein, the term "ionizable lipid" is meant to refer to a lipid, e.g., a cationic lipid, that has at least one protonatable or deprotonatable group such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and reference to charged or neutral lipids refers to the nature of the predominant species, and not all lipids need to exist in a charged or neutral form. Generally, cationic lipids have a pKa of the protonatable group in the range of about 4 to about 7. Thus, the term "cationic" as used herein encompasses both the ionized (or charged) and neutral forms of the lipids of the present invention.

[0050] As used herein, the term "neutral lipid" is meant to refer to any lipid species that exists in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0051] 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 modification groups associated with neutral lipids.

[0052] As used herein, the term "non-cationic lipid" is meant to refer to any amphipathic lipid, and any other neutral or anionic lipid.

[0053] As used herein, the term "organic lipid solution" is meant to refer to a composition comprising an organic solvent having, in whole or in part, a lipid.

[0054] As used herein, the term "liposome" is meant to refer to lipid molecules assembled in a spherical configuration that encapsulates an internal aqueous volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug delivery in the context of formulation development. They act by fusing with cell membranes and repositioning their lipid structures to deliver drugs or active formulation components. Liposome compositions for such delivery are typically composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids.

[0055] As used herein, the term "localized delivery" refers to the direct delivery of an active agent, such as an interfering RNA (e.g., siRNA), to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site, such as a tumor, or other target site, such as an inflammation site, or into a target organ, such as the liver, heart, pancreas, or kidney.

[0056] As used herein, the term "neDNA" or "nicked ceDNA" is meant to refer to closed-end DNA that has a nick or gap of 2-100 base pairs in the stem or spacer region 5' upstream of an open reading frame (e.g., an expressed promoter and transgene).

[0057] As used herein, the term "nucleic acid" is meant to refer to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. The DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, 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. The DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-end linear duplex 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), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties as the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid.Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0058] As used herein, the phrases "nucleic acid therapeutic agent", "therapeutic nucleic acid" and "TNA" are used interchangeably and refer to any modality of treatment that uses nucleic acid as an active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE)-vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), and dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). As used herein, the term "TNA LNP" refers to lipid particles containing at least one TNA, as described above.

[0059] As used herein, a "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0060] As used herein, "operably linked" refers to a juxtaposition in which the components so described are in a relationship that permits them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. A promoter can be said to drive expression or drive transcription of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence and regulates to control transcription initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in an inverted orientation, whereby what was the coding strand is now the non-coding strand and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0061] As used herein, the term "promoter" is meant to refer to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of the nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. A promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled. A promoter may also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. Within the promoter sequence, one will find a transcription initiation site, as well as protein binding domains involved in the binding of RNA polymerase. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the synthetic AAV vectors disclosed herein. The promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' orientation) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background.

[0062] A promoter may be one that is naturally associated with a gene or sequence, which may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons of a given gene or sequence. Such a promoter may be referred to as "endogenous". Similarly, in some embodiments of any of the aspects and embodiments herein, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments of any of the aspects and embodiments herein, a coding nucleic acid segment is positioned under the control of a "recombinant promoter" or a "heterologous promoter", both of which refer to a promoter that is not normally associated with an operably linked encoded nucleic acid sequence in its natural environment. Similarly, a "recombinant or heterologous enhancer" refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and synthetic promoters or enhancers that are not "naturally occurring", i.e., may contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, for the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference in its entirety). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0063] As used herein, "Rep binding site" ("RBS") and "Rep binding element" ("RBE") are used interchangeably and are meant to refer to a binding site for a Rep protein (e.g., AAV Rep78 or AAV Rep68) which, upon binding by the Rep protein, enables the Rep protein to carry out its site-specific endonuclease activity on a sequence incorporating the RBS. An RBS sequence and its reverse complement together form a single RBS. RBS sequences are well known in the art and include, for example, the RBS sequence identified in AAV2, 5'-GCGCGCTCGCTCGCTC-3'.

[0064] As used herein, the phrase "recombinant vector" is meant to refer to a vector that contains a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and therapies in some embodiments of any of the aspects and embodiments herein. In some embodiments of any of the aspects or embodiments herein, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest in a high copy number of extrachromosomal DNA in a subject, thereby eliminating the potential effects of chromosomal integration.

[0065] As used herein, the term "reporter" is meant to refer to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed.

[0066] As used herein, the terms "sense" and "antisense" are meant to refer to the orientation of a structural element on a polynucleotide. The sense and antisense versions of an element are the reverse complements of each other.

[0067] As used herein, the term "sequence identity" is meant to refer to the relatedness between two nucleotide sequences. For the purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are gap open penalty 10, gap extension penalty 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the identity percentage, calculated as follows: (identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0068] As used herein, the term "spacer region" is meant to refer to an intervening sequence that separates functional elements within a vector or genome. In some embodiments of any of the aspects or embodiments herein, the AAV spacer region holds two functional elements in a desired arrangement for optimal functionality. In some embodiments of any of the aspects or embodiments herein, the spacer region provides or increases the genetic stability of the vector or genome. In some embodiments of any of the aspects or embodiments herein, the spacer region facilitates easy genetic manipulation of the genome by providing a convenient location for cloning sites and gaps in a designed number of base pairs. For example, in certain aspects, oligonucleotide "polylinkers" or "polycloning sites" that contain several restriction endonuclease sites, or non-open reading frame sequences designed to have no known protein (e.g., transcription factor) binding sites, can be placed in the vector or genome to separate cis-acting elements, for example, inserting 6mers, 12mers, 18mers, 24mers, 48mers, 86mers, 176mers, etc.

[0069] As used herein, the term "subject" is meant to refer to a human or animal to which treatment, including prophylactic treatment, with a therapeutic nucleic acid according to the present invention is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, 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 aspects described herein, the subject is a mammal, such as a primate or a human. The subject can be male or female. Additionally, the subject may be an infant or child. In some embodiments of any of the aspects or embodiments herein, the subject may be a neonatal or fetal subject, e.g., the subject is in utero. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals may be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African American, African European, Latin American, Middle Eastern, etc. In some embodiments of any of the aspects and embodiments herein, the subject may be a patient or other subject in a clinical setting. In some embodiments of any of the aspects or embodiments herein, the subject has already undergone treatment. In some embodiments of any of the aspects or embodiments herein, the subject is an embryo, a fetus, a newborn, an infant, a child, an adolescent, or an adult.In some embodiments of any of the aspects or embodiments herein, the subject is a human fetus, a human newborn, a human infant, a human child, a human adolescent, or a human adult. In some embodiments of any of the aspects or embodiments herein, the subject is an animal embryo, or a non-human embryo or a non-human primate embryo. In some embodiments of any of the aspects or embodiments herein, the subject is a human embryo.

[0070] As used herein, the phrase "subject in need", unless the context and usage of the phrase dictates otherwise, refers to a subject: (i) who is to be administered TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention; (ii) who has received TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention; or (iii) who has received TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention.

[0071] As used herein, the terms "suppress," "reduce," "interfere," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of directly or indirectly decreasing a concentration, level, function, activity, or behavior relative to natural, expected or average, or relative to a control condition.

[0072] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vector" are meant to refer to AAV vectors and methods for their synthetic production in an entirely cell-free environment.

[0073] As used herein, the term "systemic delivery" is meant to refer to delivery of lipid particles that results in widespread biodistribution of active agents, such as interfering RNA (e.g., siRNA) in an organism. Some administration techniques may lead to systemic delivery of a particular agent, while others may not. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the agent is exposed to most parts of the body. To achieve widespread biodistribution, a blood lifetime is generally required such that the agent is not rapidly degraded or cleared (by first-pass organs (liver, lung, etc.) or by rapid non-specific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles (e.g., lipid nanoparticles) can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles (e.g., lipid nanoparticles) is by intravenous delivery.

[0074] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and are meant to refer to the region where Rep forms a tyrosine-phosphodiester bond with a 5' thymidine that generates a 3'-OH that serves as a substrate for DNA elongation via a cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex may participate in a coordinate ligation reaction.

[0075] As used herein, the terms "therapeutic amount," "therapeutically effective amount," "effective amount," "effective amount," or "pharmaceutical effective amount" of an active agent (e.g., a TNA lipid particle described herein) are used interchangeably and refer to an amount sufficient to provide the intended benefit or effect of the treatment, e.g., inhibition of expression of a target sequence compared to the expression level detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, for example, testing of protein or RNA levels using techniques known to those of skill in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays also known to those of skill in the art. Dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, patient condition, severity of condition, route of administration, and the particular active agent used. Thus, dosing regimens can vary widely, but can be routinely determined by a physician using standard methods. Additionally, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutical effective amount" include prophylactic or preventative amounts of the compositions of the invention described. In prophylactic or preventative applications of the described invention, the pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk of a disease, disorder or condition, including the biochemical, histological and / or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes manifested during the development of the disease, disorder or condition, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder or condition. In one aspect, "therapeutic amount", "effective amount", "therapeutically effective amount", and "pharmaceutical effective amount" do not include prophylactic or preventative amounts of the described compositions of the invention. It is generally preferred to use the maximum dose, i.e., the highest safe dose, according to some medical judgment. The terms "dose" and "administration" are used interchangeably herein.In one aspect of any of the aspects or embodiments herein, "therapeutic amount," "therapeutically effective amount," and "pharmaceutical effective amount" refer to non-prophylactic or non-preventative uses.

[0076] As used herein, the term "therapeutic effect" refers to the outcome of treatment, which outcome is deemed desirable and beneficial. Therapeutic effect can include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. Therapeutic effect can also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease symptoms.

[0077] For any therapeutic agent described herein, the therapeutically effective amount can be determined first from preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is within the ability of a person skilled in the art to adjust the dose to achieve maximum efficacy based on the above methods and other known methods. The following summarizes the general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0078] Pharmacokinetic principles provide the basis for modifying dosing regimens to obtain the desired degree of therapeutic effect while minimizing unacceptable side effects. In situations where plasma concentrations of a drug can be measured and are related to the therapeutic window, additional guidance regarding dosage modifications is available.

[0079] As used herein, the terms "treat", "treating" and / or "treatment" include inhibiting, slowing or reversing the progression of a condition, improving clinical symptoms of a condition, or preventing the appearance of clinical symptoms, to obtain beneficial or desired clinical results. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder, (b) limiting the onset of symptoms characteristic of the disorder being treated, (c) limiting the worsening of symptoms characteristic of the disorder being treated, (d) limiting the recurrence of the disorder in patients who previously had the disorder, and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder. In one aspect of any of the aspects or embodiments herein, the terms "treat", "treating" and / or "treatment" include inhibiting, inhibiting, slowing or reversing the progression of a condition, or improving clinical symptoms of a condition.

[0080] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, preventing the occurrence of a disease, disorder or condition in a subject who may have a predisposition to the disease, disorder or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the symptoms of the disease, disorder or condition, reducing the severity of the disease, disorder or condition, stabilizing (i.e., not worsening) the disease, disorder or condition, preventing the spread of the disease, disorder or condition, slowing or retarding the progression of the disease, disorder or condition, ameliorating or alleviating the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0081] As used herein, the term "vector" or "expression vector" is meant to refer to a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an "insert," "transgene," or "expression cassette," can be attached to effect expression or replication of the attached segment ("expression cassette") in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be of viral or non-viral origin in its final form. However, for the purposes of this disclosure, "vector" generally refers to a synthetic AAV vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that can replicate and transfer a genetic sequence to a cell when associated with the appropriate control elements. In some embodiments of any of the aspects and embodiments herein, the vector can be a recombinant vector or an expression vector.

[0082] Groupings of alternative elements or embodiments of the invention disclosed herein should not be construed as limitations. 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 deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed herein to include the modified group, thus satisfying all Markush group descriptions used in the appended claims.

[0083] In some embodiments of any of the aspects, the disclosure described herein does not pertain to human cloning processes, processes for correcting the genetic identity of human germ lines, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without providing any substantial medical benefit to humans or animals, and processes for correcting the genetic identity of animals resulting from such processes.

[0084] Other terms are defined herein within the description of various aspects of the invention.

[0085] II. Lipids In a first embodiment, a cationic lipid of formula I is provided:

[0086] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R 2 are protonated, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3~C 10 Alkylene or C3-C 10 alkenylene, R 4 But, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0087] [ka] where: R 4a and R 4b However, each independently, C1~C 16Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b However, each independently, C7~C 14 Alkyl or C7-C 14 alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; Provided is a cationic lipid, wherein n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharma- ceutically acceptable salt thereof.

[0088] In a second embodiment, in the cationic lipid according to the first embodiment, or a pharma- ceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-, and all other remaining variables are as described for Formula I or the first embodiment.

[0089] In a third embodiment, the cationic lipid of the present disclosure is represented by formula II:

[0090] [ka] or a pharma- ceutically acceptable salt thereof, where n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described in Formula I, or the first or second embodiment. In an alternative third embodiment, n is an integer selected from 1, 2, and 3, and all other remaining variables are as described in Formula I, or the first or second embodiment.

[0091] In a fourth embodiment, the cationic lipid of the present disclosure is represented by formula III:

[0092] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula I, Formula II, or any one of the first through third embodiments.

[0093] In a fifth embodiment, the cationic lipid according to the first embodiment, or a pharma- ceutically acceptable salt thereof, is 1 and R 2 are each independently hydrogen, C1-C2 alkyl, or C2-C3 alkenyl, or R', R 1 , and R 2 is each independently hydrogen, C1-C2 alkyl, and all other remaining variables are as described for Formula I, Formula II, or any one of the first through fourth embodiments.

[0094] In a sixth embodiment, the cationic lipid of the present disclosure is represented by formula IV:

[0095] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula I, Formula II, Formula III, or any one of the first through fifth embodiments.

[0096] In a seventh embodiment, in the cationic lipid according to formula I, formula II, formula III, formula IV, or any one of the first to sixth embodiments, or a pharma- ceutically acceptable salt thereof, R 5 is absent or is 1-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 does not exist or R 5 is a C6 alkylene, a C5 alkylene, a C4 alkylene, a C3 alkylene, a C2 alkylene, a C1 alkylene, a C6 alkenylene, a C5 alkenylene, a C4 alkenylene, a C3 alkenylene, or a C2 alkenylene, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, or any one of the first through sixth embodiments.

[0097] In an eighth embodiment, the cationic lipid of the present disclosure is represented by formula V:

[0098] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, or any one of the first through seventh embodiments.

[0099] In a ninth embodiment, the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to eighth embodiments, or a pharma- ceutically acceptable salt thereof, wherein R 4 But, C1~C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or

[0100] [ka] where R 4a and R 4b However, each independently, C1~C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl or R 4 But, C2~C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl or R 4 But C5~C 12 Unbranched alkyl or C5-C 12 unbranched alkenyl or R 4 But, 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 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 but,

[0101] [ka] and R 4a and R 4b However, C2 to C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl or R4 but,

[0102] [ka] where R 4a and R 4b However, each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of Embodiments 1-8.

[0103] In a tenth embodiment, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to ninth embodiments, or a pharma- ceutically acceptable salt thereof, R 3 is C3-C8 alkylene or C3-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C3-C5 alkylene or C3-C5 alkenylene, or R 3is C8 alkylene, alternatively C7 alkylene, alternatively C6 alkylene, alternatively C5 alkylene, alternatively C4 alkylene, alternatively C3 alkylene, alternatively C1 alkylene, alternatively C8 alkenylene, alternatively C7 alkenylene, alternatively C6 alkenylene, alternatively C5 alkenylene, alternatively C4 alkenylene, alternatively C3 alkenylene, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first through ninth embodiments.

[0104] In an eleventh embodiment, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to tenth embodiments, or a pharma- ceutically acceptable salt thereof, R 6a and R 6b However, each independently, C7~C 12 Alkyl or C7~C 12 alkenyl or R 6a and R 6b However, each independently, C8~C 10 Alkyl or C8~C 10 alkenyl or R 6a and R 6b However, each independently, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first through tenth embodiments.

[0105] In a twelfth embodiment, the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to eleventh embodiments, or a pharma- ceutically acceptable salt thereof, is 6a and R 6b contain an equal number of carbon atoms, or R6a and R 6b are the same or R 6a and R 6b But both are C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first through eleventh embodiments.

[0106] In a thirteenth embodiment, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to twelfth embodiments, or a pharma- ceutically acceptable salt thereof, R 6a and R 6b each contain a different number of carbon atoms from each other, or the number of carbon atoms R 6a and R 6b may differ by one or two carbon atoms, or the number of carbon atoms R 6a and R 6b differ by one carbon atom, or R 6a is C7 alkyl, and R 6a is C8 alkyl, R 6a is C8 alkyl, R 6a is C7 alkyl, and R 6a is C8 alkyl, R 6a is C9 alkyl, R 6a is C9 alkyl, R 6a is C8 alkyl, R 6a is C9 alkyl, R 6a C 10 is alkyl, R 6a C 10 is alkyl, R 6a is C9 alkyl, R 6a C 10 is alkyl, R6a C 11 is alkyl, R 6a C 11 is alkyl, R 6a C 10 is alkyl, R 6a C 11 is alkyl, R 6a C 12 is alkyl, R 6a C 12 is alkyl, R 6a C 11 is alkyl, R 6a is C7 alkyl, and R 6a is C9 alkyl, R 6a is C9 alkyl, R 6a is C7 alkyl, and R 6a is C8 alkyl, R 6a C 10 is alkyl, R 6a C 10 is alkyl, R 6a is C8 alkyl, R 6a is C9 alkyl, R 6a C 11 is alkyl, R 6a C 11 is alkyl, R 6a is C9 alkyl, R 6a C 10 is alkyl, R 6a C 12 is alkyl, R 6a C 12 is alkyl, R 6a C 10 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 first through twelfth embodiments.

[0107] In a fourteenth embodiment, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to thirteenth embodiments, or a pharma- ceutically acceptable salt thereof, R' is absent and all other remaining variables are as described for formula I or any one of the first to thirteenth embodiments. In some aspects, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to fourteenth embodiments, R' is hydrogen or C1-C6 alkyl, and R', R 1 , and R 2 The nitrogen atom to which all of the are attached is protonated, in that the nitrogen atom is positively charged.

