Cationic lipids and compositions thereof

Abstract

As used herein, lipids having formula I or formula Ia: TIFF2024517644000109.tif46128 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 1 , X 2 and n are as defined herein for each of Formula I and Formula Ia, respectively. Also provided herein is a lipid nanoparticle (LNP) composition comprising a 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 capsid-free, non-viral DNA vectors to a target site of interest (e.g., cells, tissues, organs, etc.).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 176,943, filed April 20, 2021, and U.S. Provisional Patent Application No. 63 / 217,869, filed July 2, 2021, the contents of each 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 or acquired disorders caused by abnormal gene expression profiles. Various types of gene therapy have been developed to deliver therapeutic nucleic acids into patients' cells as drugs to treat diseases.

[0003] Introduction and expression of repair genes into target cells in patients can be achieved through a number of methods, including the use of engineered viral gene delivery vectors and potentially plasmids, minigenes, oligonucleotides, minicircles, or various closed-end DNA vectors. Among the many available virus-derived vectors (e.g., recombinant retroviruses, lentiviruses, and adenoviruses), recombinant adeno-associated viruses (rAAVs) have been accepted as versatile and relatively safe vectors for gene therapy. However, viral vectors such as adeno-associated vectors are highly immunogenic and can elicit humoral and cellular immunity that may impair efficacy, especially upon re-administration.

[0004] Nonviral gene delivery avoids certain disadvantages associated with viral transduction, particularly the humoral and cellular immune responses to the viral structural proteins that form the vector particle, as well as disadvantages resulting from any de novo viral gene expression. Among the advantages of nonviral delivery techniques is the use of lipid nanoparticles (LNPs) as carriers. LNPs offer a unique opportunity to engineer cationic lipids as LNP components that can circumvent the humoral and cellular immune responses that result in significant toxicity associated with viral gene therapy.

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

[0006] Some cationic lipids (for example, DODAP and DOTAP) have two or more structurally identical hydrophobic tails in the hydrophobic domain. Some other cationic lipids have two or more hydrophobic tails that are structurally different from each other. Asymmetric cationic lipids known in the art, such as CLinDMA, are typically asymmetric in one of the following ways: (i) the hydrophobic tails are structurally different by incorporating different chemical moieties and functional groups (for example, CLinDMA incorporates cholesterol into one of the hydrophobic tails); or (ii) the hydrophobic tails are different in length. Symmetric cationic lipids are usually preferred because they present fewer challenges in synthesis.

[0007] Several widely used cationic lipids, such as CLinDMA, DLinDMA (DODAP), and DOTAP, have been used for ribonucleic acid (siRNA or mRNA) delivery, but suffer from suboptimal delivery efficiency along with toxicity at higher doses. Given the shortcomings of current cationic lipids, there is a need to provide lipid scaffolds that not only exhibit enhanced efficacy with reduced toxicity, but also improve pharmacokinetics and intracellular dynamics, such as cellular uptake and release of nucleic acids from lipid carriers. Summary of the Invention

[0008] The cationic lipids provided in the present disclosure include one hydrophobic tail containing a biodegradable group and one hydrophobic tail that does not contain a biodegradable group. Some 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 when formulated as lipid nanoparticles (LNPs) to carry therapeutic nucleic acids, the cationic lipids of the present disclosure exhibit excellent and stable in vivo expression levels of the transgene insert within the nucleic acid and are well tolerated in vivo. Many of the exemplary cationic lipids of the present disclosure, when formulated as LNPs carrying therapeutic nucleic acids, have been found to exhibit superior in vivo expression and in vivo tolerability compared to the above-mentioned reference lipid counterparts. 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 and the length of the unbranched hydrophobic tail is particularly important for achieving superior encapsulation efficiency of LNP compositions.

[0009] Thus, in one aspect, there is provided herein a lipid of formula I or Ia:

[0010] [ka] and pharmaceutically acceptable salts thereof, wherein R′, R1 , R 2 , R 3 , R 4 , R 5 , R 6a , R 6b , X 1 , X 2 , and n are as defined herein for each of Formula I or Ia, respectively.

[0011] Also provided is a pharmaceutical composition comprising a lipid described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0012] Another aspect of the present disclosure relates to a composition comprising a lipid nanoparticle (LNP) comprising a lipid described herein, or a pharmaceutically acceptable salt thereof, and a nucleic acid. In one embodiment of any of the aspects or embodiments herein, the nucleic acid is encapsulated in the LNP. In certain embodiments, the nucleic acid is closed-end DNA (ceDNA).

[0013] A further aspect of the present disclosure provides a method of treating a genetic disorder in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any of the aspects or embodiments herein. [Brief explanation of the drawings]