[0108] In some aspects, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to fourteenth embodiments, R′, R 1 , and R 2 are each C1-C6 alkyl, R', R 1 , and R 2 together with the nitrogen atom to which they are attached form a quaternary ammonium cation or a quaternary amine.

[0109] In a fifteenth embodiment, a cationic lipid of formula Ia:

[0110] [ka] or a pharma- ceutically acceptable salt thereof, wherein: R' is absent or is C1-C3 alkyl; R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3~C 10 Alkylene or C3-C 10 alkenylene, R 4 But, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl; R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b However, each independently, C7~C 14 Alkyl or C7-C 14 alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein R a is, for each occurrence, independently, hydrogen or C1-C6 alkyl; Provided is a cationic lipid, wherein n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharma- ceutically acceptable salt thereof.

[0111] In a sixteenth embodiment, in the cationic lipid according to the fifteenth embodiment, or a pharma- ceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-, and all other remaining variables are as described for Formula Ia or the fifteenth embodiment.

[0112] In a seventeenth embodiment, the cationic lipid of the present disclosure is represented by formula IIa or:

[0113] [ka] or a pharma- ceutically 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 Ia, or any one of the first through sixteenth embodiments. In an alternative third embodiment, n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula Ia, or the fifteenth or sixteenth embodiments.

[0114] In an eighteenth embodiment, the cationic lipid of the present disclosure is represented by formula IIIa or:

[0115] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula Ia, Formula IIa, or the fifteenth, sixteenth, or seventeenth embodiment.

[0116] In a nineteenth embodiment, the cationic lipid according to the first embodiment, or a pharma- ceutically acceptable salt thereof, is 1 and R 2 are each independently hydrogen, C1-C2 alkyl, or C2-C3 alkenyl, or R', R 1 , and R 2 is each independently hydrogen, C1-C2 alkyl, and all other remaining variables are as described for Formula Ia, Formula IIa, or the first through eighteenth embodiments.

[0117] In a twentieth embodiment, the cationic lipid of the present disclosure is represented by formula IVa or:

[0118] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, or nineteenth embodiments.

[0119] In a twenty-first embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, or any one of the first to twentieth embodiments, or a pharma- ceutically acceptable salt thereof, R 5 is absent or is 1-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 does not exist or R 5 is a C alkylene, a C alkylene, a C alkylene, a C alkylene, a C alkylene, a C alkylene, a C alkenylene, a C alkenylene, a C alkenylene, a C alkenylene, or a C alkenylene, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth embodiments.

[0120] In a twenty-second embodiment, the cationic lipid of the present disclosure is represented by formula Va:

[0121] [ka] or a pharma- ceutically acceptable salt thereof, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, or twenty-first embodiments.

[0122] In a twenty-third embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to twenty-second embodiments, or a pharma- ceutically acceptable salt thereof, R 4 But, C1~C 14 Unbranched alkyl or C2-C 14 unbranched alkenyl or R 4 But, C2~C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl or R 4 But C5~C 12 Unbranched alkyl or C5-C 12 unbranched alkenyl or R 4 But, 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 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, or twenty-second embodiments.

[0123] In a twenty-fourth embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to twenty-third embodiments, or a pharma- ceutically acceptable salt thereof, R 3 is 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, alternatively C7 alkylene, alternatively C6 alkylene, alternatively C5 alkylene, alternatively C4 alkylene, alternatively C3 alkylene, alternatively C1 alkylene, alternatively C8 alkenylene, alternatively C7 alkenylene, alternatively C6 alkenylene, alternatively C5 alkenylene, alternatively C4 alkenylene, alternatively C3 alkenylene, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, or twenty-third embodiments.

[0124] In a twenty-fifth embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to twenty-fourth embodiments, or a pharma- ceutically acceptable salt thereof, R 6a and R 6b However, each independently, C7~C 12 Alkyl or C7~C 12 alkenyl or R 6a and R 6b However, each independently, C8~C 10 Alkyl or C8~C 10 alkenyl or R 6a and R 6b However, each independently, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, or twenty-fourth embodiments.

[0125] In a twenty-sixth embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to twenty-fifth embodiments, or a pharma- ceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b But both are C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, or twenty-fifth embodiments.

[0126] In a twenty-seventh embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to twenty-sixth embodiments, or a pharma- ceutically acceptable salt thereof, R 6a and R 6b each contain a different number of carbon atoms from each other, or the number of carbon atoms R 6a and R 6b may differ by one or two carbon atoms, or the number of carbon atoms R 6a and R 6bdiffer by one carbon atom, or R 6a is C7 alkyl, and R 6a is C8 alkyl, R 6a is C8 alkyl, R 6a is C7 alkyl, and R 6a is C8 alkyl, R 6a is C9 alkyl, R 6a is C9 alkyl, R 6a is C8 alkyl, R 6a is C9 alkyl, R 6a C 10 is alkyl, R 6a C 10 is alkyl, R 6a is C9 alkyl, R 6a C 10 is alkyl, R 6a C 11 is alkyl, R 6a C 11 is alkyl, R 6a C 10 is alkyl, R 6a C 11 is alkyl, R 6a C 12 is alkyl, R 6a C 12 is alkyl, R 6a C 11 is alkyl, R 6a is C7 alkyl, and R 6a is C9 alkyl, R 6a is C9 alkyl, R 6a is C7 alkyl, and R 6a is C8 alkyl, R 6a C 10 is alkyl, R 6a C 10 is alkyl, R 6a is C8 alkyl, R 6a is C9 alkyl, R 6a C 11 is alkyl, R 6a C 11 is alkyl, R 6a is C9 alkyl, R 6aC 10 is alkyl, R 6a C 12 is alkyl, R 6a C 12 is alkyl, R 6a C 10 alkyl, etc., and all other remaining variables are as described in Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, or twenty-sixth embodiments.

[0127] In a twenty-eighth embodiment, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the first to twenty-seventh embodiments, or a pharma- ceutically acceptable salt thereof, R' is absent, and all other remaining variables are as described for Formula Ia, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, or twenty-seventh embodiments.

[0128] In a 29th embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to twenty-eighth embodiments, or a pharma- ceutically acceptable salt thereof, R' is absent, R', R 1 , and R 2 are protonated when the lipid is present under physiological conditions, e.g., at a pH of about 7.4 or less, e.g., at a pH of about 7.4, and all other remaining variables are as described for Formula Ia, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, or twenty-eighth embodiments.

[0129] In a 30th embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to twenty-ninth embodiments, or a pharma- ceutically acceptable salt thereof, R' is absent, R', R 1 , and R 2are attached to is protonated when the lipid is in aqueous solution, and all other remaining variables are as described for Formula Ia, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, or twenty-ninth embodiments.

[0130] In a thirty-first embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to thirtieth embodiments, or a pharma- ceutically acceptable salt thereof, R' is absent, R', R 1 , and R 2 are protonated when the lipid is present at a pH of about 7.4 or less, and all other remaining variables are as described for Formula Ia, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twentyninth, or thirtieth embodiments.

[0131] In a thirty-second embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to thirty-first embodiments, or a pharma- ceutically acceptable salt thereof, R′ is absent, and R′, R 1 , and R 2 are protonated when the lipid is present in an aqueous solution at a pH of about 7.4 or less (e.g., a pH of about 7.4), and all other remaining variables are as described for Formula Ia, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirtieth, or thirty-first embodiments.

[0132] In a thirty-third embodiment, in the cationic lipid according to formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the first to thirty-second embodiments, or a pharma- ceutically acceptable salt thereof, R′, R 1 , and R 2together with the nitrogen atom to which they are attached form a quaternary ammonium cation or a quaternary amine, and all other remaining variables are as described for Formula Ia, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirtieth, thirty-first, or thirty-second embodiments.

[0133] In some embodiments, in the cationic lipid of Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirtieth, thirty-first, thirty-second, or thirty-third embodiments, R' is hydrogen or C1-C6 alkyl, and R', R 1 , and R 2 The nitrogen atom to which all are attached is protonated if the nitrogen atom is positively charged.

[0134] In some embodiments, in the cationic lipid of formula Ia, formula IIa, formula IIIa, formula IVa, formula Va, or any one of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second, or thirty-third embodiments, R′, R 1 , and R 2 are respectively, C 1 ~C6 alkyl, R', R 1 , and R 2 together with the nitrogen atom to which they are attached form a quaternary ammonium cation or a quaternary amine.

[0135] In one embodiment, the cationic lipid of the present disclosure or of formula I or Ia is

[0136] [ka]

[0137] [ka]

[0138] [ka] or a pharma- ceutically acceptable salt thereof.

[0139] Additionally, the lipids of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or a pharma- ceutically acceptable salt thereof (e.g., a quaternary ammonium salt), or any of the exemplary lipids disclosed herein, may be converted to the corresponding lipids containing a quaternary amine or a quaternary ammonium cation, i.e., R′ ... 1 and R 2 is each C1-C6 alkyl (all are contemplated in this disclosure). The quaternary ammonium cations in such lipids are permanently charged regardless of the pH of their solution.

[0140] In some embodiments, the nitrogen atom of any of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated when the lipid is present under physiological conditions, e.g., at a pH of about 7.4 or less, e.g., at a pH of about 7.4.

[0141] In some embodiments, the nitrogen atom of any of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated when the lipid is in aqueous solution.

[0142] In some embodiments, the nitrogen atom of any of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated when the lipid is present at a pH of about 7.4 or less (e.g., a pH of about 7.4).

[0143] In some embodiments, the nitrogen atom of any of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated when the lipid is present in an aqueous solution at a pH of about 7.4 or less (e.g., a pH of about 7.4).

[0144] III. Lipid Nanoparticles (LNPs) LNPs as delivery vehicles for nucleic acids A lipid nanoparticle (LNP) comprising a cationic lipid as described herein and a capsid-free non-viral vector or a therapeutic nucleic acid (TNA) (e.g., ceDNA), or a pharmaceutical composition thereof, can be used to deliver a capsid-free non-viral DNA vector to a target site of interest (e.g., a cell, tissue, organ, etc.). Accordingly, another aspect of the present disclosure relates to a lipid nanoparticle (LNP) comprising one or more cationic lipids as described herein or a pharma- ceutically acceptable salt thereof and a therapeutic nucleic acid (TNA).

[0145] Cationic lipids are typically used to condense nucleic acid cargoes, such as ceDNA, at low pH and to drive membrane association and fusogenicity. In general, cationic lipids are lipids that contain at least one amino group that is positively charged or protonated under acidic conditions, such as pH 6.5 or lower, to form lipids that contain quaternary amines.

[0146] In one embodiment of any of the aspects or embodiments of the present specification, in the lipid nanoparticles, the cationic lipid or a pharma- ceutically acceptable salt thereof provided herein is about 30% to about 80%, for example, about 35% to about 80%, about 40% to about 80%, about 45% to about 80%, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, About 70% to about 80%, about 75% to about 80%, 30% to about 75%, about 35% to about 75%, about 40% to about 75%, about 45% to about 75%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, 30% to about 70%, about 35% to about 70%, about 40% to about 70%, about 45% to about 70%, about 50% to about 70%, about 55% to 70%, 60% to 70%, 65% to 70%, 30% to 65%, 35% to 65%, 40% to 65%, 45% to 65%, 50% to 65%, 55% to 65%, 60% to 65%, 30% to 60%, 35% to 60%, 40% to 60%, 45% to 60%, 50% to 60%, 55% to 60% , about 30% to about 55%, about 35% to about 55%, about 40% to about 55%, about 45% to about 55%, about 50% to about 55%, about 30% to about 50%, about 35% to about 50%, about 40% to about 50%, about 45% to about 50%, about 30% to about 45%, about 35% to about 45%, about 40% to about 45%, about 30% to about 40%, or about 35% to about 40%. In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the cationic lipid or pharma- ceutically acceptable salt thereof provided herein is present in a molar percentage of 40% to about 60%, or about 45% to about 60%, or about 45% to about 55%, or about 45% to about 50%, or about 50% to about 55%, or about 40% to about 50%, for example, but not limited to, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60%.

[0147] Sterols In one embodiment of any of the aspects or embodiments herein, in addition to the more cationic lipids described herein, or pharma- ceutically acceptable salts thereof, and TNA, the LNPs described herein further comprise at least one sterol to provide membrane integrity and stability of the lipid particle. In one embodiment of any of the aspects or embodiments herein, an exemplary sterol that can be used in the lipid particle is cholesterol or a derivative thereof. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol, non-polar analogs such as 5α-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate, and mixtures thereof. In some embodiments of any of the aspects or embodiments herein, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments of any of the aspects and embodiments herein, the cholesterol derivative is cholesteryl hemisuccinate (CHEMS).

[0148] Exemplary cholesterol derivatives are described in WO 2009 / 127060 and U.S. Patent Application Publication No. US2010 / 0130588, the contents of both of which are incorporated herein by reference in their entireties.

[0149] Further exemplary sterols include beta-sitosterol, campesterol, stigmasterol, ergosterol, brassicasterol, lopeol, cycloartenol, and derivatives thereof. In one embodiment of any of the aspects or embodiments herein, an exemplary sterol that may be used in the lipid particles is beta-sitosterol.

[0150] In one embodiment of any of the aspects or embodiments herein, the sterol is present in the lipid nanoparticles at a molar percentage of about 20% to about 50%, e.g., about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 50%, about 45% to about 50%, about 20% to about 45%, about 25% to about 45%, about 30% to about 45%, about 35% to about 45%, about 40% to about 45%, about 20% to about 40%, about 25% to about 40%, about 30% to about 40%, about 35% to about 40%, about 20% to about 35%, about 25% to about 35%, about 30% to about 35%, about 20% to about 30%, or about 25% to about 35%. In one embodiment of any of the aspects or embodiments herein, the sterol is present in the lipid nanoparticles in a molar percentage of about 35% to about 45%, or about 40% to about 45%, or about 35% to about 40%, for example, but not limited to, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45%.

[0151] Non-cationic lipids In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) described herein further comprise at least one non-cationic lipid. Non-cationic lipids are also known as structural lipids and can act to increase membrane fusogenicity and also increase the stability of the forming LNP to provide membrane integrity and lipid particle stability. Non-ionizable lipids include amphipathic lipids, neutral lipids, and anionic lipids. Thus, non-cationic lipids can be neutral uncharged, zwitterionic, or anionic lipids.

[0152] Exemplary non-cationic lipids include distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DS PE), monomethyl-phosphatidylethanolamine (16-O-monomethyl PE, etc.), dimethyl-phosphatidylethanolamine (16-O-dimethyl PE, etc.), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,Examples of phospholipids include, but are not limited to, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicaside, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably C, 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid is any one or more selected from dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).

[0153] Other examples of non-cationic lipids suitable for use in lipid particles (e.g., lipid nanoparticles) include non-phosphorous lipids, such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, and the like.

[0154] Additional exemplary non-cationic lipids are described in WO 2017 / 099823 and U.S. Patent Application Publication No. US2018 / 0028664, the contents of both of which are incorporated by reference in their entireties.

[0155] In one embodiment of any of the aspects or embodiments of the present specification, the non-cationic lipid in the lipid nanoparticles is about 2% to about 20%, for example, about 3% to about 20%, about 5% to about 20%, about 7% to about 20%, about 8% to about 20%, about 10% to about 20%, about 12% to about 20%, about 13% to about 20%, about 15% to about 20%, about 17% to about 20%, about 18% to about 20%, about 2% to about 18%, About 3% to about 18%, about 5% to about 18%, about 7% to about 18%, about 8% to about 18%, about 10% to about 18%, about 12% to about 18%, about 13% to about 18%, about 15% to about 18%, about 17% to about 18%, about 2% to about 17%, about 3% to about 17%, about 5% to about 17%, about 7% to about 17%, about 8% to about 17%, about 10% to about 17%, about 12% to about 17%, about 13% to about 17%, about 15% to about 17 %, about 2% to about 15%, about 3% to about 15%, about 5% to about 15%, about 7% to about 15%, about 8% to about 15%, about 10% to about 15%, about 12% to about 15%, about 13% to about 15%, about 2% to about 13%, about 3% to about 13%, about 5% to about 13%, about 7% to about 13%, about 8% to about 13%, about 10% to about 13%, about 12% to about 13%, about 2% to about 12%, about 3% to about 12%, about 5% to about 12%, It is present in a molar percentage of about 7% to about 12%, about 8% to about 12%, about 10% to about 12%, about 2% to about 10%, about 3% to about 10%, about 5% to about 10%, about 7% to about 10%, about 8% to about 10%, about 2% to about 8%, about 3% to about 8%, about 5% to about 8%, about 7% to about 8%, about 2% to about 7%, about 3% to about 7%, about 5% to about 7%, about 2% to about 5%, about 3% to about 5%, or about 2% to about 3%.In one embodiment of any of the aspects or embodiments of the present specification, the non-cationic lipid in the lipid nanoparticles is about 5% to about 15%, about 7% to about 15%, about 8% to about 15%, about 10% to about 15%, about 12% to about 15%, about 13% to about 15%, 5% to about 13%, about 7% to about 13%, about 8% to about 13%, about 10% to about 13%, about 12% to about 13%, about 5% to about 12%, about 7% about 12%, about 8% to about 12%, about 10% to about 12%, about 5% to about 10%, about 7% to about 10%, about 8% to about 10%, about 5% to about 8%, about 7% to about 8%, or about 5% to about 7%, for example, but not limited to, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 11%, about 12%, about 13%, about 14%, or about 15%.

[0156] PEGylated lipids In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) described herein further comprise at least one (e.g., one, two, or three) PEGylated lipid. A PEGylated lipid is a lipid, as defined herein, that is covalently or non-covalently attached to one or more polyethylene glycol (PEG) polymer chains, and is therefore a class of conjugated lipids. In general, PEGylated lipids are incorporated into LNPs to inhibit particle aggregation and / or provide steric stabilization. In one embodiment of any of the aspects or embodiments herein, the lipid is covalently attached to one or more PEG polymer chains.

[0157] PEG molecules suitable for use in PEGylated lipids include, but are not limited to, those having a molecular weight of about 500 to about 10,000, or about 1,000 to about 7,500, or about 1,000 to about 5,000, or about 2,000 to about 5,000, or about 2,000 to about 4,000, or about 2,000 to about 3,500, or about 2,000 to about 3,000, for example, but are not limited to, PEG 2000, PEG 2500, PEG 3000, PEG 3350, PEG 3500, and PEG 4000.

[0158] The lipid to which one or more PEG chains are linked may be a sterol, a non-cationic lipid, or a phospholipid. Exemplary PEGylated lipids include PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1, Examples of PEGylated lipids include, but are not limited to, 2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEGylated lipids are described in, for example, U.S. Patent Nos. 5,885,613 and 6,287,591, and U.S. Patent Application Publication Nos. US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904, the contents of each of which are incorporated herein by reference in their entirety.

[0159] In one embodiment of any of the aspects or embodiments herein, the at least one PEGylated lipid in the lipid nanoparticle (LNP) provided herein is selected from the group consisting of PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, PEG-distearyloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol-PEG (DMG-PEG), distearoyl-rac-glycerol-PEG (DSG-PEG), PEG-dilauryloxypropyl, ... PEG-glycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol); 3,4-ditetradecoxybenzyl-[omega]-methyl-poly( In one embodiment of any of the aspects or embodiments herein, the at least one PEGylated lipid is selected from the group consisting of DMG-PEG, DSPE-PEG, DSPE-PEG-OH, DSG-PEG, or a combination thereof. In one embodiment of any of the aspects or embodiments herein, the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSG-PEG2000, or a combination thereof. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticle (LNP) provided herein comprises DMG-PEG2000 and DSPE-PEG2000.In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) provided herein comprise DMG-PEG2000 and DSG-PEG2000. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) provided herein comprise DSPE-PEG2000 and DSPE-PEG2000-OH.

[0160] In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the at least one PEGylated lipid is present in a total amount of about 1% to about 10%, for example, about 1.5% to about 10%, about 2% to about 10%, about 2.5% to about 10%, about 3% to about 10%, about 3.5% to about 10%, about 4% to about 10%, about 4.5% to about 10%, about 5% to about 10%, about 5.5% to about 10%, about 6% to about 10%, about 6.5% to about 10%, about 7% to about 10%, about 7.5% to about 10%, about 8% to about 10%, about 8.5% to about 10%, about 9% to about 10%, about 9.5% to about 10%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about 5%, about 5% to about 5 ...5% to about 5%, about 6% to about 10%, about 6% to about 10%, about 6.5% to about 10%, about 7% to about 10%, about 7.5% to about 10%, about 8% to about 10%, about %, about 2.5% to about 5%, about 3% to about 5%, about 3.5% to about 5%, about 4% to about 5%, about 4.5% to about 5%, about 1% to about 4%, about 1.5% to about 4%, about 2% to about 4%, about 2.5% to about 4%, about 3% to about 4%, about 3.5% to about 4%, about 1% to about 3.5%, about 1.5% to about 3.5%, about 2% to about 3.5%, It is present in a molar percentage of about 2.5% to about 3.5%, about 3% to about 3.5%, about 1% to about 3%, about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 1% to about 2.5%, about 1.5% to about 2.5%, about 2% to about 2.5%, about 1% to about 2%, about 1.5% to about 2%, or about 1% to about 1.5%. In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticle, the at least one PEGylated lipid is present in a total molar percentage of about 1% to about 2%, about 1.5% to about 2%, or about 1% to about 1.5%, for example, but not limited to, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0161] In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticle, the at least one PEGylated lipid is present in a total amount of about 2.1% to about 10%, e.g., about 2.5% to about 10%, about 3% to about 10%, about 3.5% to about 10%, about 4% to about 10%, about 4.5% to about 10%, about 5% to about 10%, about 5.5% to about 10%, about 6% to about 10%, about 6.5% to about 10%, about 7% to about 10%, about 7.5% to about 10%, about 8% to about 10%, about 8.5% to about 10%, about 9% to about 10%, about 9.5% to about 10%, about 2.1% to about 7%, about 2.5% to about 7%, about It is present in a molar percentage of from 3% to about 7%, from about 3.5% to about 7%, from about 4% to about 7%, from about 4.5% to about 7%, from about 5% to about 7%, from about 5.5% to about 7%, from about 6% to about 7%, from about 6.5% to about 7%, from about 2.1% to about 5%, from about 2.5% to about 5%, from about 3% to about 5%, from about 3.5% to about 5%, from about 4% to about 5%, from about 4.5% to about 5%, from about 2.1% to about 4%, from about 2.5% to about 4%, from about 3% to about 4%, from about 3.5% to about 4%, from about 2.1% to about 3.5%, from about 2.5% to about 3.5%, from about 3% to about 3.5%, from about 2.1% to about 3%, from about 2.5% to about 3%, or from about 2.1% to about 2.5%. In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticle, the at least one PEGylated lipid is, in total, about 2.1% to about 5%, about 2.5% to about 5%, about 3% to about 5%, about 3.5% to about 5%, about 4% to about 5%, about 4.5% to about 5%, about 2.1% to about 4%, about 2.5% to about 4%, about 3% to about 4%, about 3.5% to about 4%, about 2.1% to about 3.5%, about 2.5% to about 3.5%, about 3% to about 3.5%, about 2.1% to about 3%, about 2.5% to about 3%, or about 2.1%. % to about 2.5%, for example, but not limited to, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, or about 5%.

[0162] Tissue-specific targeting ligands and PEGylated lipid conjugates In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) described herein further comprise at least one tissue-specific targeting ligand to aid, enhance, and / or increase delivery of the LNP to a target site of interest. The ligand can be any biomolecule, such as a peptide, protein, antibody, glycan, sugar, nucleic acid, lipid, or conjugate comprising any of the foregoing, that recognizes a receptor or surface antigen that is unique to certain cells and tissues.

[0163] In one embodiment of any of the aspects or embodiments herein, at least one tissue-specific targeting ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. The term "GalNAc derivative" encompasses modified GalNAc, functionalized GalNAc, and GalNAc conjugates, in which one or more GalNAc molecules (natural or modified) are covalently attached to one or more functional groups or one or more classes of exemplary biomolecules, such as, but not limited to, peptides, proteins, antibodies, glycans, sugars, nucleic acids, lipids, etc. The biomolecule itself to which one or more GalNAc molecules can be attached typically aids in increasing stability and / or inhibiting aggregation. In one embodiment of any of the aspects or embodiments herein, the molar ratio of the tissue-specific targeting ligand, such as GalNAc, to the biomolecule to which the ligand is conjugated is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. In one embodiment of any of the aspects or embodiments herein, the molar ratio of the tissue-specific targeting ligand, such as GalNAc, to the biomolecule to which the ligand is conjugated is 1:1 (e.g., mono-antennary GalNAc), 2:1 (bi-antennary GalNAc), 3:1 (tri-antennary GalNAc), and 4:1 (tetra-antennary GalNAc).Conjugated GalNAc, such as tri-antennary GalNAc (GalNAc3) or tetra-antennary GalNAc (GalNAc4), can be synthesized as known in the art (see WO 2017 / 084987 and WO 2013 / 166121) and can be chemically conjugated to lipids or PEG as known in the art (see Resen et al., J. Biol. Chem. (2001) "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo" 276:375577-37584).

[0164] In one embodiment of any of the aspects or embodiments herein, the tissue-specific targeting ligand is covalently attached to a PEGylated lipid as defined and described herein to form a PEGylated lipid conjugate. Exemplary PEGylated lipids are described above and include PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, PEG-distearyloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (DMG-PEG), PEG-dilauryl glycerol, PEG-dipalmitoyl glycerol, PEG-disteryl glycerol, PEG-dilauryl glycamide, PEG-dimyristyl glycerol ... Likamide, PEG-dipalmitoyl glycamide, PEG-disteryl glycamide, (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-ditetradecoxybenzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) provided herein comprise DMG-PEG2000 and DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the tissue-specific targeting ligand is covalently attached to GalNAc or a GalNAc derivative. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is mono-, bi-, tri-, or tetra-antennary GalNAc-DSPE-PEG. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is mono-, bi-, tri-, or tetra-antennary GalNAc-DSG-PEG.In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is mono-, bi-, tri-, or tetra-antennary GalNAc-DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is mono-, bi-, tri-, or tetra-antennary GalNAc-DSG-PEG2000. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is tri-antennary GalNAc-DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is tri-antennary GalNAc-DSG-PEG2000. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is tetra-antennary GalNAc-DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is tetra-antennary GalNAc-DSG-PEG2000.

[0165] In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the PEGylated lipid conjugate is present in an amount of about 0.1% to about 10%, for example, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, about 2% to about 10%, about 2.5% to about 10%, about 3% to about 10%, about 3.5% to about 10%, about 4% to about 10%, about 4.5% to about 10%, about 5% to about 10%, about 5.5% to about 10%, about 6% to about 10%, about 6.5% to about 10%, about 7% to about 10%, about 7.5% to about 10%, about 8% to about 10%, about 8.5% to about 10%, about 9% to about 10%, about 9.5% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to about 10%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 2.5% to about 5%, about 3% to about 5%, about 3.5% ~ about 5%, about 4% to about 5%, about 4.5% to about 5%, about 0.1% to about 3%, about 0.2% to about 3%, about 0.3% to about 3%, about 0.4% to about 3%, about 0.5% to about 3%, about 0.6% to about 3%, about 0.7% to about 3%, about 0.8% to about 3%, about 0.9% to about 3%, about 1% to about 3%, about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 0.1% to about 2%, about 0.2% to about 2%, about 0.3% to about 2%, about 0.4% to about 2%, about 0.5% to about 2%, about 0.6% to about 2%, about 0.7% to about 2%, about 0.8% to about 2%, about 0.9% to about 2%, It is present in a molar percentage of from 1% to about 2%, from about 1.5% to about 2%, from about 0.1% to about 1.5%, 0.2% to about 1.5%, from about 0.3% to about 1.5%, from about 0.4% to about 1.5%, from about 0.5% to about 1.5%, from about 0.6% to about 1.5%, from about 0.7% to about 1.5%, from about 0.8% to about 1.5%, from about 0.9% to about 1.5%, from about 1% to about 1.5%, from about 0.1% to about 1%, 0.2% to about 1%, from about 0.3% to about 1%, from about 0.4% to about 1%, from about 0.5% to about 1%, from about 0.6% to about 1%, from about 0.7% to about 1%, from about 0.8% to about 1%, or from about 0.9% to about 1%.In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the PEGylated lipid conjugate is about 0.1% to about 1.5%, about 0.2% to about 1.5%, about 0.3% to about 1.5%, about 0.4% to about 1.5%, about 0.5% to about 1.5%, about 0.6% to about 1.5%, about 0.7% to about 1.5%, about 0.8% to about 1.5%, about 0.9% to about 1.5%, about 1% to about 1.5%, about 0.1% to about 1%, about 0.2% to about 1%. , about 0.3% to about 1%, about 0.4% to about 1%, about 0.5% to about 1%, about 0.6% to about 1%, about 0.7% to about 1%, about 0.8% to about 1%, or about 0.9% to about 1%, for example, but not limited to, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, or about 1.5%.

[0166] Other components of lipid nanoparticles (LNPs) Additional components of the LNP, such as conjugated lipids, are also contemplated in the present disclosure. Exemplary conjugated lipids include, but are not limited to, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof.

[0167] Furthermore, in one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) described herein further comprise an immunomodulatory compound, for example, by co-encapsulation in the LNP or by conjugation to a therapeutic nucleic acid or any one of the components of the LNPs described above. An immunomodulatory compound, such as dexamethasone or modified dexamethasone, can help minimize immune responses. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) described herein further comprise dexamethasone palmitate.

[0168] In some embodiments of any of the aspects and embodiments herein, in addition to the cationic lipid, the lipid nanoparticle comprises an agent for condensing and / or encapsulating nucleic acid cargo, such as ceDNA. Such an agent is also referred to herein as a condensing agent or encapsulating agent. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acid can be used as long as it is non-fusogenic. In other words, the agent can condense and / or encapsulate nucleic acid cargo, such as ceDNA, but has little or no fusogenic activity. Without wishing to be bound by theory, the condensing agent may have some fusogenic activity when it does not condense / encapsulate nucleic acid, such as ceDNA, but the nucleic acid encapsulated in the lipid nanoparticle formed with the condensing agent may be non-fusogenic.

[0169] Total lipid to nucleic acid ratio In general, lipid particles (e.g., lipid nanoparticles) have a final particle ratio of about 10:1 to 60:1, for example, about 15:1 to about 60:1, about 20:1 to about 60:1, about 25:1 to about 60:1, about 30:1 to about 60:1, about 35:1 to about 60:1, about 40:1 to about 60:1, about 45:1 to about 60:1, about 50:1 to about 60:1, about 55:1 to about 60:1, about 10:1 to about 55:1, about 15:1 to about 55:1, about 20 ... about 55:1, about 25:1 to about 55:1, about 30:1 to about 55:1, about 35:1 to about 55:1, about 40:1 to about 55:1, about 45:1 to about 55:1, about 50:1 to about 55:1, about 10:1 to about 50:1, about 15:1 to about 50:1, about 20:1 to about 50:1, about 25:1 to about 50:1, about 30:1 to about 50:1, about 35:1 to about 50:1, about 40:1 to about 50:1, about 45:1 to about 50:1, about 10:1 to about 4 5:1, about 15:1 to about 45:1, about 20:1 to about 45:1, about 25:1 to about 45:1, about 30:1 to about 45:1, about 35:1 to about 45:1, about 40:1 to about 45:1, about 10:1 to about 40:1, about 15:1 to about 40:1, about 20:1 to about 40:1, about 25:1 to about 40:1, about 30:1 to about 40:1, about 35:1 to about 40:1, about 10:1 to about 35:1, about 15:1 to about 35:1, about 20:1 to about 35 1, about 25:1 to about 35:1, about 30:1 to about 35:1, about 10:1 to about 30:1, about 15:1 to about 30:1, about 20:1 to about 30:1, about 25:1 to about 30:1, about 10:1 to about 25:1, about 15:1 to about 25:1, about 20:1 to about 25:1, about 10:1 to about 20:1, about 15:1 to about 20:1, or about 10:1 to about 15:1 total lipid to therapeutic nucleic acid (mass or weight).

[0170] The amounts of lipid and nucleic acid can be adjusted to provide a desired N / P ratio (i.e., the ratio of positively chargeable polymeric amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups), for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. Generally, the total lipid content of a lipid particle formulation can range from about 5 mg / ml to about 30 mg / mL.

[0171] Lipid Nanoparticle (LNP) Size According to some embodiments of any of the aspects or embodiments herein, the LNP has a diameter of about 40 nm to about 120 nm, e.g., about 45 nm to about 120 nm, about 50 nm to about 120 nm, about 55 nm to about 120 nm, about 60 nm to about 120 nm, about 65 nm to about 120 nm, about 70 nm to about 120 nm, about 75 nm to about 120 nm, about 80 nm to about 120 nm, about 85 nm to about 120 nm, about 90 nm to about 120 nm, about 95 nm to about 120 nm, about 100 nm to about 120 nm, about 105 nm to about 120 nm, about 110 nm to about 120 nm, about 115 nm to about 120 nm, about 40 nm to about 110 nm, about 45 nm to about 110 nm, about 50 nm to about 1 ... 10nm, approximately 60nm ~ approximately 110nm, approximately 65nm ~ approximately 110nm, approximately 70nm ~ approximately 110nm, approximately 75nm ~ approximately 110nm, approximately 80nm ~ approximately 110nm, approximately 85nm ~ Approximately 110nm, approximately 90nm to approximately 110nm, approximately 95nm to approximately 110nm, approximately 100nm to approximately 110nm, approximately 105nm to approximately 110nm, approximately 40nm to approximately 100nm, approximately 4 The diameter ranges from 5 nm to about 100 nm, from about 50 nm to about 100 nm, from about 55 nm to about 100 nm, from about 60 nm to about 100 nm, from about 65 nm to about 100 nm, from about 70 nm to about 100 nm, from about 75 nm to about 100 nm, from about 80 nm to about 100 nm, from about 85 nm to about 100 nm, from about 90 nm to about 100 nm, or from about 95 nm to about 100 nm.

[0172] According to some embodiments of any of the aspects or embodiments herein, the LNP is less than about 100 nm, e.g., about 40 nm to about 90 nm, about 45 nm to about 90 nm, about 50 nm to about 90 nm, about 55 nm to about 90 nm, about 60 nm to about 90 nm, about 65 nm to about 90 nm, about 70 nm to about 90 nm, about 75 nm to about 90 nm, about 80 nm to about 90 nm, about 85 nm to about 90 nm, about 40 nm to about 85 nm, about 45 nm to about 85 nm, about 50 nm to about 85 nm, about 55 nm to about 85 nm, about 60 nm to about 85 nm, about 65 nm to about 85 nm, about 70 nm to about 85 nm, about 75 nm to about 85 nm, about The diameter is about 80 nm to about 85 nm, about 40 nm to about 80 nm, about 45 nm to about 80 nm, about 50 nm to about 80 nm, about 55 nm to about 80 nm, about 60 nm to about 80 nm, about 65 nm to about 80 nm, about 70 nm to about 80 nm, about 75 nm to about 80 nm, about 40 nm to about 75 nm, about 45 nm to about 75 nm, about 50 nm to about 75 nm, about 55 nm to about 75 nm, about 60 nm to about 75 nm, about 65 nm to about 75 nm, about 70 nm to about 75 nm, about 40 nm to about 70 nm, about 45 nm to about 70 nm, about 50 nm to about 70 nm, about 55 nm to about 70 nm, about 60 nm to about 70 nm, or about 65 nm to about 70 nm. In one embodiment of any of the aspects or embodiments herein, the LNP is about 60 nm to about 85 nm, about 65 nm to about 85 nm, about 70 nm to about 85 nm, about 75 nm to about 85 nm, about 80 nm to about 85 nm, about 60 nm to about 80 nm, about 65 nm to about 80 nm, about 70 nm to about 80 nm, about 75 nm to about 80 nm, about 60 nm to about 75 nm, about 65 nm to about 75 nm, about 70 nm to about 75 nm, about 60 nm to about 70 nm, or The diameter may be about 65 nm to about 70 nm, for example, but not limited to, about 60 mm, about 61 mm, about 62 mm, about 63 mm, about 64 mm, about 65 mm, about 66 mm, about 67 mm, about 68 mm, about 69 mm, about 70 mm, about 71 mm, about 72 mm, about 73 mm, about 74 mm, about 75 mm, about 76 mm, about 77 mm, about 78 mm, about 79 mm, about 80 mm, about 81 mm, about 82 mm, about 83 mm, about 84 mm, or about 85 mm.