[0014] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to exemplary embodiments of the present disclosure, which are depicted in the accompanying drawings. However, the accompanying drawings illustrate only typical embodiments of the present disclosure and therefore should not be considered limiting in scope, as the present disclosure may admit of other equally effective embodiments. [Figure 1A]1 is a graph showing the total amount of luciferase expression measured by fluorescence in mice 4 days after administration of luciferase-encoding ceDNA formulated in LNP1, LNP2, and LNP3. LNP1 is a lipid nanoparticle formulated with reference lipid A and used as a positive control, while LNP2 and LNP3 are lipid nanoparticles formulated with lipid 20 listed in Table 4. PBS was used as a negative control. [Figure 1B] 1 is a graph showing the weight change on day 1 in mice administered LNP1, LNP2, LNP3, and luciferase-encoding ceDNA formulated in PBS as described above. [Figure 2A] 1 is a graph showing total luciferase expression as measured by fluorescence in mice 4 days after administration of luciferase-encoding ceDNA formulated in LNP4 and LNP5. LNP4 is a lipid nanoparticle formulated with reference lipid A and GalNAc4 and used as a positive control, while LNP5 is a lipid nanoparticle formulated with lipid 20 and GalNAc4 as described in Table 5. PBS was used as a negative control. [Figure 2B] 1 is a graph showing the weight change on day 1 in mice administered LNP4, LNP5, and cdDNA encoding luciferase formulated in PBS as described above. [Figure 3A] 1 is a graph showing the total amount of luciferase expression as measured by fluorescence in mice 4 days after administration of luciferase-encoding ceDNA formulated in LNPs comprising lipids of the invention as described in Table 6, using PBS as a negative control. [Figure 3B] 1 is a graph showing the total amount of luciferase expression as measured by fluorescence in mice 7 days after administration of luciferase-encoding ceDNA formulated in LNPs containing lipids of the invention as described in Table 6, using PBS as a negative control. [Figure 3C]10 is a graph showing total luciferase expression as measured by fluorescence in mice 4 and 7 days after administration of luciferase-encoding ceDNA formulated in LNPs as described in Table 6. [Figure 3D] 1 is a graph showing the weight change in mice one day after administration of luciferase-encoding ceDNA formulated in lipid-containing LNPs of the present invention as described in Table 6. [Figure 4A] 7 is a graph showing the total amount of luciferase expression in mice as measured by fluorescence 4 days after administration of luciferase-encoding ceDNA formulated in LNPs comprising lipids of the invention as described in Table 7, using PBS as a negative control. [Figure 4B] 1 is a graph showing the total amount of luciferase expression as measured by fluorescence in mice 7 days after administration of luciferase-encoding ceDNA formulated in LNPs comprising lipids of the invention as described in Table 7, using PBS as a negative control. [Figure 4C] 10 is a graph showing total luciferase expression in mice 4 and 7 days after administration of luciferase-encoding ceDNA formulated in LNPs as described in Table 7. [Figure 4D] 1 is a graph showing weight change in mice one day after administration of luciferase-encoding ceDNA formulated in lipid-containing LNPs of the present invention as described in Table 7. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs), such as non-viral vectors (e.g., closed-ended DNA), 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 patient re-administration to titrate to an effective level for each patient or to maintain efficacy over time. Furthermore, other nucleic acid modalities suffer significantly from immunogenicity due to innate DNA or RNA sensing mechanisms that trigger a cascade of immune responses. Due to the lack of pre-existing immunity, the TNA lipid particles (e.g., lipid nanoparticles) described herein allow for additional doses of TNAs, such as mRNA, siRNA, synthetic vectors, or ceDNA, as needed, further expanding patient access to include pediatric populations, who may require subsequent administrations depending on tissue growth. Furthermore, it is a discovery of the present disclosure that lipid compositions containing one or more tertiary amino groups and TNA lipid particles (e.g., lipid nanoparticles) containing disulfide bonds, in particular, provide more efficient delivery of TNAs (e.g., ceDNA), better tolerability, and an improved safety profile. Because the TNA lipid particles (e.g., lipid nanoparticles) described herein are free from the packaging constraints imposed by the space within a viral capsid, theoretically the only size limitation of the TNA lipid particles (e.g., lipid nanoparticles) resides in the host cell's expression (e.g., DNA replication or RNA translation) efficiency.

[0016] One of the biggest hurdles in developing therapies, especially for rare diseases, is the large number of individual pathologies. Approximately 350 million people worldwide live with a rare disorder, and the National Institutes of Health defines a rare disorder as a disorder or condition diagnosed in fewer than 200,000 people. Approximately 80% of these rare disorders are genetic in origin, and approximately 95% of them do not have FDA-approved treatments (rarediseases.info.nih.gov / diseases / pages / 31 / faqs-about-rare-diseases). Among the advantages of the TNA lipid particles (e.g., lipid nanoparticles) described herein is that they provide an approach that can be rapidly adapted to multiple diseases that can be treated with specific TNA modalities, particularly rare monogenic diseases, which could meaningfully change the current state of treatment for many genetic disorders or diseases.

[0017] I. Definition The term "alkyl" refers to a saturated, straight-chain (i.e., unbranched) or branched-chain hydrocarbon monovalent radical. 16 As used herein, the term "alkyl" applies to both branched and unbranched alkyl groups, unless specifically stated to be an unbranched alkyl. Exemplary alkyl groups include C1-C 16 Unbranched alkyl, C7-C 16 Alkyl, C8-C 14 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 12), 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.

[0018] The term "alkylene" refers to a divalent radical of a saturated, straight-chain, or branched hydrocarbon. Alkylene can be unbranched, e.g., C1-C 12 Unless specifically stated to be an unbranched alkylene, the term "alkylene" as used herein applies to both branched and unbranched alkylene groups. Exemplary alkylene groups include C1-C 12 Examples include, but are not limited to, alkylene, C1-C9 alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C2-C8 alkylene, C3-C7 alkylene, C5-C7 alkylene, C7 alkylene, C5 alkylene, and alkenylene corresponding to any of the above exemplary alkyl groups.

[0019] The term "alkenyl" refers to a linear or branched hydrocarbon monovalent radical having one or more (e.g., one or two) carbon-carbon double bonds; alkenyl radicals include groups having "cis" and "trans" orientations, or, alternatively, "E" and "Z" orientations. An alkenyl can be unbranched, e.g., C2-C 16 Unless specifically stated to be an unbranched alkenyl, the term "alkenyl" as used herein applies to both branched and unbranched alkenyl groups. 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 These include, but are not limited to, alkenyls and the alkenyls corresponding to any of the above exemplary alkyl groups containing two or more carbon atoms.