[0173] In one embodiment of any of the aspects or embodiments herein, lipid particle (e.g., lipid nanoparticle) size can be determined by quasi-elastic light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, UK) system.

[0174] LNPs comprising a cationic lipid, a sterol, a non-cationic lipid, a PEGylated lipid, and optionally a tissue-specific targeting ligand. According to some embodiments of any of the aspects or embodiments herein, the lipid nanoparticles provided herein comprise at least one cationic lipid, at least one sterol, at least one non-cationic lipid, and at least one PEGylated lipid as described herein.In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles provided herein essentially consist of at least one cationic lipid, at least one sterol, at least one non-cationic lipid, and at least one PEGylated lipid as described herein.In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles provided herein consist of at least one cationic lipid, at least one sterol, at least one non-cationic lipid, and at least one PEGylated lipid as described herein. In one embodiment of any of the aspects or embodiments herein, the molar ratio of cationic lipid:sterol:non-cationic lipid:PEGylated lipid is about 48(±5):10(±3):41(±5):2(±2), e.g., about 47.5:10.0:40.7:1.8, or about 47.5:10.0:40.7:3.0.

[0175] According to some embodiments of any of the aspects or embodiments herein, the lipid nanoparticles provided herein comprise at least one cationic lipid, at least one sterol, at least one non-cationic lipid, at least one PEGylated lipid, and a tissue-specific targeting ligand as described herein. In one embodiment of any of the aspects or embodiments herein, the tissue-specific targeting ligand is GalNac. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles provided herein essentially consist of at least one cationic lipid, at least one sterol, at least one non-cationic lipid, at least one PEGylated lipid, and a tissue-specific targeting ligand as described herein. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles provided herein consist of at least one cationic lipid, at least one sterol, at least one non-cationic lipid, at least one PEGylated lipid, and a tissue-specific targeting ligand as described herein. In one embodiment of any of the aspects or embodiments herein, the tissue-specific targeting ligand is conjugated to the PEGylated lipid to form a PEGylated lipid conjugate. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is mono-, bi-, tri-, or tetra-antennary GalNAc-DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is tetra-antennary GalNAc-DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the molar ratio of cationic lipid:sterol:non-cationic lipid:PEGylated lipid:PEGylated lipid conjugate is about 48(±5):10(±3):41(±5):2(±2), 1.5(±1), e.g., 47.5:10.0:40.2:1.8:0.5 or 47.5:10.0:39.5;2.5:0.5.

[0176] IV. Therapeutic Nucleic Acids (TNA) The present disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs). Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimally immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). Thus, aspects of the present disclosure generally provide ionizable lipid particles (e.g., lipid nanoparticles) that include TNAs.

[0177] siRNA or miRNA, which can downregulate the intracellular level of a specific protein through a process called RNA interference (RNAi), are also contemplated by the present invention as nucleic acid therapeutics. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of siRNA or miRNA is removed by the RISC complex. When the RISC complex binds to complementary mRNA, it cleaves the mRNA and releases the cleaved strand. RNAi is by inducing specific destruction of mRNA, which leads to the downregulation of the corresponding protein.

[0178] Antisense oligonucleotides (ASOs) and ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxynucleic acids have sequences complementary to the sequences of target protein mRNA and can bind to mRNA by Watson-Crick base pairing. This binding prevents the translation of the target mRNA and / or induces RNase H degradation of the mRNA transcript. As a result, antisense oligonucleotides have enhanced specificity of action (i.e., downregulation of specific disease-related proteins).

[0179] In any of the methods and compositions provided herein, the therapeutic nucleic acid (TNA) can be a therapeutic RNA. The therapeutic RNA can be an inhibitor of mRNA translation, an agent of RNA interference (RNAi), a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), or an RNA that binds to an mRNA transcript (ASO), a protein, or other molecular ligand (aptamer). In any of the methods provided herein, the agent of RNAi can be a double-stranded RNA, a single-stranded RNA, a microRNA, a short interfering RNA, a small hairpin RNA, or a triple-helix forming oligonucleotide.

[0180] In any of the method compositions provided herein, the therapeutic nucleic acid (TNA) is a therapeutic DNA such as a closed-end double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministring"), doggybone™, protelomeric closed-end DNA, dumbbell linear DNA, plasmid, minicircle, etc.). Some embodiments of the present disclosure are based on methods and compositions comprising closed-end linear duplexes (ceDNA) capable of expressing a transgene (e.g., a therapeutic nucleic acid). ceDNA vectors as described herein do not have the packaging constraints imposed by the limited space within a viral capsid. ceDNA vectors represent a versatile eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors.

[0181] The ceDNA vector preferably has a linear, continuous structure rather than a discontinuous structure. A linear, continuous structure is believed to be more stable against attack by cellular endonucleases and at the same time less likely to be recombined to cause mutagenesis. Thus, a linear, continuous structure ceDNA vector is a preferred embodiment. A continuous, linear, single-stranded intramolecular duplex ceDNA vector may be covalently linked at the termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. Complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, whereas, conversely, a ceDNA vector has complementary strands but is a single DNA molecule and therefore remains a single molecule even when denatured. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector, unlike a plasmid, can be produced without prokaryotic cell-type DNA base methylation. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (in particular linear vs. circular) and in terms of the methods used to produce and purify these different objects, and also in terms of their DNA methylation, which in the case of ceDNA-plasmids is of prokaryotic cell type and in the case of ceDNA vectors is of eukaryotic cell type.

[0182] Provided herein are non-viral capsid-free ceDNA molecules (ceDNA) with covalently closed ends. These non-viral capsid-free ceDNA molecules can be produced in permissive host cells from expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrative cell lines) that contain a heterologous gene (e.g., a transgene, particularly a therapeutic transgene) located between two different inverted terminal repeat (ITR) sequences, the ITRs being different with respect to each other. In some embodiments of any of the aspects and embodiments herein, one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs includes a functional terminal resolution site (TRS) and a Rep binding site. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vectors have covalently closed ends and are therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) for, for example, more than 1 hour at 37°C.

[0183] In one aspect of any of the aspects and embodiments herein, the ceDNA vector comprises, in a 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR. In one embodiment of any of the aspects and embodiments herein, the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric with respect to each other, i.e., they have a different three-dimensional spatial configuration from each other. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutant or modified ITR, or vice versa, where the first ITR can be a mutant or modified ITR and the second ITR can be a wild-type ITR. In one embodiment of any of the aspects and embodiments herein, the first ITR and the second ITR are both modified, but are different sequences, or have different modifications, or are not the same modified ITRs, but have different three-dimensional spatial configurations. In other words, a ceDNA vector with asymmetric ITRs has an ITR where any change in one ITR relative to the WT-ITR is not reflected in the other ITR, or alternatively, the asymmetric ITRs may have different sequences and different three-dimensional shapes relative to each other when the asymmetric ITRs have a modified asymmetric ITR pair.

[0184] In one embodiment of any of the aspects and embodiments herein, the ceDNA vector comprises, in the 5' to 3' direction, 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, where the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical with respect to each other, i.e., the ceDNA vector may comprise ITR sequences that have a symmetrical three-dimensional spatial organization, whereby 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 an embodiment, the symmetrical ITR pair or the substantially symmetrical ITR pair may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair may have the same sequence that has one or more modifications from the wild-type ITR and is the reverse complement (inversion) of each other. In one embodiment of any of the aspects and embodiments herein, the modified ITR pair is substantially symmetrical as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetrical three-dimensional shapes. In some embodiments of any of the aspects and embodiments herein, the symmetrical ITR or the substantially symmetrical ITR may be wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs of the same AAV serotype. In one embodiment of any of the aspects and embodiments herein, one WT-ITR may be derived from one AAV serotype and the other WT-ITR may be derived from a different AAV serotype. In such an embodiment, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they may have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial configuration.

[0185] The wild-type or mutant or otherwise modified ITR sequences provided herein represent DNA sequences contained in an expression construct (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus) for the production of a ceDNA vector. Thus, the ITR sequences actually contained in a ceDNA vector produced from a ceDNA-plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring variations (e.g., replication errors) that occur during the production process.

[0186] In one embodiment of any of the aspects and embodiments herein, the ceDNA vector described herein, which comprises an expression cassette having a transgene that is a therapeutic nucleic acid sequence, can be operably linked to one or more regulatory sequences that allow or control the expression of the transgene. In one embodiment of any of the aspects and embodiments herein, the polynucleotide comprises a first ITR sequence and a second ITR sequence, the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetric with respect to each other or symmetric with respect to each other.

[0187] In one embodiment of any of the aspects and embodiments herein, the expression cassette is located between two ITRs and comprises one or more of a promoter operably linked to a transgene, a post-transcriptional regulatory element, and a polyadenylation and termination signal, in that order. In one embodiment of any of the aspects or embodiments herein, the promoter is regulatable-inducible or repressible. The promoter can be any sequence that promotes transcription of the transgene. In one embodiment of any of the aspects or embodiments herein, the promoter is a CAG promoter or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates the expression of the transgene, and by way of non-limiting example, any sequence that creates a tertiary structure that enhances the expression of the transgene, which is a therapeutic nucleic acid sequence.

[0188] In one embodiment of any of the aspects or embodiments herein, the post-transcriptional regulatory element comprises WPRE. In one embodiment of any of the aspects or embodiments herein, the polyadenylation and termination signal comprises BGHpolyA. Any cis-regulatory element known in the art, or combinations thereof, may additionally be used, such as, but not limited to, the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements, including, but not limited to, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV). In one embodiment of any of the aspects or embodiments herein, the expression cassette length in the 5' to 3' direction exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment of any of the aspects or embodiments herein, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0189] In one embodiment of any of the aspects or embodiments herein, the expression cassette can contain more than 4000 nucleotides, e.g., about 5000 nucleotides, about 10,000 nucleotides, or about 20,000 nucleotides, or about 30,000 nucleotides, or about 40,000 nucleotides, or about 50,000 nucleotides, or any range of about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides.

[0190] In one embodiment of any of the aspects or embodiments herein, the expression cassette may also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type specific promoters, and enhancers. In some embodiments of any of the aspects or embodiments herein, the ITRs may act as promoters of the transgene. In some embodiments of any of the aspects or embodiments herein, the ceDNA vector may include additional components for regulating expression of the transgene, such as a regulatory switch for controlling and regulating expression of the transgene, and, if desired, a regulatory switch that is a kill switch that allows for controlled cell death of cells containing the ceDNA vector.

[0191] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is capsid-free and may be obtained from a plasmid encoding, in that order, a first ITR, an expressible transgene cassette and a second ITR, wherein at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence.

[0192] In one embodiment of any of the aspects or embodiments herein, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary uses) or immunogenic polypeptides.

[0193] The expression cassette can include any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector includes any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0194] In one embodiment of any of the aspects or embodiments herein, the sequences provided in the expression cassette, expression construct, or donor sequence of the ceDNA vector described herein can be codon-optimized for the host cell. As used herein, the term "optimized codons" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a substantial number of codons of a native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of a vertebrate of interest, such as a mouse or human, for enhanced expression in the cell of the vertebrate of interest. Different species exhibit certain biases for certain codons of specific amino acids.

[0195] Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another public database.

[0196] Many organisms show a bias to use certain codons to code for the insertion of certain amino acids in the growing peptide chain. Codon preference or codon bias, which is the difference in codon usage between organisms, is brought about by the degeneracy of the genetic code and is well documented among many organisms. Codon bias is often correlated with the efficiency of messenger RNA (mRNA) translation and is thought to depend, among other things, on the properties of the codon being translated and on the availability of certain transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0197] Given the large number of gene sequences available in a wide variety of animal, plant, and microbial species, it is possible to calculate the relative frequency of codon usage (Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000)).

[0198] inverted terminal repeat (ITR) As described herein, a ceDNA vector is a non-encapsid-containing linear double-stranded DNA molecule formed from continuous strands of complementary DNA with covalent ends (linear continuous non-encapsid structure) and includes 5' inverted terminal repeat (ITR) and 3' ITR sequences that are different or asymmetric with respect to each other. At least one of the ITRs includes a functional terminal resolution site and a replication protein binding site (RPS) (sometimes called a replicative protein binding site), e.g., a Rep binding site. In general, a ceDNA vector contains at least one modified AAV inverted terminal repeat (ITR), i.e., a deletion, insertion, and / or substitution with respect to the other ITR, and an expressible transgene.

[0199] In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is an AAV ITR, for example, a wild-type AAV ITR.In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is a modified ITR relative to the other ITRs, i.e., the ceDNA comprises ITRs that are asymmetric relative to each other.In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is a non-functional ITR.

[0200] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector comprises (1) an expression cassette comprising a cis-regulatory element, a promoter and at least one transgene, or (2) a promoter operably linked to at least one transgene, and (3) two self-complementary sequences, e.g., ITRs, flanking the expression cassette, and the ceDNA vector is not associated with a capsid protein. In some embodiments of any of the aspects or embodiments herein, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, at least one of which comprises an operational Rep-binding element (RBE) and a terminal resolution site (TRS) or a functional variant of the RBE of AAV, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector may comprise additional components for regulating expression of the transgene, e.g., a regulatory switch for controlling and regulating expression of the transgene, and a regulatory switch that is a kill switch that allows for controlled cell death of cells containing the ceDNA vector.

[0201] In one embodiment of any of the aspects or embodiments herein, the two self-complementary sequences can be ITR sequences from any known parvovirus, such as a depend virus, such as AAV (e.g., AAV1-AAV12). Any AAV serotype can be used, including, but not limited to, modified AAV2 ITR sequences that retain a Rep binding site (RBS) and a terminal resolution site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to variable palindromic sequences that allow for hairpin secondary structure formation. In some embodiments of any of the aspects and embodiments herein, the ITRs can be synthetic. In one embodiment of any of the aspects and embodiments herein, the synthetic ITRs are based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITRs do not include AAV base sequences. In yet another embodiment, the synthetic ITRs preserve the ITR structure described above, but have little or no AAV source sequence. In some aspects, the synthetic ITRs may preferentially interact with wild-type Rep or Rep of a particular serotype, or in some cases, are not recognized by wild-type Rep, but are recognized only by mutant Rep. In some embodiments of any of the aspects and embodiments herein, the ITR is a synthetic ITR sequence that retains a functional Rep binding site (RBS) and terminal resolution site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to a variable palindrome sequence that allows for hairpin secondary structure formation. In some examples, the modified ITR sequence retains the sequence of the RBS, TRS, and the structure and position of the Rep binding element that forms the terminal loop portion of one ITR hairpin secondary structure from the corresponding sequence of the wild-type AAV2 ITR. Exemplary ITR sequences for use in ceDNA vectors are disclosed in Tables 2-9, 10A and 10B, SEQ ID NOs: 2, 52, 101-449 and 545-547, and partial ITR sequences are shown in Figures 26A-B of International Application No. PCT / US2018 / 049996, filed September 7, 2018.In some embodiments of any of the aspects and embodiments herein, the ceDNA vector may include an ITR with a modification in the ITR corresponding to any of the modifications in the ITR sequence or ITR subsequences set forth in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B of International Application No. PCT / US2018 / 049996, filed Sep. 7, 2018.

[0202] In one embodiment of any of the aspects and embodiments herein, the ceDNA vector may be produced from an expression construct that further comprises a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type specific promoters, and enhancers. In some embodiments of any of the aspects or embodiments herein, the ITRs may act as promoters of the transgene. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector comprises an additional component for regulating the expression of the transgene, such as a regulatory switch described in International Application No. PCT / US2018 / 049996, filed September 7, 2018, to regulate the expression of the transgene, or a kill switch that may kill cells containing the ceDNA vector.

[0203] In one embodiment of any of the aspects or embodiments herein, the expression cassette may also include a posttranscriptional element to increase expression of the transgene. In one embodiment of any of the aspects or embodiments herein, the Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE) is used to increase expression of the transgene. Other posttranscriptional processing elements, such as posttranscriptional elements from the thymidine kinase gene of Herpes Simplex Virus or Hepatitis B Virus (HBV), can be used. Secretory sequences may be linked to the transgene, such as VH-02 and VK-A26 sequences. The expression cassette may include polyadenylation sequences or variations thereof known in the art, such as natural sequences isolated from bovine BGHpA or viral SV40pA, or synthetic sequences. Some expression cassettes may also include an SV40 late polyA signal upstream enhancer (USE) sequence. USE may be used in combination with SV40pA or a heterologous polyA signal.

[0204] 1A-1C of International Application No. PCT / US2018 / 050042, filed September 7, 2018, show schematic diagrams of non-limiting exemplary ceDNA vectors, or corresponding sequences of ceDNA plasmids, which are incorporated herein by reference in their entirety. The ceDNA vectors are capsid-free and may be obtained from a plasmid encoding a first ITR, an expressible transgene cassette, and a second ITR, in that order, where at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence. The expressible transgene cassette preferably comprises one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA), in that order.

[0205] promoter Suitable promoters, including those mentioned above, can be derived from viruses and therefore can be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVTE), the rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6, e.g., (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31 (17)), the human H1 promoter (H1), the CAG promoter, the human alpha 1-antitrypsin (human alpha l-antitrypsin (HAAT) promoters (e.g., and the like). In one embodiment of any of the aspects or embodiments herein, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites. In one embodiment of any of the aspects or embodiments herein, the DNA containing the nuclease cleavage sites is foreign to the promoter DNA.

[0206] In one embodiment of any of the aspects or embodiments herein, the promoter can include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. The promoter can also include distal enhancer or repressor elements that can be located as far away as several thousand base pairs from the start site of transcription. The promoter can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter can regulate the expression of genetic components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to an external stimulus such as a physiological stress, a pathogen, a metal ion, or an inducer. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter and the CMV IE promoter, as well as the promoters listed below. Such promoters and / or enhancers can be used to express any gene of interest, e.g., a therapeutic protein. For example, a vector can include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein.In one embodiment of any of the aspects or embodiments herein, the promoter operably linked to the therapeutic protein coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as the long terminal repeat (LTR) promoter of the bovine immunodeficiency virus (BIV), a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter, an Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. In one embodiment of any of the aspects or embodiments herein, the promoter may also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a natural or synthetic tissue-specific promoter, such as a liver-specific promoter, such as human alpha 1-antitrypsin (HAAT) or transthyretin (TTR). In one embodiment of any of the aspects or embodiments herein, delivery to the liver may be achieved using endogenous ApoE-specific targeting of the composition comprising the ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0207] In one embodiment of any of the aspects or embodiments herein, the promoter used is the native promoter of the gene encoding the therapeutic protein. The promoter and other regulatory sequences of each gene encoding the therapeutic protein are known and characterized. The promoter region used may further include one or more additional regulatory sequences (e.g., naturally occurring), such as enhancers known in the art (e.g., Serpin enhancers).

[0208] Non-limiting examples of suitable promoters for use in accordance with the present invention include, for example, the CAG promoter, the HAAT promoter, the human EF1-α promoter, or fragments of the EF1-α promoter and the rat EF1-α promoter.

[0209] Polyadenylation sequence A sequence encoding a polyadenylation sequence may be included in the ceDNA vector to stabilize the mRNA expressed from the ceDNA vector and to aid in nuclear transport and translation. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector does not include a polyadenylation sequence. In other embodiments, the vector includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenine dinucleotides. In some embodiments of any of the aspects or embodiments herein, the polyadenylation sequence includes about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0210] In one embodiment of any of the aspects or embodiments herein, the ceDNA can be obtained from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two different inverted terminal repeats (ITRs) (e.g., AAV ITRs), where at least one of the ITRs comprises a terminal resolution site and a replication protein binding site (RPS), e.g., a Rep binding site (e.g., wt AAV ITR), and one of the ITRs comprises a deletion, insertion, and / or substitution relative to the other ITR, e.g., a functional ITR.

[0211] In one embodiment of any of the aspects or embodiments herein, the host cell does not express viral capsid proteins and the polynucleotide vector template lacks any viral capsid coding sequence. In one embodiment of any of the aspects or embodiments herein, the polynucleotide vector template lacks the AAV capsid gene, but also the capsid genes of other viruses). In one embodiment of any of the aspects or embodiments herein, the nucleic acid molecule also lacks the AAV Rep protein coding sequence. Thus, in some embodiments of any of the aspects or embodiments herein, the nucleic acid molecule of the invention lacks both functional AAV cap and AAV rep genes.

[0212] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector does not have modified ITRs.

[0213] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector comprises a regulatory switch disclosed herein (or in International Application No. PCT / US2018 / 049996, filed September 7, 2018).

[0214] Production of V.ceDNA vector Methods for the production of ceDNA vectors described herein comprising asymmetric or symmetric ITR pairs as defined herein are described in Section IV of International Application No. PCT / US2018 / 049996, filed September 7, 2018, which is incorporated herein by reference in its entirety. As described herein, ceDNA vectors can be obtained, for example, by a process comprising: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), which in the presence of Rep proteins lacks viral capsid coding sequences under conditions effective to induce production of the ceDNA vector in the host cells, and for a time sufficient therefor, and b) harvesting and isolating the ceDNA vector from the host cells. The presence of Rep proteins induces replication of the vector polynucleotide with modified ITRs to produce the ceDNA vector in the host cell.

[0215] However, no viral particles (e.g., AAV virions) are expressed, and therefore there are no size limitations such as those naturally imposed on AAV or other viral-based vectors.

[0216] The presence of a ceDNA vector isolated from a host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0217] In one embodiment of any of the aspects or embodiments herein, the invention provides for the use of host cell lines that have stably integrated a DNA vector polynucleotide expression template (ceDNA template) into their own genome in the production of non-viral DNA vectors, e.g., as described in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cells at an MOI of about 3. If the host cell line is a mammalian cell line, e.g., HEK293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector, such as a herpes virus, may be used to introduce Rep proteins into the cells, allowing excision and amplification of ceDNA in the presence of Rep and a helper virus.

[0218] In one embodiment of any of the aspects or embodiments herein, the host cells used to generate the ceDNA vectors described herein are insect cells, and baculoviruses are used to deliver both the polynucleotides encoding the Rep proteins and the non-viral DNA vector polynucleotide expression construct template for the ceDNA. In some embodiments of any of the aspects and embodiments herein, the host cells are engineered to express the Rep proteins.

[0219] The ceDNA vector is then harvested and isolated from the host cells. The time for harvesting and collecting the ceDNA vector described herein from the cells can be selected and optimized to achieve high yield production of the ceDNA vector. For example, the harvest time can be selected taking into consideration cell viability, cell morphology, cell growth, etc. In one embodiment of any of the aspects and embodiments herein, the cells are grown under sufficient conditions and harvested after sufficient time has passed since baculovirus infection to produce the ceDNA vector, but before most of the cells begin to die due to the toxicity of the baculovirus. The DNA vector can be isolated using a plasmid purification kit, such as the Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation can also be adapted for DNA vectors. In general, any nucleic acid purification method can be employed.

[0220] The DNA vector may be purified by any means known to those skilled in the art for purifying DNA. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is purified as a DNA molecule. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is purified as an exosome or microparticle. The presence of the ceDNA vector may be confirmed by digesting the vector DNA isolated from the cell with a restriction enzyme that has a single recognition site on the DNA vector, and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0221] VI. Preparation of lipid particles Lipid particles (e.g., lipid nanoparticles) can form spontaneously upon mixing of TNA (e.g., ceDNA) and lipids. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane (e.g., 100 nm cutoff) using, for example, 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.

[0222] 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 the methods described in, for example, US Patent Application Publication Nos. US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129, and US2010 / 0130588, the contents of each of which are incorporated herein by reference in their entirety.In some embodiments of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. Processes and apparatus for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in U.S. Patent Application Publication No. 2007 / 0042031, the contents of which are incorporated herein by reference in their entirety. Processes and apparatus for preparing lipid nanoparticles using serial dilution processes are described in U.S. Patent Application Publication No. US2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0223] In one embodiment of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) can be prepared by an impinging jet process. Generally, the particles are formed by mixing lipid 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 mixture ratio can be about 45-55% lipid and about 65-45% ceDNA.

[0224] The lipid solution can contain the disclosed cationic lipids, non-cationic lipids (e.g., phospholipids such as DSPC, DOPE, and DOPC), one or more PEGylated lipids, and a sterol (e.g., cholesterol) in a total lipid concentration of 5-30 mg / mL, more likely 5-15 mg / mL, most likely 9-12 mg / mL in an alcohol, e.g., ethanol. In the lipid solution, the molar ratio of lipids can range from about 25-98%, e.g., about 35-65%, for cationic lipids, about 0-15%, e.g., about 0-12%, for non-ionic lipids, about 0-15%, e.g., about 1-6%, for PEGylated lipids, and about 0-75%, e.g., about 30-50%, for sterols.

[0225] The ceDNA solution can contain ceDNA in a buffer solution having a pH in the range of 3.5 to 5, at a concentration in the range of 0.3 to 1.0 mg / mL, preferably 0.3 to 0.9 mg / mL.

[0226] To form the LNPs, in one exemplary but non-limiting embodiment, the two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impinging jet mixer, to instantly form the LNPs. The mixing flow rate can be in the range of 10-600 mL / min. The tube ID can have a range of 0.25-1.0 mm, with a total flow rate of 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 nm-200 nm. The solution can then be mixed with a buffer solution at a higher pH, in a mixing ratio ranging from 1:1 to 1:3 volume:volume, preferably about 1:2 volume:volume. Optionally, this buffer solution can be at a temperature in the range of 15-40°C or 30-40°C. The mixed LNPs can then undergo an anion exchange filtration step. Prior to anion exchange, the mixed LNPs can be incubated for a period of time, for example, 30 minutes to 2 hours. The temperature during incubation can 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. Tube IDs ranging from 1 mm ID to 5 mm ID and flow rates from 10 to 2000 mL / min can be used in this process.

[0227] After formation, the LNPs can be concentrated and ultrafiltered via an ultrafiltration process in which the alcohol is removed and the buffer is exchanged for a final buffer solution, e.g., phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0228] The ultrafiltration process can use a tangential flow filtration format (TFF) with a membrane nominal molecular weight cutoff range of 30-500 kD. The membrane format is hollow fiber or flat sheet cassette. In a TFF process with the appropriate molecular weight cutoff, the LNPs can be retained in the retentate, while the filtrate or permeate contains alcohol, citrate buffer, and final buffer waste. The TFF process is a multi-step process that results in an initial concentration of 1-3 mg / mL ceDNA. After concentration, the LNP solution is ultrafiltered with 10-20 volumes against the final buffer to remove alcohol and perform a buffer exchange. The material can then be further concentrated 1-3 times. The concentrated LNP solution can be sterile filtered.

[0229] VII. Pharmaceutical Compositions and Formulations Also provided herein is a pharmaceutical composition comprising a TNA lipid particle and a pharma- ceutically acceptable carrier or excipient. In one embodiment of any of the aspects or embodiments herein, the present invention further relates to a pharmaceutical composition comprising a cationic lipid as described in any of the aspects or embodiments herein, or a lipid nanoparticle as described in any of the aspects or embodiments herein, and a pharma- ceutically acceptable excipient.

[0230] Generally, the lipid particles (eg, lipid nanoparticles) of the present invention have an average diameter selected to provide the intended therapeutic effect.

[0231] Depending on the intended use of the lipid particle (e.g., lipid nanoparticle), the ratio of components may vary, and the delivery efficiency of a particular formulation may be measured, for example, using an endosomal release parameter (ERP) assay.

[0232] In one embodiment of any of the aspects or embodiments herein, the ceDNA may be complexed with the lipid portion of the particle or may be encapsulated in the lipid portion of the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments herein, the ceDNA may be fully encapsulated in the lipid portion of the lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by nucleases, for example, in aqueous solution. In one embodiment of any of the aspects or embodiments herein, the ceDNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after exposure of the lipid particle (e.g., lipid nanoparticle) to nucleases for at least about 20, 30, 45, or 60 minutes at 37°C. In some embodiments of any of the aspects and embodiments herein, the ceDNA in the lipid particles (e.g., lipid nanoparticles) is not substantially degraded after incubation of the particles in serum at 37° C. for at least about 30, about 45, or about 60 minutes, or for at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, or about 36 hours.

[0233] In one embodiment of any of the aspects or embodiments herein, the lipid particle (eg, lipid nanoparticle) is substantially non-toxic to a subject, e.g., a mammal, such as a human.

[0234] In one embodiment of any of the aspects or embodiments herein, the pharmaceutical composition comprising the therapeutic nucleic acid of the present disclosure may be formulated into a lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects or embodiments herein, the lipid particle comprising the therapeutic nucleic acid may be formed from the disclosed cationic lipids. In some other embodiments, the lipid particle comprising the therapeutic nucleic acid may be formed from a non-cationic lipid. In a preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles formed from the disclosed cationic lipids containing a therapeutic nucleic acid selected from the group consisting of mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), 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 DNA minimal vectors ("dumbbell DNA").

[0235] In another preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles, which are formed from non-cationic lipids and, optionally, PEGylated lipids or other forms of conjugated lipids that prevent aggregation of the particles.

[0236] In one embodiment of any of the aspects or embodiments herein, the lipid particle formulation is an aqueous solution. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation is a lyophilized powder.

[0237] According to some aspects, the present disclosure provides lipid particle formulations further comprising one or more pharmaceutical excipients. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation further comprises sucrose, tris, trehalose, and / or glycine.

[0238] In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to a cell, tissue, or organ of the subject. Typically, the pharmaceutical composition comprises the TNA lipid particles (e.g., lipid nanoparticles) disclosed herein and a pharma- ceutically acceptable carrier. In one embodiment of any of the aspects or embodiments herein, the TNA lipid particles (e.g., lipid nanoparticles) of the present disclosure can be incorporated into a pharmaceutical composition suitable for a desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high pressure intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compounds in an appropriate buffer with one or a combination of the above-listed ingredients, as appropriate, and sterilized by filtration.

[0239] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intra-subarachnoid, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intra-subarachnoid, intravesical, conjunctival (e.g., extra-orbital, intra-orbital, retro-orbital, intra-retinal, sub-retinal, choroidal, subchoroidal, interstitial, intracameral, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intra-arterial infusion is also contemplated, as well as intracellular injections such as intranuclear microinjection or intracytoplasmic injection.

[0240] Pharmaceutically active compositions comprising TNA lipid particles (e.g., lipid nanoparticles) can be formulated to deliver a transgene in a nucleic acid to a recipient's cells, resulting in therapeutic expression of the transgene therein. The compositions can also comprise a pharma- ceutically acceptable carrier.

[0241] The pharmaceutical composition for therapeutic purposes is typically sterile and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high ceDNA vector concentration.A sterile injectable solution can be prepared by incorporating the required amount of ceDNA vector compound in a suitable buffer with one or a combination of the above-listed components as necessary, and sterilizing by filtration.

[0242] In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) is a solid core particle having at least one lipid bilayer. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) has a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) morphology. Without limitation, the non-bilayer morphology may include, for example, three-dimensional tube, rod, cubic symmetry, etc. The non-lamellar morphology (i.e., non-bilayer structure) of the lipid particle (e.g., lipid nanoparticle) may be determined using analytical techniques known and used by those skilled in the art. Such techniques include, but are not limited to, cryo-Transmission Electron Microscopy ("Cryo-TEM"), Differential Scanning calorimetry ("DSC"), X-ray diffraction, etc. For example, the morphology of lipid particles (lamellar vs. non-lamellar) can be readily assessed and characterized using Cryo-TEM analysis, for example, as described in U.S. Patent Application Publication No. 2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0243] In one embodiment of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) having a non-lamellar morphology are electron dense.

[0244] In one embodiment of any of the aspects or embodiments herein, the present disclosure provides lipid particles (e.g., lipid nanoparticles) that are either unilamellar or multilamellar structures. In some aspects, the present disclosure provides lipid particle (e.g., lipid nanoparticle) formulations that include multivesicular particles and / or effervescent base particles. By controlling the composition and concentration of the lipid components, the rate at which the conjugated lipids exchange out of the lipid particle (e.g., lipid nanoparticle), which in turn, the rate at which the lipid nanoparticle becomes fusogenic, can be controlled. In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid particle (e.g., lipid nanoparticle) becomes fusogenic. Other methods that can be used to control the rate at which the lipid particle (e.g., lipid nanoparticle) becomes fusogenic will be apparent to one of skill in the art based on the present disclosure. It will also be apparent that by controlling the composition and concentration of the conjugated lipids, the lipid particle size can be controlled.

[0245] In one embodiment of any of the aspects or embodiments herein, the pKa of the formulated cationic lipid can be correlated with the effectiveness of the LNP for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entireties). In one embodiment of any of the aspects or embodiments herein, the preferred range of pKa is about 5 to about 8. In one embodiment of any of the aspects or embodiments herein, the preferred range of pKa is about 6 to about 7. In one embodiment of any of the aspects or embodiments herein, the preferred pKa is about 6.5. In one embodiment of any of the aspects or embodiments herein, the pKa of a cationic lipid may be determined in a lipid particle (e.g., a lipid nanoparticle) using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0246] In one embodiment of any of the aspects or embodiments herein, the encapsulation of ceDNA in lipid particles (e.g., lipid nanoparticles) can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or the PicoGreen® assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. In general, encapsulation is determined by adding a dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ceDNA, allowing it to interact with the membrane-impermeable dye. The encapsulation of ceDNA can be calculated as E=(Io-I) / Io, where I and Io refer to the fluorescence intensity before and after the addition of detergent.

[0247] Unit Dose In one embodiment of any of the aspects or embodiments herein, the pharmaceutical composition can be provided in a unit dosage form. The unit dosage form will typically be adapted for one or more routes of administration of the pharmaceutical composition. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by inhalation. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by an inhaler. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by a nebulizer. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by an aerosolizer. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for oral administration, buccal administration, or sublingual administration. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for intrathecal or intraventricular administration. In some embodiments of any of the aspects and embodiments herein, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

[0248] VIII. Treatment method The lipid nanoparticles and methods described herein (e.g., the TNA lipid particles (e.g., lipid nanoparticles) described herein) can be used to introduce a nucleic acid sequence (e.g., a therapeutic nucleic acid sequence) into a host cell. In one embodiment of any of the aspects and embodiments herein, the introduction of a nucleic acid sequence into a host cell using a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) can be monitored with appropriate biomarkers from the treated patient to assess gene expression.

[0249] The LNP compositions provided herein can be used to deliver transgenes (nucleic acid sequences) for a variety of purposes. In one embodiment of any of the aspects and embodiments herein, the ceDNA vectors (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be used in a variety of ways, including, for example, ex situ, in vitro and in vivo applications, methodologies, diagnostic procedures, and / or gene therapy regimens.