[0020] 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 groups having "cis" and "trans" orientations, or, alternatively, "E" and "Z" orientations. Alkenylene can be unbranched, e.g., C2-C 12 Unless specifically stated to be an unbranched alkylene, the term "alkenylene" as used herein applies to both branched and unbranched alkenylene groups. Exemplary alkenylene groups include C2-C 12 Examples include, but are not limited to, alkenylene, C2-C9 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 two or more carbon atoms.

[0021] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt 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, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0022] As used in this specification and the appended claims, the term "about," when referring to a measurable value such as an amount, temporal duration, etc., is meant to encompass variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.5%, and even more preferably ±0.1%, from the specified value as appropriate to practice the disclosed methods.

[0023] As used herein, "comprise," "comprising," and "comprises," and "comprised of" are meant to be synonymous with the terms "include," "including," "includes," or "contain," "containing," "contains," e.g., are inclusive or open-ended terms specifying the presence of what follows the components, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps that are known in the art or disclosed therein.

[0024] 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.

[0025] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the invention.

[0026] As used herein, the terms "administration," "administering," and variations thereof refer to the introduction of a composition or agent (e.g., a nucleic acid, particularly ceDNA) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. Introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another. Administration can be performed by any suitable route. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein. In one aspect of any of the aspects or embodiments herein, "administration" refers to therapeutic administration.

[0027] As used herein, phrases such as "anti-therapeutic nucleic acid immune response," "anti-transfer vector immune response," "immune response to a therapeutic nucleic acid," "immune response to a transfer vector," and the like are meant to refer to any unwanted immune response to a therapeutic nucleic acid, whether viral or non-viral in origin. In some embodiments of any of the aspects and embodiments herein, the unwanted immune response is an antigen-specific immune response to the viral transfer vector itself. In some embodiments of any of the aspects 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.

[0028] 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 pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0029] As used herein, the term "ceDNA" refers to capsid-free, closed-ended, linear, double-stranded (ds) duplex DNA for synthetic or other non-viral gene transfer. A detailed description of ceDNA is provided in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for producing ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application Nos. PCT / US2018 / 049996, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018, the contents of each of which are incorporated herein by reference in their entirety. Certain methods for producing synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Patent Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference. As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a closed-end linear double-stranded (CELiD) CELiD DNA. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a DNA-based minicircle. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a 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 comprising 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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., which provide for and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0035] As used herein, the term "exogenous" is meant to refer to a substance present in a cell other than its natural source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that is not normally found and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that is found in relatively low amounts and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to result in ectopic expression or levels. In contrast, as used herein, the term "endogenous" refers to a substance that is native to a biological system or cell.

[0036] As used herein, the term "expression" is meant to refer to the cellular processes involved in the production of RNA and proteins, and, where appropriate, secreted proteins, including, for example, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0037] As used herein, the term "expression vector" is meant to refer to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the host cell. Expression vectors can contain additional elements; for example, an expression vector can have two replication systems, allowing it to be maintained in two organisms, such as human cells for expression and prokaryotic hosts for cloning and amplification. An expression vector may be a recombinant vector.

[0038] 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.

[0039] As used herein, the term "flanking" is meant to refer to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is adjacent to A and C. Similarly for the sequence AxBxC. Thus, a flanking sequence precedes or follows the flanked sequence, but need not be contiguous with or immediately adjacent to the flanked sequence. In one embodiment of any of the aspects or embodiments herein, the term flanking refers to the terminal repeats at each end of a linear, single-stranded synthetic AAV vector.

[0040] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid involved in the expression of a given RNA or protein in vitro or in vivo. Thus, a gene includes a region that encodes the expressed RNA (usually including a polypeptide-coding sequence) and often the regulatory sequences required for their expression. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and may include sequences specifically designed to have desired parameters.

[0041] As used herein, the phrase "genetic disease" or "genetic disorder" is meant to refer to a disease or deficiency that is caused, directly or indirectly, in part or in whole, by one or more abnormalities in the genome, particularly conditions that are present from birth. The abnormality can be a mutation, insertion, or deletion in a gene. The abnormality can affect the coding sequence of the gene or its regulatory sequence.

[0042] 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.

[0043] 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 a human cell, an induced pluripotent stem cell, or any of several immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be a cell in situ or in vivo in a tissue, organ, or organism. Furthermore, the host cell may be, for example, a target cell in a mammalian subject (e.g., a human patient in need of gene therapy).

[0044] As used herein, an "inducible promoter" is meant to refer to a promoter that can initiate or enhance transcriptional activity when in the presence of, affected by, or contacted by an inducer or inducer agent. As used herein, an "inducer" or "inducing agent" can be endogenous or can be a compound or protein, usually exogenous, administered in such a way that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments of any of the aspects or embodiments herein, the inducer or inducer, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of the inducible promoter. In some embodiments of any of the aspects 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.

[0045] 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.

[0046] As used herein, the term "in vivo" is meant to refer to an assay or process that occurs in or within an organism, such as a multicellular animal. In some of the aspects described herein, the method or use may be said to occur "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses that are carried out using living cells with intact membranes outside the body of a multicellular animal or plant, for example, explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells), among others.

[0047] As used herein, the term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. They are generally classified into at least three classes: (1) "simple lipids," including fats, oils, and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids. 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.

[0048] 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).

[0049] As used herein, the terms "lipid particle" or "lipid nanoparticle" 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 a "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 an 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 another preferred embodiment, 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 an LNP formulated with a cationic lipid as described herein.

[0050] As used herein, the term "ionizable lipid" refers to a lipid, e.g., a cationic lipid, that has at least one protonatable or deprotonatable group such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and reference to charged or neutral lipids refers to the nature of the predominant species, and not all lipids need 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.

[0051] 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.

[0052] As used herein, the term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0053] As used herein, the term "non-cationic lipid" is meant to refer to any amphipathic lipid, and any other neutral or anionic lipid.

[0054] 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.