[0250] Provided herein is a method of treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) into a target cell (e.g., a hepatic cell, a muscle cell, a renal cell, a neuronal cell, or other diseased cell type) of a subject in need of treatment, optionally with a pharma- ceutically acceptable carrier. The implemented TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) comprises a nucleotide sequence of interest useful for treating a disease. In particular, the TNA may comprise a desired exogenous DNA sequence operably linked to a control element capable of directing transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into a subject. The TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) may be administered via any suitable route described herein and known in the art. In one embodiment of any of the aspects or embodiments herein, the target cell is within a human subject.

[0251] Provided herein is a method of providing a diagnostically or therapeutically effective amount of a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to a subject in need thereof, comprising providing an amount of the TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to a tissue or organ of the subject in need thereof, and providing to the subject a diagnostically or therapeutically effective amount of a protein, peptide, or nucleic acid expressed by the TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) for a time effective to allow expression of a transgene from the TNA LNP. In one embodiment of any of the aspects or embodiments herein, the subject is a human.

[0252] Provided herein is a method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or trauma in a subject. In general, the method includes at least the step of administering a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to a subject in need thereof in an amount and for a time sufficient to diagnose, prevent, treat, or ameliorate one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the subject. In one embodiment of any of the aspects or embodiments herein, the subject is a human.

[0253] Provided herein are methods for using TNA LNPs as tools to treat one or more symptoms of a disease or disease state. There are several genetic diseases with known defective genes, typically divided into two classes: enzyme deficiency states, which are usually inherited recessively, and imbalance states, which may involve regulatory or structural proteins, but are typically not always inherited dominantly. In the case of deficiency disease, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be used to deliver transgenes to carry normal genes to affected tissues for replacement therapy, and in some embodiments of any of the aspects or embodiments herein, antisense mutations can be used to create animal models of the disease. In the case of imbalance disease states, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to create disease states in model systems, which can then be used to attempt to counteract the disease state. Thus, the TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) and methods disclosed herein allow for the treatment of genetic diseases. As used herein, a disease state is treated by partially or totally correcting the deficiency or imbalance that causes the disease or makes it more severe.

[0254] In general, any transgene can be delivered using TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) as described above to treat, prevent, or ameliorate symptoms associated with any disorder related to gene expression. Exemplary disease states include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes, muscular dystrophies (e.g., Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondriopathy (e.g., Leber's hereditary optic neuropathy (LHON), Leigh's syndrome, and subacute sclerosing encephalitis), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like. In some embodiments of any of the aspects or embodiments herein, the ceDNA vectors as disclosed herein may be advantageously used in the treatment of individuals with metabolic disorders (e.g., ornithine transcarbamylase deficiency).

[0255] In one embodiment of any of the aspects or embodiments herein, the TNA LNPs described herein can be used to treat, ameliorate, and / or prevent a disease or disorder caused by a mutation in a gene or gene product. Exemplary diseases or disorders that can be treated with TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage diseases); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC)); blood diseases or disorders (e.g., hemophilia A and B, thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (e.g., cystic fibrosis).

[0256] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to deliver heterologous nucleotide sequences in situations where it is desirable to regulate the expression levels of a transgene (e.g., a transgene encoding a hormone or growth factor).

[0257] In one embodiment of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to correct abnormal levels and / or function of gene products (e.g., absence or deficiency in a protein) that results in a disease or disorder. The TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can produce functional proteins and / or restore levels of proteins to alleviate or reduce symptoms or confer a benefit from a particular disease or disorder caused by the absence or deficiency in the protein. For example, treatment of OTC deficiency can be achieved by producing a functional OTC enzyme, treatment of hemophilia A and B can be achieved by altering the levels of factors VIII, IX, and X, treatment of PKU can be achieved by altering the levels of phenylalanine hydroxylase enzyme, treatment of Fabry disease or Gaucher disease can be achieved by producing functional alpha-galactosidase or beta-glucocerebrosidase, respectively, treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively, treatment of cystic fibrosis can be achieved by producing a functional cystic fibrosis transmembrane regulator, treatment of glycogen storage diseases can be achieved by restoring functional G6Pase function, and treatment of PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

[0258] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to provide RNA-based therapeutics to cells in vitro or in vivo. Examples of RNA-based therapeutics include, but are not limited to, mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). For example, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to provide antisense nucleic acids to cells in vitro or in vivo. For example, if the transgene is an RNAi molecule, expression of the antisense nucleic acid or RNAi in the target cell reduces expression of a particular protein by the cell. Thus, a transgene that is an RNAi molecule or an antisense nucleic acid can be administered to reduce expression of a particular protein in a subject in need thereof. Antisense nucleic acids can also be administered to cells in vitro to modulate cell physiology, e.g., to optimize a cell or tissue culture system.

[0259] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver DNA-based therapeutics to cells in vitro or in vivo. Examples of DNA-based therapeutics include, but are not limited to, minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE)-vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). For example, in one embodiment of any of the aspects or embodiments herein, ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver minicircles to cells in vitro or in vivo. For example, if the transgene is minicircle DNA, expression of the minicircle DNA in a target cell reduces expression of a particular protein by the cell. Therefore, the transgene, which is a minicircle DNA, can be administered to reduce the expression of a particular protein in a subject in need thereof. Minicircle DNA can also be administered to cells in vitro to regulate cell physiology, for example, to optimize cell or tissue culture systems.

[0260] In one embodiment of any of the aspects or embodiments herein, exemplary transgenes encoded by the TNA vector comprising the expression cassette include, but are not limited to, X, lysosomal enzymes (e.g., hexosaminidase A associated with Tay-Sachs disease, or iduronate sulfatase associated with Hunter syndrome / MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony stimulating factor, lymphotoxin, and the like), 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 (GF), and the like). In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, two or more transgenes are encoded by a ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects or embodiments herein, the transgene encodes an antibody, including a full-length antibody or an antibody fragment, as defined herein. In some embodiments of any of the aspects or embodiments herein, 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, oxycytidine kinase, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, and tumor suppressor gene products.

[0261] In one embodiment of any of the aspects or embodiments herein, there is provided a method of treating a genetic disorder in a subject (e.g., a human), the method comprising administering to the subject an effective amount of a lipid nanoparticle or a pharmaceutical composition thereof, as described in any of the aspects or embodiments herein. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I HS), Hunter syndrome (MPS II), Sanfilippo types A, B, C, and D (MPS III), A, B, C, and D), Morquio A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick disease A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase Lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidoses, galactosialidosis, amyotrophic lateral sclerosissclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is hemophilia A. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is hemophilia B, and in one embodiment of any of the aspects or embodiments herein, the genetic disorder is phenylketonuria (PKU). In one embodiment of any of the aspects or embodiments herein, the genetic disorder is Wilson's disease. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is Gaucher disease types I, II, and III. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is Stargardt's macular dystrophy. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is LCA10. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is Usher syndrome. In one embodiment of any of the aspects or embodiments herein, the genetic disorder is wet AMD.

[0262] In one embodiment of any of the aspects or embodiments herein, the present disclosure relates to the use of lipid nanoparticles or pharmaceutical compositions thereof as described in any of the aspects or embodiments herein for the manufacture of a medicament for treating a genetic disorder in a subject (e.g., a human). Exemplary genetic disorders are as described above. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the medicament is Stargardt's macular dystrophy. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the medicament is LCA10. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the medicament is Usher syndrome. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the medicament is wet AMD.

[0263] In one embodiment of any of the aspects or embodiments herein, the present disclosure relates to a lipid nanoparticle or pharmaceutical composition thereof according to any of the aspects or embodiments herein for use in treating a genetic disorder in a subject (e.g., human). Exemplary genetic disorders are as described above. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the use is Stargardt's macular dystrophy. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the use is LCA10. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the use is Usher syndrome. In one embodiment of any of the aspects or embodiments herein, the genetic disorder treated by the use is wet AMD.

[0264] Administration In one embodiment of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA vector lipid particles described herein) can be administered to an organism for transduction of cells in vivo. In one embodiment of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA vector lipid particles) can be administered to an organism for transduction of cells ex vivo.

[0265] Generally, administration is by any of the routes that are normally used to ultimately contact molecules with blood or tissue cells.Suitable methods for administering such nucleic acids are available and well known by those skilled in the art, and more than one route may be used to administer a particular composition, although a particular route may often provide a more immediate and effective response than another route. Exemplary modes of administration of TNA LNPs (e.g., ceDNA vector lipid particles) include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intradermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to the skeleton, diaphragm, and / or myocardium), intrapleural, intracerebral, and intra-arterial), topical (e.g., to skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., into the liver, eye, skeletal muscle, myocardium, diaphragm, muscle, or brain).

[0266] Administration of TNA LNP-like ceDNA vectors (e.g., ceDNA LNPs) can be to any site of a subject, including, but not limited to, a site selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. In one embodiment of any of the aspects or embodiments herein, administration of ceDNA LNPs can also be to a tumor (e.g., in or near a tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition to be treated, ameliorated, and / or prevented, as well as the nature of the particular ceDNA LNP being used. Additionally, ceDNA allows for the administration of two or more transgenes in a single vector, or in multiple ceDNA vectors (e.g., ceDNA cocktails).

[0267] In one embodiment of any of the aspects or embodiments herein, administration of ceDNA LNPs to skeletal muscles includes, but is not limited to, administration to skeletal muscles of the limbs (e.g., upper arms, lower arms, upper legs, and / or lower legs), lower back, neck, head (e.g., tongue), pharynx, abdomen, pelvis / perineum, and / or fingers. ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be delivered to skeletal muscles by intravenous administration, intraarterial administration, intraperitoneal, limb perfusion (optionally isolated limb perfusion of the legs and / or arms, see, e.g., Arruda et al., (2005) Blood 105:3458-3464), and / or direct intramuscular injection. In certain embodiments, ceDNA LNPs are administered to a subject (e.g., a subject with a muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intraarterial administration). In one embodiment of any of the aspects or embodiments herein, the ceDNA LNPs can be administered without the use of "hydrodynamic" techniques.

[0268] Administration of TNA LNPs (e.g., ceDNA LNPs) to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. TNA LNPs (e.g., ceDNA LNPs) can be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion. Administration to the diaphragm muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to smooth muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment of any of the aspects or embodiments herein, administration can be to endothelial cells present in, near, and / or on smooth muscle.

[0269] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA LNPs) are administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (e.g., to treat, ameliorate, and / or prevent muscular dystrophy or cardiac disease (e.g., PAD or congestive heart failure).

[0270] TNA LNPs (e.g., ceDNA LNPs) can be administered to the CNS (e.g., brain or eye). TNA LNPs (e.g., ceDNA LNPs) can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (cerebrum including striatum, occipital, temporal, parietal, and frontal lobes, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. TNA LNPs (e.g., ceDNA LNPs) can also be administered to different regions of the eye, such as the retina, cornea, and / or optic nerve. TNA LNPs (e.g., ceDNA LNPs) can be delivered to the cerebrospinal fluid (e.g., by lumbar puncture). TNA LNPs (eg, ceDNA vector lipid particles) can also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (eg, brain tumors or cerebral infarction).

[0271] In one embodiment of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA LNPs) can be administered to the desired region of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's area) delivery, and intramuscular delivery with retrograde delivery to motor neurons.

[0272] According to some embodiments of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA LNPs) are administered in a liquid formulation by direct injection (e.g., stereotactic injection) into the desired region or compartment in the CNS. According to other embodiments, the TNA LNPs (e.g., ceDNA LNPs) may be provided by topical application to the desired region or by intranasal administration of an aerosol formulation. Administration to the eye may be by topical application of liquid drops. As a further alternative, the ceDNA vectors may be administered as a solid sustained release formulation (see, e.g., U.S. Pat. No. 7,201,898, which is incorporated by reference in its entirety). In one embodiment of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA LNPs) may be used for retrograde transport to treat, ameliorate, and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, TNA LNPs (eg, ceDNA LNPs) can be delivered to muscle tissue and from there translocate into neurons.

[0273] In one embodiment of any of the aspects or embodiments herein, repeated administration of the therapeutic product can be performed until an appropriate level of expression is achieved. Thus, in one embodiment of any of the aspects or embodiments herein, 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 about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, about 18 years, about 20 years, about 22 years, about 24 years, about 26 years, about 28 years, about 29 years, about 30 years, about 31 years, about 32 years, about 33 years, about 34 years, about 35 years, about 36 years, about 37 years, about 38 years, about 39 years, about 40 years, about 41 years, about 42 years, about 43 years, about 44 years, about 45 years, about 46 years, about 47 years, about 48 years, about 49 years, about 50 years, about 51 years, about 52 years, about 53 years, about 54 years, about 55 years, about 56 years, about 57 years, about 58 years, about 59 years, about 60 years, about 61 years, about 6 A second administration (readministration) can be administered after about 18 years, about 19 years, about 20 years, about 21 years, about 22 years, about 23 years, about 24 years, about 25 years, about 26 years, about 27 years, about 28 years, about 29 years, about 30 years, about 31 years, about 32 years, about 33 years, about 34 years, about 35 years, about 36 years, about 37 years, about 38 years, about 39 years, about 40 years, about 41 years, about 42 years, about 43 years, about 44 years, about 45 years, about 46 years, about 47 years, about 48 years, about 49 years, or about 50 years.

[0274] In one embodiment of any of the aspects or embodiments herein, one or more additional compounds can also be included. These compounds can be administered separately, or the additional compounds can be included in the lipid particles (e.g., lipid nanoparticles) of the present invention. In other words, the lipid particles (e.g., lipid nanoparticles) can contain other compounds in addition to TNA, or at least a second TNA that is different from the first one. Without limitation, the other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and their derivatives, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0275] In one embodiment of any of the aspects or embodiments herein, the one or more additional compounds may be therapeutic agents. The therapeutic agents may be selected from any class suitable for therapeutic purposes. Thus, the therapeutic agents may be selected from any class suitable for therapeutic purposes. The therapeutic agents may be selected according to the desired therapeutic purpose and biological effect. For example, in one embodiment of any of the aspects or embodiments herein, if the TNAs in the LNPs are useful for treating cancer, the additional compounds may be anti-cancer agents (e.g., chemotherapeutic agents, targeted cancer therapies (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates). In one embodiment of any of the aspects or embodiments herein, if the LNPs containing the TNAs are useful for treating infectious diseases, the additional compounds may be anti-microbial agents (e.g., antibiotics or anti-viral compounds). In one embodiment of any of the aspects or embodiments herein, if the LNPs containing the TNAs are useful for treating immune diseases or disorders, the additional compounds may be anti-cancer agents (e.g., chemotherapeutic agents, targeted cancer therapies (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates). The additional compound can be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In one embodiment of any of the aspects or embodiments herein, different cocktails of different lipid particles containing different compounds, such as TNAs encoding different proteins or different compounds (such as therapeutic agents), can be used in the compositions and methods of the invention. In one embodiment of any of the aspects or embodiments herein, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressant. In some embodiments of any of the aspects or embodiments herein, the additional compound is an immunostimulatory compound. EXAMPLES

[0276] The following examples are provided by way of illustration and not by way of limitation. It will be understood by those skilled in the art that the lipids contemplated in this disclosure can be designed and synthesized using the general synthetic methods described below.

[0277] Example 1: General synthesis Lipids of formula I were designed and synthesized using the same synthetic methodology as shown in Scheme 1 below. All variables in the compounds shown in Scheme 1, i.e., R 1 , R 2 , R 3 , R 4 , R 5 , R 6a , R 6b , X, and n are as defined in formula I. x is defined as R 4 but with one less carbon atom in the aliphatic chain.

[0278] [ka]

[0279] Monoester lipids of the present disclosure, i.e., Formula I, where X is -C(=O)-, were designed and synthesized using a similar synthetic methodology as shown in Scheme 2 below. All variables in the compounds shown in Scheme 1, i.e., R 1 , R 2 , R 3 , R 4 , R 5 , R 6a , R 6b , X, and n are as defined in formula I. x is defined as R 4 but with one less carbon atom in the aliphatic chain.

[0280] [ka]

[0281] Scheme 1 and Scheme 2 Referring to Scheme 1 and Scheme 2, in step 1, to a stirred solution of acid 2 in dichloromethane (DCM) was added 4-dimethylaminopyridine (DMAP) followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). The resulting mixture was stirred at room temperature under nitrogen (N2) atmosphere for 15 minutes. Compound 1 was then added dropwise and the mixture was stirred overnight. The next day, the reaction was diluted with DCM and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and evaporated to dryness. The crude product was purified by silica gel column chromatography using 0-10% methanol in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give compound 3 (0.78 g, 54%).

[0282] In step 2, to a solution of 3 in tetrahydrofuran (THF) was added lithium aluminum hydride (LiAlH4). The reaction mixture was heated at 50° C. overnight. The next day, the reaction was cooled to 0° C. and quenched by dropwise addition of water. The reaction was then filtered through Celite to give the crude product 4, which was used in the next step without further purification.

[0283] In step 3, compound 5 or 5' (synthesized according to the procedure described in WO 2017 / 049245, incorporated herein by reference in its entirety) was dissolved in dimethylformamide / methanol mixture DMF:MeOH (1:1) and 4 was added. The reaction was stirred at room temperature overnight. The product was extracted with ethyl acetate (EtOAc) and the organic layer was washed with saturated aqueous sodium bicarbonate (NaHCO3(aq)) and brine, and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% methanol in DCM as eluent to obtain the cationic lipid of formula I.

[0284] Compounds 5 or 5' may alternatively be synthesized according to the procedure shown in Scheme 3 below. y is defined as R 5 but with one less carbon atom in the aliphatic chain.

[0285] [ka]

[0286] Referring to Scheme 3, to an ice-cold solution of 9-heptadecanone 6 in tetrahydrofuran (anhydrous) was added dropwise neat phosphoric anhydride solution 7. The reaction was stirred for 30 min, followed by portionwise addition of NaH. The reaction mixture was refluxed, cooled to 0° C., quenched with water, and extracted with ether. The organic layer was washed several times with water, brine, dried over Na2SO4, and concentrated. The crude product was purified by column chromatography to provide 7.1 g (93% yield) of pure 8.