[0055] As used herein, the term "liposome" refers to lipid molecules assembled in a spherical configuration that encloses an internal aqueous volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug delivery in the context of formulation development. They act by fusing with cell membranes and repositioning their lipid structure to deliver drugs or active pharmaceutical ingredients. Liposome compositions for such delivery are typically composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids.

[0056] As used herein, the term "local delivery" refers to the direct delivery of an active agent, such as an interfering RNA (e.g., siRNA), to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site, such as a tumor, or other target site, such as a site of inflammation, or into a target organ, such as the liver, heart, pancreas, or kidney.

[0057] As used herein, the term "neDNA" or "nicked ceDNA" is meant to refer to closed-end DNA that has a nick or gap of 2 to 100 base pairs in the stem or spacer region 5' upstream of the open reading frame (e.g., the promoter and transgene to be expressed).

[0058] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. The DNA may be in the form of, for example, an antisense molecule, a plasmid DNA, a DNA-DNA duplex, a precondensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. The DNA may be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear, covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), doggybone™ DNA, dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0059] As used herein, the phrases "nucleic acid therapeutic agent," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any modality of treatment that uses nucleic acids as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., lentivirus or AAV genome) or non-viral DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, doggybone™ DNA vector, minimal immunologically defined gene expression (MIDGE)-vector, non-viral ministring DNA vector (linear covalently closed DNA vector), and dumbbell-shaped DNA minimal vector ("dumbbell DNA"). As used herein, the term "TNA LNP" refers to a lipid particle containing at least one TNA, as described above.

[0060] 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.

[0061] As used herein, "operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if it affects its transcription or expression. A promoter can be said to drive expression, or drive transcription, of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence and controls transcription initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in the reverse orientation, such that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences can be used in various embodiments to modulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0062] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. A promoter can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter can also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Within the promoter sequence, a transcription initiation site will be found, 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 levels detectable above background.

[0063] A promoter may be one naturally associated with a gene or sequence, which may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons of a given gene or sequence. Such a promoter may be referred to as "endogenous." Similarly, in some embodiments of any of the aspects and embodiments herein, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments of any of the aspects and embodiments herein, a coding nucleic acid segment is positioned under the control of a "recombinant promoter" or a "heterologous promoter," both of which refer to a promoter that is not normally associated with an operably linked encoded nucleic acid sequence in its natural environment. Similarly, a "recombinant or heterologous enhancer" refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and synthetic promoters or enhancers that are not "naturally occurring," i.e., may contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in connection with the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference in its entirety). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria and chloroplasts, can similarly be used.

[0064] As used herein, the terms "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) that, 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'.

[0065] As used herein, the phrase "recombinant vector" is meant to refer to a vector containing a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and therapies in some embodiments of any of the aspects and embodiments herein. In some embodiments of any of the aspects or embodiments herein, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining a high copy number of the nucleotide of interest in a subject as extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0066] 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.

[0067] 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.

[0068] As used herein, the term "sequence identity" refers to the relationship between two nucleotide sequences. For purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle labeled "Longest Identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment - total number of gaps in the alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0069] 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 maintains 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 of a designed number of base pairs. For example, in certain aspects, an oligonucleotide "polylinker" or "polycloning site" containing several restriction endonuclease sites, or a non-open reading frame sequence designed to lack known protein (e.g., transcription factor) binding sites, can be positioned in the vector or genome to separate cis-acting elements, e.g., inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc.

[0070] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, with a therapeutic nucleic acid according to the present invention is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, 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, and 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 may 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 newborn or fetal subject, e.g., the subject is in utero. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals may be advantageously used as subjects representing animal models of diseases and disorders. In addition, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African-American, African-European, Latino, Middle Eastern, etc. In some embodiments of any of the aspects or 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 received treatment. In some embodiments of any of the aspects or embodiments herein, the subject is an embryo, fetus, newborn, infant, child, adolescent, or 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.

[0071] As used herein, the phrase "subject in need", unless the context and usage of the phrase dictates otherwise, refers to a subject: (i) who is to be administered TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention; (ii) who is receiving TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention; or (iii) who has received TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention.

[0072] 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.

[0073] 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.

[0074] As used herein, the term "systemic delivery" refers to the delivery of lipid particles that results in widespread biodistribution of an active agent, such as an interfering RNA (e.g., siRNA), within an organism. Some administration techniques result in systemic delivery of a particular agent, while others do not. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. Widespread biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or excreted (by first-pass organs (liver, lung, etc.) or by rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles (e.g., lipid nanoparticles) can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles (e.g., lipid nanoparticles) is by intravenous delivery.

[0075] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and are meant to refer to the region where Rep forms a tyrosine-phosphodiester bond with the 5' thymidine that generates a 3'-OH that serves as a substrate for DNA elongation via a cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex can participate in a coordinate ligation reaction.

[0076] As used herein, the terms "therapeutic amount," "therapeutically effective amount," "effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent (e.g., a TNA lipid particle described herein) are used interchangeably and refer to an amount sufficient to provide the intended benefit or effect of treatment, e.g., inhibition of expression of a target sequence compared to the expression level detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include, for example, examination of protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays also known to those skilled in the art. Dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, patient condition, severity of the condition, route of administration, and the specific active agent used. Thus, dosing regimens can vary widely but can be routinely determined by a physician using standard methods. Additionally, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include prophylactic or preventative amounts of the described compositions of the present invention. In prophylactic or preventative uses of the described invention, a pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk of a disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes manifested during the development of the disease, disorder, or condition, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder, or condition. It is generally preferred to use the maximum dose, i.e., the highest safe dose, according to some medical judgment. The terms "dose" and "administration" are used interchangeably herein. In one aspect of any of the aspects or embodiments herein, "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" refer to non-prophylactic or non-preventative uses.

[0077] As used herein, the term "therapeutic effect" refers to a result of treatment, which result is deemed to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. A therapeutic effect can also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease symptoms.

[0078] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjusting the dose to achieve maximum efficacy based on the above methods and other well-known methods is within the ability of one skilled in the art. The following summarizes general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference.

[0079] Pharmacokinetic principles provide the basis for modifying dosing regimens to achieve the desired degree of therapeutic effect while minimizing unacceptable side effects. In situations where the plasma concentration of a drug can be measured and is related to the therapeutic window, additional guidance regarding dosage modifications is available.

[0080] As used herein, the terms "treat," "treating," and / or "treatment" include suppressing, inhibiting, slowing, or reversing the progression of a condition, improving clinical symptoms of a condition, or preventing the appearance of clinical symptoms, or achieving a beneficial or desired clinical result. 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 suppressing, inhibiting, slowing, or reversing the progression of a condition, or improving clinical symptoms of a condition.

[0081] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, preventing a disease, disorder, or condition from occurring in a subject who may be predisposed to the disease, disorder, or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the symptoms of the disease, disorder, or condition, reducing the severity of the disease, disorder, or condition, stabilizing (i.e., not worsening) the disease, disorder, or condition, preventing the spread of the disease, disorder, or condition, delaying or slowing the progression of the disease, disorder, or condition, ameliorating or alleviating the disease, disorder, or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0082] 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 bring about the expression or replication of the attached segment ("expression cassette") in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be of viral or non-viral origin in its final form. However, for the purposes of this disclosure, "vector" generally refers to a synthetic AAV vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that, when associated with the appropriate control elements, is capable of replicating and transferring a gene sequence to a cell. In some embodiments of any of the aspects and embodiments herein, the vector can be a recombinant vector or an expression vector.

[0083] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to include the group as modified, thus satisfying all Markush group descriptions used in the appended claims.

[0084] 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.

[0085] Other terms are defined herein within the description of various aspects of the invention.

[0086] II. Lipids In a first embodiment, a cationic lipid of formula I:

[0087] [ka] or a pharmaceutically acceptable salt thereof, wherein: R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 The nitrogen atoms to which all are bonded are protonated, R 1 and R 2 are each independently hydrogen or C1-C6 alkyl, R 3 However, C1~C 12 Alkylene or C2-C 12 is alkenylene, R 4 However, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0088] [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-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b However, independently, C7~C 16 Alkyl or C7-C 16 alkenyl, provided that R 6a and R 6b the total number of carbon atoms combined is greater than 15, X 1 and X 2 are each independently -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; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0089] In a second embodiment, in the cationic lipid according to the first embodiment or a pharmaceutically acceptable salt thereof, X 1 and X 2 is the same, and all other remaining variables are as described for Formula I or the first embodiment.

[0090] In a third embodiment, the cationic lipid according to the first or second embodiment, or a pharmaceutically acceptable salt thereof, is 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-; or X 1 and X 2 are each independently -C(=O)O-, -C(=O)S-, or -SS-, or X 1 and X 2 is each independently —C(═O)O or —SS—, and all other remaining variables are as described for Formula I or the first or second embodiment.

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

[0092] [ka] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for Formula I or any one of the first through third embodiments.

[0093] In a fifth embodiment, the cationic lipid of the present disclosure is represented by formula III:

[0094] [ka] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula I, Formula II, or any one of the first through fourth embodiments.

[0095] In a sixth embodiment, the cationic lipid of the present disclosure is represented by formula IV:

[0096] [ka] or a pharmaceutically 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.

[0097] In a seventh embodiment, the cationic lipid of formula I, formula II, formula III, formula IV, or any one of embodiments 1-6, or a pharmaceutically acceptable salt thereof, is 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C1-C3 alkyl or C2-C3 alkenyl, or C1-C2 alkyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl, 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.

[0098] In an eighth embodiment, the cationic lipid of the present disclosure is represented by Formula V:

[0099] [ka] or a pharmaceutically 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.

[0100] 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 pharmaceutically acceptable salt thereof, is 3is C1-C9 alkylene or C2-C9 alkenyl, or C1-C7 alkylene or C2-C7 alkynylene, or C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 But C 12 Alkylene, C 11 Alkylene, C 10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first through eighth embodiments. Alternatively, as part of the ninth embodiment, in a cationic lipid according to Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first through eighth embodiments, or a pharmaceutically acceptable salt thereof, R 3 is C1-C9 alkylene or C2-C9 alkenyl, or C1-C7 alkylene or C2-C7 alkynylene, or C1-C6 alkylene or C2-C6 alkenylene, or C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 But C 12 Alkylene, C 11 Alkylene, C 10Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first through eighth embodiments.

[0101] 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 pharmaceutically acceptable salt thereof, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene, or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene, or R 5 does not exist or R 5 is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C 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.

[0102] 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 pharmaceutically acceptable salt thereof, R 4 However, C1~C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or

[0103] [ka] and R 4a and R 4b are independently C1 to 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 is a C2-C7 unbranched alkyl or a C2-C7 unbranched alkenyl, or R 4 is a C3-C7 unbranched alkyl or a C3-C7 unbranched alkenyl, or R 4 is a C4-C7 unbranched alkyl or a C4-C7 unbranched alkenyl, or R 4 is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 is a C6-C7 unbranched alkyl or a C6-C7 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 10unbranched 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,

[0104] [ka] and R 4a and R 4b However, independently, C2~C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl, or R 4 but,

[0105] [ka] where R 4a and R 4b However, 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 10unbranched 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 1-10.