[0287] Compound 8 was dissolved in an EtOAc / MeOH mixture and subjected to reduction with H2 using wet 10% Pd / C catalyst to afford compound 9 with clean conversion.

[0288] Compound 9 (THF) was cooled and LiAlH was added dropwise. The reaction mixture was left stirring overnight, warmed to room temperature and then quenched using a THF / H2O mixture (1:1 by volume). The reaction mixture was extracted with EtOAc and filtered through Celite. The organic phase was washed twice with water, brine, dried over Na2SO4 and concentrated. Purification by column chromatography (CH2Cl2-EtOAc) afforded compound 10.

[0289] Compound 10 and alkanoic acid 11 were dissolved in DCM, then DMAP and EDCI were added to the solution at room temperature. After stirring overnight, the reaction was quenched with water, diluted with DCM, washed with NaHCO3 (sat. aq.) and brine. The organic phase was dried over Na2SO4 and concentrated. Column chromatography purification (hexane-EtOAc) provided 3.8 g of compound 5 or 5'.

[0290] Example 2: Synthesis of lipid 6 The procedure for synthesizing lipid 6 is described below with reference to Scheme 4 and is also provided below.

[0291] [ka]

[0292] Step 1: Synthesis of N-(2-(dimethylamino)ethyl)nonanamide (3a) To a stirred solution of nonanoic acid (2a) (1.0 g, 6.3 mmol) in 60 mL of DCM was added DMAP (0.91 g, 7.5 mmol) followed by EDCI (1.44 g, 7.5 mmol). The resulting mixture was stirred at room temperature under N2 for 15 min. Then, 1 ,N 1 -Dimethylethane-1,2-diamine (1a) (0.66 g, 7.5 mmol) was added dropwise and the mixture was stirred overnight. The next day, the reaction was diluted with DCM and washed with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using 0-10% methanol in DCM as eluent. Fractions containing the desired compound were pooled and evaporated to give 3a (0.78 g, 54%).

[0293] Step 2:N 1 ,N 1 Synthesis of -Dimethyl-N2-nonylethane-1,2-diamine (4a) To a solution of 3a (0.78 g, 3.4 mmol) in THF was added LiAlH4. The reaction mixture was heated at 50° C. overnight. The next day, the reaction was cooled to 0° C. and quenched by dropwise addition of water. The reaction was then filtered through Celite to give the crude product 4a (0.6 g, 82%). The product was used in the next step without further purification.

[0294] Step 3: Synthesis of heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoate or lipid 6 Compound 5a (synthesized according to the procedure described in WO 2017 / 049245, incorporated herein by reference in its entirety) (0.6 g, 1.3 mmol) was dissolved in 20 mL of DMF:MeOH (1:1) and 4a (0.35 g, 1.5 mmol) was added. The reaction was stirred at room temperature overnight. The product was extracted with EtOAc (200 mL) and the organic layer was washed with saturated NaHCO3 (aq) and brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% methanol in DCM as eluent to give lipid 6 (0.062 g, 10%). 1 H NMR (300 MHz, chloroform-d) δ 4.85 (quint, J = 6.2 Hz, 1H), 2.57-2.48 (m, 2H), 2.43-2.32 (m, 6H), 2.31-2.25 (m, J = 7.5 Hz, 2H), 2.23 (s, 6H), 1.66-1.34 (m, 8H), 1.24 (s, 47H), 0.86 (t, J = 6.6 Hz, 9H). Example 3: Synthesis of lipid 1 The procedure for synthesizing lipid 1 is described below with reference to Scheme 5 and is also provided below.

[0295] [ka]

[0296] Steps 1 and 2 of Scheme 5 are as described in Example 2.

[0297] Synthesis of Henicosane-11-yl 8-bromooctanoate (5b) To a stirred solution of henicosane-11-ol (10.0 g, 32.0 mmol) and 8-bromooctanoic acid (7.1 g, 44.8 mmol) (both commercially available) in 250 mL of dichloromethane (DCM) was added EDCI (6.1 g, 32.1 mmol) followed by DMAP (392 mg, 3.21 mmol). The resulting mixture was left stirring overnight at room temperature under N2 atmosphere. The next day, the reaction was diluted with DCM and washed with aqueous NaHCO3 (250 mL) and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using 0-10% EtOAc in hexanes as eluent. Fractions containing the desired compound were pooled and evaporated to give 5b (6.3 g, 38%). 1H NMR (300 MHz, chloroform-d) δ 4.84-4.88 (m, 1H), 3.39 (t, J = 6.0 Hz, 2H), 2.28 (t, J = 6.0 Hz, 2H), 1.80-1.89 (m, 2H), 1.25-1.62 (m, 43H), 0.86 (t, J = 6.0 Hz, 6H). Step 3: Synthesis of Henicosan-11-yl 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoate or lipid 1 Compound 5b (4.34 g, 8.41 mmol) was dissolved in 5.0 mL of DMF:MeOH (1:1) and 4a (2.0 g, 9.35 mmol) was added. The reaction was stirred at room temperature overnight. The solvent was evaporated under vacuum and the residue was purified by column chromatography using 0-10% methanol in DCM as eluent to give lipid 1 (330 mg, 11%). 1 H NMR(300MHz,chloroform-d)δ 4.84-4.93 m,1H),3.51-3.55(m,4H),2.98-3.03(m,4H),2.83(s,6H),2.26(t,J=6.0Hz,2H),1.48-1.77(m,8H),1.23-1.44(m,57H),0.86(t,J=6.0Hz,9H). Example 4: Synthesis of lipid 3 The procedure for synthesizing lipid 3 is described below with reference to Scheme 6 and is also provided below.

[0298] [ka]

[0299] Steps 1 and 2 of Scheme 6 are as described in Example 3.

[0300] Synthesis of pentacosan-13-yl 8-bromooctanoate (5c) Using a procedure similar to that described above for the synthesis of henicosane-11-yl 8-bromooctanoate (5b), compound 5c was synthesized by substituting commercially available pentacosan-13-ol for the starting material henicosane-11-ol.

[0301] Step 3: Synthesis of Pentacosan-13-yl 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoate or lipid 3 Using a procedure similar to that described above for the synthesis of lipid 1, lipid 3 was prepared by substituting compound 5c for starting material 5b.

[0302] Example 5: Synthesis of lipid 7 The procedure for synthesizing lipid 7 is described below with reference to Scheme 7 and is also provided below.

[0303] [ka]

[0304] Step 1: Synthesis of N-(2-(dimethylamino)ethyl)heptanamide (3b) To a stirred solution of enanthic acid (2b) (7.0 g, 80 mmol) in 20 mL of DCM was added EDCI (20 g, 104 mmol). The resulting mixture was stirred at room temperature under N2 atmosphere for 15 min. 1a (7.1 g, 80 mmol) dissolved in 10 mL of DCM was then added, followed by DMAP (0.3 g, 2.5 mmol) and stirring was continued overnight. The next day, the reaction was diluted with DCM and washed with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using 0-10% methanol in DCM as eluent. Fractions containing the desired compound were pooled and evaporated to give compound 3b (9.5 g, 59%). 1 H NMR (300MHz, chloroform-d) δ 6.0(broad s,1H),3.3(dd,2H),2.4(dd,J=6,2H),2.2(s,6H),2.16(dd,J=6,2H),1.9-1.5(m,4H),1.3-1.2(m,7H),087(t,3H).

[0305] Step 2:N 1 -Heptyl-N 2 ,N 2 Synthesis of -dimethylethane-1,2-diamine (4b) To a solution of 3b (3 g, 15 mmol) in THF (80 mL) was added LiAlH4 2 M in THF (15 mL, 30 mmol) at 0 °C. The reaction mixture was heated at reflux overnight. The next day, the reaction was cooled to 0 °C and quenched by dropwise addition of water (3 mL). The reaction was then filtered through Celite to give the crude product 4b. The crude product was purified by silica gel column chromatography using 0-10% methanol / NH3 (0.1%) in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 4b (1.3 g, 46%). 1 H NMR(300MHz,chloroform-d)δ 2.67(dd,J=6,2H),2.59(dd,J=7,2H),2.40(dd,J=6,2H),2.20(s,6H),1.50-1.40(m,3H),1.30-1.15(m,9H),0.87(t,3H).MS actual value 187.2[M+H]+ , calculated value 186.3 ([C 11 H 26 N2]).

[0306] Step 3: Synthesis of heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(heptyl)amino)octanoate or lipid 7 Compound 5a (6 g, 13 mmol) was dissolved in 20 mL of DMF:MeOH (1:1) and 4b (2.65 g, 14 mmol) was added. The reaction was stirred at room temperature overnight. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% methanol in DCM as eluent to give lipid 7 (0.5 g, 6%). 1 H NMR(300MHz,chloroform-d)δ 4.85(quint,J=6.2Hz,1H),3.10-2.90(m,2H),2.88-2.80(m,6H),2.43(s,6H),2. 27(dd,2H),1.67-1.34(m,8H),1.30-1.2(m,45H),0.86(t,9H).MS actual value 567.5[M+H] + , calculated value 566.6 ([C 36 H 74 (N2O2).

[0307] Example 6: Synthesis of lipid 10 The procedure for synthesizing lipid 10 is described below with reference to Scheme 8 and is also provided below.

[0308] [ka]

[0309] Step 1: Synthesis of N-(2-(dimethylamino)ethyl)undecaneamide (3c) To a stirred solution of undecanoic acid (2c) (5.27 g, 28.3 mmol) in 250 mL of DCM was added DMAP (4.49 g, 36.8 mmol) followed by EDCI (6.3 g, 36.0 mmol). The resulting mixture was stirred at room temperature under N2 atmosphere for 15 min. 1a (3.03 g, 34.4 mmol) was then added dropwise and stirring was continued overnight. The next day the reaction was diluted with DCM and washed with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using 0-10% methanol in DCM as eluent. Fractions containing the desired compound were pooled and evaporated to give 3 (6.94 g, 95% yield). 1 H NMR(300MHz,chloroform-d)δ ppm:3.25-3.35(m,2H),2.38-2.44(m,2H),2.22(s,6H),2.12-2.22(m,2H),1.55-1.62(m,2H),1.18-1.32(br s, 14H), 0.80-0.90(m, 3H).

[0310] Step 2:N 1 ,N 1 -Dimethyl-N 2 Synthesis of -undecylethane-1,2-diamine (4c) To an ice-cold solution of 3c (5.97 g, 23.3 mmol) in 90 mL of THF was added 23.3 mL of 2 N LiAlH4 (46.6 mmol) in THF. The reaction mixture was stirred at 80° C. overnight. The reaction was cooled to 0° C. and quenched by dropwise addition of water. The reaction was then filtered through Celite, and the filtrate was concentrated and purified by chromatography (DMC-MeOH-NH3) to give 4.2 g of compound 4c (4.2 g, 75% yield). 1 H NMR(300MHz,chloroform-d)δ:2.66(t,J=6.3Hz,2H),2.58(t,J=7.14Hz,2H),2.40(t,J=6.3H z,2H),2.21(s,6H),1.42-1.54(m,2H),1.41-1.54(m,16H),1.24(0.86(t,J=6.3Hz,3H).

[0311] Step 3: Synthesis of henicosan-11-yl 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoate of lipid 10 Compound 5a (0.91 g, 2.0 mmol) was dissolved in 50 mL of EtOH and 4c (1.6 g, 7.0 mmol) was added. The reaction was stirred at 65-75 °C overnight. The reaction mixture was concentrated and purified by column chromatography using DCM-MeOH-NH3 as the eluent to give lipid 10 (142 mg, 12%). 1 H NMR(300MHz,dmso-d6)δ:4.70-4.82(m,1H),2.90-3.0(m,2H),2.78-2.88(m,2H),2.52-2.62(m,10 H),2.20-2.30(m,2H),1.55-1.35(m,10H),1.15-1.35(m,46H),0.75-0.90(9H).MS actual value 623.6[M+H] + , calculated value 622.6 (exact mass) ([C 40 H 82 (N2O2).

[0312] Example 7: Synthesis of lipid 11 The procedure for synthesizing lipid 11 is described below with reference to Scheme 9 and is also provided below.

[0313] [ka]

[0314] Steps 1 and 2 of Scheme 9 are as described in Example 3.

[0315] Step 3: Synthesis of 3-Octylundecyl 6-((2-(dimethylamino)ethyl)(nonyl)amino)hexanoate or Lipid 11 Compound 5d (1.36 g, 2.95 mmol - synthesis described below) was dissolved in 13 mL of EtOH and 4a (1.21 g, 5.89 mmol) was added. The reaction was stirred overnight at 65-75 °C. The reaction mixture was concentrated and purified twice by column chromatography using DCM-MeOH-NH3 as eluent to give lipid 11 (142 mg, 12%). 1 H NMR(300MHz,dmso-d6)δ:4.02(t,J=6.6Hz,2H),2.90-3.00(m,2H),2.75-2.85(m,2H),2.61(s,6H), 2.50-2.60(m,4H),2.27(t,J=7.4Hz,2H),1.15-1.60(m,53H),0.80-0.90(m,9H).MS actual value 595.2[M+H] + , calculated value 594.61 (exact mass) ([C 38 H 78 (N2O2).

[0316] Synthesis of ethyl 3-octylundec-2-enoate (8a) To an ice-cold solution of 9-heptadecanone (6a, 5.98 g, 23.5 mmol) in 200 mL of THF (anhydrous) was added neat ethyl 2-(diethoxyphosphoryl)acetate (7a) (40.0 g, 178 mmol) dropwise. The reaction was stirred for 30 min, followed by portionwise addition of NaH (6.25 g, 157 mmol, 60% in oil). The reaction mixture was refluxed for 18 h, cooled to 0 °C, quenched with 300 mL of water, and extracted with ether. The organic layer was washed several times with water, brine, dried over Na2SO4, and concentrated. The crude product was purified by column chromatography to provide 7.1 g (93% yield) of pure 8a. 1 H NMR (300 MHz, d-chloroform) δ ppm: 5.60 (s, 1H), 4.14 (q, J=7.1 Hz, 2H), 2.60-2.54 (m, 2H), 2.12-2.08 (m, 2H), 1.50-1.20 (m, 27H), 0.95-0.82 (m, 6H).

[0317] Synthesis of ethyl 3-octylundecanoate (9a) Compound 9a (7.05 g, 21.7 mmol) was dissolved in 220 mL of EtOAc and 100 mL of MeOH and subjected to reduction with H2 (1 atm) using 1.2 g of wet 10% Pd / C catalyst. Clean conversion afforded 7.0 g (99% yield) of compound 7. 1 H NMR (300 MHz, d-chloroform) δ (ppm), J (Hz): 4.12 (q, J = 7.1 Hz, 2H), 2.20 (d, J = 6.9, 2H), 1.90-1.80 (m, 1H), 1.35-1.20 (m, 33H), 1.90-1.81 (m, 6H).

[0318] Synthesis of 3-octylundecan-1-ol (10a) Compound 9a (7.0 g, 21.4 mmol) was dissolved in 16 mL of THF, cooled to 0° C., and LiAlH4 (16 mL, 2 M in THF, 32.2 mmol) was added dropwise. The reaction mixture was left stirring overnight, warmed to room temperature, and then quenched at 0° C. by addition of 30 mL of a THF / H2O mixture (1:1 by volume). The reaction mixture was extracted with EtOAc and filtered through Celite. The organic phase was washed twice with water, brine, dried over Na2SO4, and concentrated. Purification by column chromatography (CH2Cl2-EtOAc) provided 6.0 g of compound 10a in 97% yield. 1 H NMR (300 MHz, d-chloroform) δ ppm: 3.66 (t, J = 6.9 Hz, 2H), 1.51 (m, 2H), 1.41 (br s, 1H), 1.10-1.29 (m, 29H), 1.81-1.90 (m, 6H).

[0319] Synthesis of 3-octylundecyl 6-bromohexanoate (5d) Compound 10a (3.5 g, 12.3 mmol) and 11a (2.9 g, 14.9 mmol-commercially available) were dissolved in 25 mL of dichloromethane, then DMAP (190 mg, 1.55 mmol) and EDCI (2.95 g, 15.4 mmol) were added to the solution at room temperature. After stirring overnight, the reaction was quenched with water, diluted with dichloromethane, washed with NaHCO3 (sat. aq.) and brine. The organic phase was dried over Na2SO4 and concentrated. Column chromatography purification (hexane-EtOAc) provided 3.8 g of compound 5d in 67% yield. 1 H NMR(300MHz,d-chloroform)δ ppm:4.08(t,J=7.14Hz,2H),3.40(t,J=6.6Hz,2H),2.30(t,J=7.14Hz,2H),1.92-1.80(m,2H),1.70-1.20(m,36H),1.92-1.80(m,6H)

[0320] Example 8: Synthesis of cationic lipids containing quaternary amines or quaternary ammonium cations Each of the lipids 1-11 above and the lipid of formula I can be converted to its corresponding lipid containing a quaternary amine or a quaternary ammonium cation by treatment with chloromethane (CH3Cl) in acetonitrile (CH3CN) and chloroform (CHCl3).

[0321] Example 9: Preparation of lipid nanoparticles Lipid nanoparticles (LNPs) were prepared with a total lipid to ceDNA weight ratio of about 10:1 to 30:1. Briefly, cationic lipids of the present disclosure, non-cationic lipids (e.g., distearoylphosphatidylcholine (DSPC)), components for providing membrane fusogenicity (such as sterols, e.g., cholesterol), and complex lipid molecules (such as PEGylated lipid conjugates), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (having an average PEG molecular weight of 2000 ("PEG-DMG")), were solubilized in alcohol (e.g., ethanol) at a molar ratio of, e.g., 47.5:10.0:40.7:1.8, 47.5:10.0:39.5:3.0, or 47.5:10.0:40.2:2.3. The ceDNA was diluted to the desired concentration with a buffer solution. For example, ceDNA was diluted to a concentration of 0.1 mg / ml to 0.25 mg / ml in a buffer solution containing sodium acetate, sodium acetate and magnesium chloride, citric acid, malic acid, or malic acid and sodium chloride. In one example, ceDNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate buffer (pH 4). The alcohol lipid solution was mixed with the ceDNA aqueous solution in a ratio of about 1:5 to 1:3 (volume / volume) at a total flow rate of more than 10 ml / min, for example using a syringe pump or an impingement jet mixer. In one example, the alcohol lipid solution was mixed with the ceDNA aqueous solution in a ratio of about 1:3 (volume / volume) at a flow rate of 12 ml / min. The alcohol was removed and the buffer replaced with PBS by dialysis. Alternatively, the buffer was replaced with PBS using a centrifuge tube. Alcohol removal and simultaneous buffer exchange was achieved, for example, by dialysis or tangential flow filtration. The resulting lipid nanoparticles are filtered through a 0.2 μm pore sterile filter.

[0322] In one study, a lipid solution containing reference lipid A, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 47.5:10.0:40.7:1.8) was used as a control to prepare lipid nanoparticles containing exemplary ceDNA. In some studies, a tissue-specific targeting ligand such as N-acetylgalactosamine (GalNAc) was included in the formulation containing reference lipid A, reference lipid B, MC3, or a cationic lipid of the present disclosure. MC3 is (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate, also known as DLin-MC3-DMA, and has the following structure:

[0323] [ka]

[0324] GalNAc ligands, such as tri-antennary GalNAc (GalNAc3) or tetra-antennary GalNAc (GalNAc4), can be synthesized as known in the art (see WO 2017 / 084987 and WO 2013 / 166121) and chemically conjugated to lipids or PEG as known in the art (see Resen et al., J. Biol. Chem. (2001) "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo" 276:375577-37584). An aqueous solution of ceDNA in a buffer solution was prepared. The lipid and ceDNA solutions were mixed using an in-house procedure on a NanoAssembler at a lipid to ceDNA ratio of 1:3 (v / v) and a total flow rate of 12 mL / min.

[0325] [Table 1A]

[0326] [Table 1B] No. = number, IV = intravenous, ROA = route of administration, LNP = lipid nanoparticles, IVIS = in vivo imaging session, BW = body weight

[0327] [Table 2A] DSPC = distearoylphosphatidylcholine, Chol = cholesterol, DMG-PEG2000 = 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG 2000 -DMG), GalNAc = N-acetylgalactosamine, GalNAc4 = tetraantennary GalNAc

[0328] [Table 2B] DSPC = distearoylphosphatidylcholine, Chol = cholesterol, DMG-PEG2000 = 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG 2000 -DMG), GalNAc = N-acetylgalactosamine, GalNAc4 = tetraantennary GalNAc

[0329] LNPs containing reference lipid A, reference lipid B, and MC3 were used as positive controls.

[0330] Example 10: Preclinical in vivo studies of lipid nanoparticles Preclinical studies were performed to evaluate the in vivo expression and tolerability of ceDNA-luciferase formulated LNPs in mice. These LNPs contained either Reference Lipid A, Reference Lipid B, or MC3 as positive controls, or the cationic lipids of the present disclosure. The study design and procedures involved in these preclinical studies are as follows:

[0331] Materials and Methods Species (number, sex, age): CD-1 mice, male, approximately 4 weeks old upon arrival in study 1 and approximately 6–8 weeks old in study 2.

[0332] Cageside Observations: Cageside observations were conducted daily.

[0333] Clinical Observations: Clinical observations were performed on days 0, 1, 2, 3, 4, and 7 (prior to euthanasia) in both Study 1 and Study 2. Additional observations were performed for each exception. Body weights of all animals were recorded on the same days as above, if applicable. Additional body weights were recorded as necessary.

[0334] Dose Administration: Test article (LNP:ceDNA-Luc) was administered by intravenous administration into the lateral tail vein in a volume of 5 mL / kg to all groups on day 0. Dose levels were 0.25 mg / kg in Study 1 and 0.5 mg / kg in Study 2.

[0335] In-life imaging: On day 4, all animals received 150 mg / kg (60 mg / mL) luciferin via intraperitoneal (IP) injection at 2.5 mL / kg. ≦15 min after each luciferin administration. All animals underwent an IVIS imaging session according to the in vivo imaging protocol described below.

[0336] In vivo imaging protocol Luciferin stock powder was stored at -20°C according to labeling. • Formulated luciferin was stored in 1 mL aliquots at 2-8°C, protected from light. ●Formulated luciferin was stable for up to 3 weeks at 2-8°C and for approximately 12 hours at room temperature (RT), protected from light. Luciferin was dissolved in a sufficient volume of PBS to a target concentration of 60 mg / mL and adjusted to pH=7.4 with 5 M NaOH (approximately 0.5 μl / mg luciferin) and HCl (approximately 0.5 μl / mg luciferin) as needed. • The appropriate amount was prepared according to protocol, including at least approximately 50% overage.

[0337] Injection and imaging • Shaved the animal's coat (if necessary). ● Following the protocol, 150 mg / kg luciferin in 60 mg / mL PBS was injected via intraperitoneal cavity. Imaging was performed immediately after administration or up to 15 minutes after administration. Set the isoflurane vaporizer to 1-3% (usually 2.5%) to anesthetize the animal during the imaging session. Isoflurane anesthesia for imaging sessions: ○The animal was placed in the isoflurane chamber and allowed to sit for approximately 2-3 minutes for the isoflurane to take effect. ○Make sure the anesthesia level on the side of the IVIS machine is in the "on" position. ○The animal was placed in the IVIS machine.

[0338] The desired acquisition protocol was performed at the highest sensitivity setting.

[0339] Results and Discussion Research 1 Study 1 was conducted to evaluate the ability of an exemplary lipid of the present disclosure, i.e., lipid 6, to be formulated as an LNP, as well as the in vivo expression and tolerability, when the LNP-ceDNA-luciferase composition was administered to mice at a dose of 0.25 mg / kg.

[0340] As a general rule, a polydispersity index (PDI) of 0.15 or less indicates good size uniformity of the formed LNPs, and an encapsulation efficiency (EE) of 90% indicates a satisfactory encapsulation rate. LNP2, LNP3, and LNP4, each formulated with lipid 6 but with various DMG-PEG2000 amounts and appropriately adjusted cholesterol amounts, showed excellent PDI values ​​of less than 0.1 and EE values ​​of more than 95%.

[0341] As shown in Figure 1, LNP2, LNP3, and LNP4 (i.e., LNPs containing lipid 6 as the cationic lipid and ceDNA-luciferase as the nucleic acid cargo) showed good in vivo luciferase expression levels at day 4 that were comparable to that of LNP1 formulated with reference lipid A and ceDNA-luciferase.

[0342] Research 2 Study 2 was conducted to evaluate the ability of several exemplary lipids of the present disclosure, namely lipid 1, lipid 7, and lipid 11, to be formulated as LNPs (i.e., LNP10, and LNP8, and LNP9, respectively), as well as the in vivo expression and tolerability, when the LNP-ceDNA-luciferase composition was administered to mice at a dose of 0.5 mg / kg. The expression and tolerability of these LNP compositions of the present invention were also compared to LNP compositions formulated with reference lipid A, reference lipid B, and MC3 (all of which have a different head group than the formula (I) lipid). All LNP compositions were formulated with satisfactory encapsulation efficiency and polydispersity index.

[0343] As shown in FIG. 2A, LNP8, LNP9, and LNP10 (i.e., LNPs containing lipid 7, lipid 11, and lipid 1, respectively) showed good in vivo luciferase expression levels on day 4. Notably, the luciferase expression levels of LNP8 and LNP9 formulated with lipid 7 and lipid 11, respectively, were higher than that of LNP6 formulated with MC3. Furthermore, FIG. 2B shows that even at 0.5 mg / kg, which is twice the dose level applied in Study 1, LNP8, LNP9, and LNP10, each formulated with a cationic lipid of the present disclosure, all achieved full body weight recovery by day 4 after treatment, thereby indicating that these LNP compositions were well tolerated in mice.

[0344] References and Equivalents All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0345] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified as appropriate to provide further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, some changes can be made to protein structures without affecting the type or amount of biological or chemical action, due to considerations of biological functional equivalence. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0346] Particular elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily must exhibit such advantages, to be within the scope of the present disclosure.

[0347] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It is to be understood that the present invention is not limited in any manner to the specific methodology, protocols, and reagents, etc. described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

Claims

1. A lipid represented by Formula I, 【Chemical 31】 or a pharmaceutically acceptable salt thereof, wherein in the formula, R' is absent, hydrogen, or C １ -C ３ -alkyl, provided that when R' is hydrogen or C １ -C ３ -alkyl, the nitrogen atom to which R', R １ , and R ２ are all attached is positively charged R １ and R ２ each independently is hydrogen or C １ to C ３ alkyl, R ３ is C ３ to C １０ alkylene or C ３ to C １０ alkenylene, and R ４ is C １ to C １６ unbranched alkyl, C ２ to C １６ unbranched alkenyl, or 【Chemical 32】 wherein in the formula, R ４ａ and R ４ｂ are each independently C １ to C １６ unbranched alkyl or C ２ to C １６ unbranched alkenyl, R ５ is absent, or C １ to C ８ alkylene, or C ２ to C ８ alkenylene, R ６ａ and R ６ｂ are each independently C ７ to C １４ alkyl or C ７ to C １４ alkenyl, and X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(R ａ )=N-, -N=C(R ａ )-, -C(R ａ )=NO-, -O-N=C(R ａ )-, -C(=O)NR ａ -, -NR ａ C(=O)-, -NR ａ C(=O)NR ａ -, -OC(=O)O-, -OSi(R ａ ), ２ O-, -C(=O)(CR ａ ２ ),C(=O)O-, or OC(=O)(CR ａ ２ ),C(=O)-, where, R ａ is, for each occurrence, independently hydrogen or C １ to C ６ alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6, the lipid, or a pharmaceutically acceptable salt thereof.

2. The lipid according to Claim 1, or a pharmaceutically acceptable salt thereof, wherein X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -S-S-.

3. The lipid according to Claim 1 or Claim 2, wherein the lipid is represented by Formula II, 【Chemical 33】 or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4.

4. The lipid according to Claim 1, wherein the lipid is represented by Formula III, 【Chemical 34】 or a pharmaceutically acceptable salt thereof.

5. R １ and R ２ are each independently hydrogen, C １ to C ２ alkyl, or C ２ to C ３ alkenyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

6. R', R １ , and R ２ are each independently hydrogen or C １ -C ２ alkyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

7. The lipid according to Claim 1, wherein the lipid is represented by Formula IV, 【Chemical 35】 or a pharmaceutically acceptable salt thereof.

8. R ５ is absent or is C １ to C ８ alkylene, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

9. R ５ is absent or is C ２ alkylene, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

10. The lipid according to Claim 1, wherein the lipid is represented by Formula V, 【Chemical 36】 or a pharmaceutically acceptable salt thereof.

11. R ４ is C １ to C １４ unbranched alkyl, C ２ to C １４ unbranched alkenyl, or 【Chemical 37】 and in the formula, R ４ａ and R ４ｂ each independently represents C １ to C １２ unbranched alkyl or C ２ to C １２ unbranched alkenyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

12. R ４ is C ２ to C １２ unbranched alkyl or C ２ to C １２ unbranched alkenyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

13. R ４ is C ５ -C １２ to C non-branched alkyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

14. R ４ is C ６ unbranched alkyl, C ７ unbranched alkyl, C ８ unbranched alkyl, C ９ unbranched alkyl, C １０ unbranched alkyl, C １１ unbranched alkyl, or C １２ unbranched alkyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

15. R ４ is C ９ The lipid according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is an unbranched alkyl.

16. R ３ is C ３ -C ８ alkylene or C ３ -C ８ alkenylene, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

17. R ３ is C ３ to C ７ an alkylene, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

18. R ３ is C ７ alkylene or C5 alkylene, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

19. R ６ａ and R ６ｂ are each independently C ７ to C １２ alkyl or C ７ to C １２ alkenyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

20. R ６ａ and R ６ｂ are each independently C ７ alkyl, C ８ alkyl, C ９ alkyl, C １０ alkyl, C １１ alkyl, C １２ alkyl, C ８ alkenyl, C １０ alkenyl, C １１ alkenyl, or C １２ alkenyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

21. R ６ａ and R ６ｂ are each independently C ７ alkyl, C ８ alkyl, C ９ alkyl, C １０ alkyl, C １１ alkyl, or C １２ alkyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

22. R ６ａ and R ６ｂ are each a lipid according to claim 1, or a pharmaceutically acceptable salt thereof, containing the same number of carbon atoms.

23. R ６ａ and R ６ｂ are the same, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

24. R ６ａ and R ６ｂ are both C ７ alkyl, or C ８ alkyl, or C ９ alkyl, or C １０ alkyl, or C １１ alkyl, or C １２ alkyl, or a pharmaceutically acceptable salt thereof, of the lipid according to claim 1.

25. R ６ａ and R ６ｂ are both C ８ alkyl, C9 alkyl, C10 alkyl, C11 alkyl or C12 alkyl, the lipid according to claim 1, or a pharmaceutically acceptable salt thereof.

26. R ６ａ and R ６ｂ are each a lipid according to claim 1, or a pharmaceutically acceptable salt thereof, containing a different number of carbon atoms.

27. The lipid according to Claim 1, or a pharmaceutically acceptable salt thereof, wherein R' is absent.

28. The lipid according to Claim 1, wherein the lipid is as follows, 【Chemical 38】 【Chemical 39】 【Chemical 40】 or a pharmaceutically acceptable salt thereof.

29. A lipid nanoparticle (LNP) comprising the lipid according to Claim 1, or a pharmaceutically acceptable salt thereof, and a therapeutic nucleic acid.

30. The lipid nanoparticle according to Claim 29, wherein the therapeutic nucleic acid is encapsulated within the lipid.

31. The lipid nanoparticle according to claim 29, wherein the therapeutic nucleic acid is selected from the group consisting of a minigene, a plasmid, a minicircle, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), ribozyme, cDNA, ministring, doggybone (trademark), telomere-capped DNA, dumbbell linear DNA, dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, a DNA virus vector, a viral RNA vector, a non-viral vector, and any combination thereof.

32. The lipid nanoparticle according to claim 29, wherein the therapeutic nucleic acid is mRNA or siRNA.

33. The lipid nanoparticle according to claim 29, further comprising a sterol.

34. The lipid nanoparticle according to claim 33, wherein the sterol is cholesterol or beta-sitosterol.

35. The lipid nanoparticle according to claim 29, further comprising a non-cationic lipid.

36. The non-cationic lipid is distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dieleoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,The lipid nanoparticles according to claim 35, selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof.

37. The lipid nanoparticle according to claim 35, wherein the non-cationic lipid is selected from the group consisting of dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).

38. The lipid nanoparticle according to claim 29, further comprising at least one PEGylated lipid.

39. The at least one PEGylated lipid is PEG-dilauroxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, PEG-distearyloxypropyl, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol-PEG (DMG-PEG), distearoyl-rac-glycerol-PEG (DSG-PEG), The lipid nanoparticles according to claim 38, selected from the group consisting of PEG-dilauryl glycerol, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauryl glycamide, PEG-dimyristyl glycamide, PEG-dipalmitoyl glycamide, PEG-distearyl glycamide, (1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)) (PEG-cholesterol), 3,4-ditetradecyloxybenzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE-PEG-OH).

40. The lipid nanoparticles according to claim 29, further comprising a tissue-specific targeting ligand.

41. The lipid nanoparticles according to claim 40, wherein the tissue-specific targeting ligand is N-acetylgalactosamine (GalNAc), a GalNAc derivative, or an antibody.

42. The lipid nanoparticles according to claim 40, wherein the tissue-specific targeting ligand is covalently bound to the at least one PEGylated lipid to form a PEGylated lipid conjugate.

43. The cationic lipid is present in the lipid nanoparticles in a molar percentage of about 30% to about 80%, and / or The sterol is present in the lipid nanoparticles in a molar percentage of about 20% to about 50%, and / or The non-cationic lipid is present in the lipid nanoparticles in a molar percentage of about 2% to about 20%, and / or The at least one PEGylated lipid is present in the lipid nanoparticles in a molar percentage of about 2.1% to about 10%, and / or The PEGylated lipid conjugate is present in the lipid nanoparticles in a molar percentage of about 0.1% to about 10%, the lipid nanoparticles according to claim 33.

44. A pharmaceutical composition comprising the lipid according to claim 1, or lipid nanoparticles comprising the lipid or a pharmaceutically acceptable salt thereof and a therapeutic nucleic acid, and a pharmaceutically acceptable excipient. A composition comprising the lipid nanoparticles according to claim 29 or a pharmaceutical composition according to claim 44 for use in a method of treating a genetic disorder.

46. A composition or pharmaceutical composition for use according to claim 45, wherein the subject is a human.

47. The genetic disorder is melanoma, hemophilia A (factor VIII (FVIII) deficiency), hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR deficiency), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary liver metabolic disorder, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS IH-S), Hunter syndrome (MPS II), Sanfilippo syndrome type A (MPS IIIA), Sanfilippo syndrome type B (MPS IIIB), Sanfilippo syndrome type C (MPS IIIC), Sanfilippo syndrome type D (MPS IIID), Morquio syndrome type A (MPS IVA), Morquio syndrome type B (MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease A / B, Niemann-Pick disease C1, Niemann-Pick disease C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis type I, mucolipidosis II / III, mucolipidosis IV, sialidosis type I, sialidosis type II, glycogen storage disease type I, glycogen storage disease type II (Pompe disease), Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4 deficiency), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), progressive familial intrahepatic cholestasis (PFIC) type II (ABCB11 deficiency), progressive familial intrahepatic cholestasis (PFIC) type III (ABCB4 deficiency), progressive familial intrahepatic cholestasis (PFIC) type IV (TJP2), and compositions or pharmaceutical compositions for use according to claim 45 selected from the group consisting of cathepsin A deficiency.

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