[0106] In a twelfth embodiment, in 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 pharmaceutically acceptable salt thereof, R 6a and R 6b However, independently, C7~C 14 Alkyl or C7~C 14 alkenyl, or R 6a and R 6b However, each independently, C8~C 12 Alkyl or C8-C 12 alkenyl, or R 6a and R 6b However, independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b wherein the total number of carbon atoms combined is greater than 15, 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.

[0107] 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 pharmaceutically acceptable salt thereof, R 6a and R 6b are greater than C8 alkyl or C8 alkenyl, respectively, i.e., R 6a and R 6b However, each independently, C9~C 16 Alkyl or C9-C 16 alkenyl, or R 6a and R 6b However, each independently, C9~C 15 Alkyl or C9-C 15 alkenyl, or R 6a and R 6b However, each independently, C9~C 14 Alkyl or C9-C 14 alkenyl, or R 6a and R 6b However, each independently, C9~C 13 Alkyl or C9-C 13 alkenyl, or R 6a and R 6b However, each independently, C9~C 12 Alkyl or C9-C 12 alkenyl, or R 6a and R 6b However, independently, C 10 ~C 12 Alkyl or C 10 ~C 12 alkenyl, or R 6a and R 6b However, independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, or C9 alkenyl, provided that R 6a and R 6b 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. In a fourteenth embodiment, in a cationic lipid according to Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first through thirteenth embodiments, or a pharmaceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms to each other, or R 6a and R 6b are the same or R 6a and R 6b But both are C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b wherein the total number of carbon atoms combined is greater than 15, 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 thirteenth embodiments.

[0108] In a fifteenth embodiment, 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, or a pharmaceutically acceptable salt thereof, R6a 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 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, and R 6a is C8 alkyl and R 6a is a C7 alkyl, and R 6a is C8 alkyl and R 6a is a C9 alkyl, and R 6a is C9 alkyl and R 6a is C8 alkyl, and R 6a is C9 alkyl and R 6a C 10 alkyl, and R 6a C 10 alkyl and R 6a is a C9 alkyl, and R 6a C 10 alkyl and R 6a C 11 alkyl, and R 6a C 11 alkyl and R 6a C 10 alkyl, and R 6a C 11 alkyl and R 6a C 12 alkyl, and R 6a C 12 alkyl and R 6a C 11 alkyl, and R 6a is C7 alkyl and R 6a is a C9 alkyl, and R 6a is C9 alkyl and R 6a is a C7 alkyl, and R 6a is C8 alkyl and R 6a C 10 alkyl, and R 6aC 10 alkyl and R 6a is C8 alkyl, and R 6a is C9 alkyl and R 6a C 11 alkyl, and R 6a C 11 alkyl and R 6a is a C9 alkyl, and R 6a C 10 alkyl and R 6a C 12 alkyl, and R 6a C 12 alkyl and R 6a C 10 alkyl, and R 6a C 11 alkyl and R 6a C 13 alkyl, or R 6a C 13 alkyl and R 6a C 11 alkyl, etc., 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 fourteenth embodiments.

[0109] In a sixteenth embodiment, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to fifteenth embodiments, or a pharmaceutically acceptable salt thereof, R' is absent.

[0110] In a seventeenth embodiment, in the cationic lipid according to formula I, formula II, formula III, formula IV, formula V, or any one of the first to sixteenth embodiments, or a pharmaceutically acceptable salt thereof, R 4 is alkyl which is unbranched alkyl of C7 or less or alkenyl which is unbranched alkenyl of C7 or less, and R 6a and R 6b is an alkyl greater than C8 alkyl or an alkenyl greater than C8 alkenyl, i.e., or R 4is a C2-C7 unbranched alkyl or a C2-C7 unbranched alkenyl, or R 4 is a C3-C7 unbranched alkyl or a C3-C7 unbranched alkenyl, or R 4 is a C4-C7 unbranched alkyl or a C4-C7 unbranched alkenyl, or R 4 is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 is a C6-C7 unbranched alkyl or a C6-C7 unbranched alkenyl, or R 4 is a C7 unbranched alkyl, a C6 unbranched alkyl, a C5 unbranched alkyl, a C4 unbranched alkyl, a C3 unbranched alkyl, a C2 unbranched alkyl, a C1 unbranched alkyl, a C7 unbranched alkenyl, a C6 unbranched alkenyl, a C5 unbranched alkenyl, a C4 unbranched alkenyl, a C3 unbranched alkenyl, or a C2 alkenyl, or R 6a and R 6b However, each independently, C9~C 16 Alkyl or C9-C 16 alkenyl, or R 6a and R 6b However, each independently, C9~C 15 Alkyl or C9-C 15 alkenyl, or R 6a and R 6b However, each independently, C9~C 14 Alkyl or C9-C 14 alkenyl, or R 6a and R 6b However, each independently, C9~C 13 Alkyl or C9-C 13 alkenyl, or R 6a and R 6b However, each independently, C9~C 12 Alkyl or C9-C 12 alkenyl, or R 6a and R 6b However, independently, C 10 ~C 12 Alkyl or C 10 ~C 12 alkenyl, or R6a and R 6b However, independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, or C9 alkenyl, provided that R 6a and R 6b wherein the total number of carbon atoms combined is greater than 15, 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 sixteenth embodiments.

[0111] In some aspects, in the cationic lipid of Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first to sixteenth embodiments, R' is hydrogen or C1-C6 alkyl, and R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated in that the nitrogen atoms are positively charged.

[0112] In some aspects, in the cationic lipid of Formula I, Formula II, Formula III, Formula IV, Formula V, or any one of the first to sixteenth embodiments, R′, R 1 , and R 2 are each C1-C6 alkyl, and R', R 1 , and R 2 together with the nitrogen atom to which they are attached to form a quaternary ammonium cation or a quaternary amine.

[0113] In an eighteenth embodiment, a cationic lipid of formula Ia:

[0114] [ka] or a pharmaceutically acceptable salt thereof, wherein: R' is absent or is C1-C6 alkyl; R 1 and R 2 are each independently hydrogen or C1-C6 alkyl, R 3 However, C1~C 12 Alkylene or C2-C 12 is alkenylene, R 4 However, C1~C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or

[0115] [ka] wherein: R 4a and R 4b However, each independently, C1~C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b However, independently, C7~C 16 Alkyl or C7-C 16 alkenyl, provided that R 6a and R 6b the total number of carbon atoms combined is greater than 15, X 1 and X 2 are each independently -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)NRa -, -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; n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0116] In a nineteenth embodiment, in the cationic lipid according to the eighteenth embodiment, or a pharmaceutically acceptable salt thereof, X in formula Ia 1 and X 2 is the same, and all other remaining variables are as described for Formula Ia or the eighteenth embodiment.

[0117] In a twentieth embodiment, in the cationic lipid according to the eighteenth or nineteenth embodiment, or a pharmaceutically acceptable salt thereof, X in formula Ia 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-; or X 1 and X 2 are each independently -C(=O)O-, -C(=O)S-, or -SS-, or X 1 and X 2 is each independently —C(═O)O or —SS—, and all other remaining variables are as described for Formula Ia or any one of the eighteenth or nineteenth embodiments.

[0118] In a twenty-first embodiment, the cationic lipid of the present disclosure is represented by formula IIa or

[0119] [ka] or a pharmaceutically acceptable salt thereof, wherein 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 eighteenth, nineteenth, or twentieth embodiments.

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

[0121] [ka] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula Ia, Formula IIa, or any one of the eighteenth, nineteenth, twentieth, or twenty-first embodiments.

[0122] In a twenty-third embodiment, the cationic lipid of the present disclosure is represented by formula IVa:

[0123] [ka] or a pharmaceutically acceptable salt thereof, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, or any one of the eighteenth, nineteenth, twentieth, twenty-first, or twenty-second embodiments.

[0124] In a twenty-fourth embodiment, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, or any one of the eighteenth, nineteenth, twentieth, twenty-first, or twenty-second embodiments, or a pharmaceutically acceptable salt thereof, R 1 and R 2are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C1-C3 alkyl or C2-C3 alkenyl, or C1-C2 alkyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, or any one of the eighteenth, nineteenth, twentieth, twenty-first, twenty-second, or twenty-third embodiments.

[0125] In a twenty-fifth embodiment, the cationic lipid of the present disclosure is represented by formula Va:

[0126] [ka] or a pharmaceutically 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 eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, or twenty-fourth embodiments.

[0127] In a 26th embodiment, in the cationic lipid of Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, or 25th embodiments, or a pharmaceutically acceptable salt thereof, R 3 is C1-C9 alkylene or C2-C9 alkenylene, or C1-C7 alkylene or C2-C7 alkenylene, or C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 But C 12 Alkylene, C 11 Alkylene, C10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described in Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, or 25th embodiments. Alternatively, as part of the 26th embodiment, in a cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, or 25th embodiments, or a pharmaceutically acceptable salt thereof, R 3 is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2-C7 alkenylene, C1-C6 alkylene or C2-C6 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, C2-C8 alkylene or C2-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 But C 12 Alkylene, C 11 Alkylene, C 10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described in Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, or 25th embodiments.

[0128] In a 27th embodiment, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, or 26th embodiments, or a pharmaceutically acceptable salt thereof, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene, or R 5 is absent, C1-C4 alkylene, or C2-C4 alkenylene, or R 5 does not exist or R 5 is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described in Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, or twenty-sixth embodiments.

[0129] In a 28th embodiment, in the cationic lipid of Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, or 27th embodiments, or a pharmaceutically acceptable salt thereof, R 4 However, C1~C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or

[0130] [ka] and 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 is a C2-C7 unbranched alkyl or a C2-C7 unbranched alkenyl, or R 4 is a C3-C7 unbranched alkyl or a C3-C7 unbranched alkenyl, or R 4 is a C4-C7 unbranched alkyl or a C4-C7 unbranched alkenyl, or R 4 is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 is a C6-C7 unbranched alkyl or a C6-C7 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 10unbranched 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,

[0131] [ka] and R 4a and R 4b However, independently, C2~C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl, or R 4 but,

[0132] [ka] and R 4a and R 4b However, 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 10unbranched 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 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, or 27th embodiments.

[0133] In a 29th embodiment, in the cationic lipid of Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, or 28th embodiments, or a pharmaceutically acceptable salt thereof, R 6a and R 6b However, independently, C7~C 14 Alkyl or C7~C 14 alkenyl, or R 6a and R 6b However, each independently, C8~C 12 Alkyl or C8~C 12 alkenyl, or R 6a and R 6b However, independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6bthe total number of carbon atoms combined is greater than 15, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, or 28th embodiments.

[0134] In a thirtieth embodiment, in the cationic lipid of Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, or twenty-ninth embodiments, or a pharmaceutically acceptable salt thereof, R 6a and R 6b are each greater than C8 alkyl or C8 alkenyl, i.e., R 6a and R 6b However, each independently, C9~C 16 Alkyl or C9-C 16 alkenyl, or R 6a and R 6b However, each independently, C9~C 15 Alkyl or C9-C 15 alkenyl, or R 6a and R 6b However, each independently, C9~C 14 Alkyl or C9-C 14 alkenyl, or R 6a and R 6b However, each independently, C9~C 13 Alkyl or C9-C 13 alkenyl, or R 6a and R 6b However, each independently, C9~C 12 Alkyl or C9-C 12 alkenyl, or R 6a and R 6b However, independently, C 10 ~C 12 Alkyl or C 10 ~C 12 alkenyl, or R 6a and R 6b However, independently, C16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, or C9 alkenyl, provided that R 6a and R 6b the total number of carbon atoms combined is greater than 15, and all other remaining variables are as described for Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, or 29th embodiments.

[0135] In a 31st embodiment, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, or 30th embodiments, or a pharmaceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms to each other, or R 6a and R 6b are the same or R 6a and R 6b But both are C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b the total number of carbon atoms combined is greater than 15, and all other remaining variables are as defined in Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, or 30th embodiments.

[0136] In a 32nd embodiment, in a cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, or 31st embodiments, or a pharmaceutically acceptable salt thereof, R is as defined in any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, or 31st embodiments. 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, and R 6a is C8 alkyl and R 6a is a C7 alkyl, and R 6a is C8 alkyl and R 6a is a C9 alkyl, and R 6a is C9 alkyl and R 6a is C8 alkyl, and R 6a is C9 alkyl and R 6a C 10 alkyl, and R 6a C 10 alkyl and R 6ais a C9 alkyl, and R 6a C 10 alkyl and R 6a C 11 alkyl, and R 6a C 11 alkyl and R 6a C 10 alkyl, and R 6a C 11 alkyl and R 6a C 12 alkyl, and R 6a C 12 alkyl and R 6a C 11 alkyl, and R 6a is C7 alkyl and R 6a is a C9 alkyl, and R 6a is C9 alkyl and R 6a is a C7 alkyl, and R 6a is C8 alkyl and R 6a C 10 alkyl, and R 6a C 10 alkyl and R 6a is C8 alkyl, and R 6a is C9 alkyl and R 6a C 11 alkyl, and R 6a C 11 alkyl and R 6a is a C9 alkyl, and R 6a C 10 alkyl and R 6a C 12 alkyl, and R 6a C 12 alkyl and R 6a C 10 alkyl, and R 6a C 11 alkyl and R 6a C 13 alkyl, or R 6a C 13 alkyl and R 6a C 11alkyl, etc., with the proviso that all other remaining variables are as defined in Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 embodiments.

[0137] 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 eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-first, or thirty-second embodiments, or a pharmaceutically acceptable salt thereof, R' is absent.

[0138] In a 34th embodiment, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, or 33rd embodiments, or a pharmaceutically acceptable salt thereof, R 4 is alkenyl that is an unbranched alkyl of up to C7 or an unbranched alkenyl of up to C7, and R 6a and R 6b is an alkenyl that is a greater than C alkyl or a greater than C alkenyl, i.e., or R 4 is a C2-C7 unbranched alkyl or a C2-C7 unbranched alkenyl, or R 4 is a C3-C7 unbranched alkyl or a C3-C7 unbranched alkenyl, or R 4 is a C4-C7 unbranched alkyl or a C4-C7 unbranched alkenyl, or R 4 is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 is a C6-C7 unbranched alkyl or a C6-C7 unbranched alkenyl, or R 4is a C7 unbranched alkyl, a C6 unbranched alkyl, a C5 unbranched alkyl, a C4 unbranched alkyl, a C3 unbranched alkyl, a C2 unbranched alkyl, a C1 unbranched alkyl, a C7 unbranched alkenyl, a C6 unbranched alkenyl, a C5 unbranched alkenyl, a C4 unbranched alkenyl, a C3 unbranched alkenyl, or a C2 alkenyl, or R 6a and R 6b However, each independently, C9~C 16 Alkyl or C9-C 16 alkenyl, or R 6a and R 6b However, each independently, C9~C 15 Alkyl or C9-C 15 alkenyl, or R 6a and R 6b However, each independently, C9~C 14 Alkyl or C9-C 14 alkenyl, or R 6a and R 6b However, each independently, C9~C 13 Alkyl or C9-C 13 alkenyl, or R 6a and R 6b However, each independently, C9~C 12 Alkyl or C9-C 12 alkenyl, or R 6a and R 6b However, independently, C 10 ~C 12 Alkyl or C 10 ~C 12 alkenyl, or R 6a and R 6b However, independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, or C9 alkenyl, provided that R 6a and R 6b , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 embodiment, and the total number of carbon atoms in the aryl group is greater than 15, and all other remaining variables are as defined in Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 embodiments.

[0139] In some embodiments, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 embodiments, R' is absent and R', R 1 , and R 2 are all bound to nitrogen atoms are protonated when the lipid is present at physiological conditions at a pH of about 7.4 or less, for example, a pH of about 7.4.

[0140] In some embodiments, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 embodiments, R' is absent and R', R 1 , and R 2 The nitrogen atoms to which all are attached are protonated when the lipid is in aqueous solution.

[0141] In some embodiments, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 embodiments, R' is absent and R', R 1 , and R 2The nitrogen atoms to which all are attached are protonated when the lipid is present at a pH below about 7.4.

[0142] In some embodiments, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 embodiments, R' is absent and R', R 1 , and R 2 are all attached to nitrogen atoms are protonated when the lipid is in aqueous solution and at a pH below about 7.4 (eg, a pH of about 7.4).

[0143] In some embodiments, in the cationic lipid according to Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or any one of the 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 embodiments, R′, R 1 and R 2 are each C1-C6 alkyl, and R', R 1 and R 2 together with the nitrogen atom to which they are attached to form a quaternary ammonium cation or a quaternary amine.

[0144] In one embodiment, the cationic lipid of the present disclosure or the cationic lipid of Formula I or Formula Ia is any one lipid selected from:

[0145] [ka]

[0146] [ka]

[0147] [ka]

[0148] [ka]

[0149] [ka] or a pharmaceutically acceptable salt thereof.

[0150] Additionally, a lipid of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va, or a pharmaceutically acceptable salt thereof (e.g., a quaternary ammonium salt), or any of the exemplary lipids disclosed herein, may be converted to the corresponding lipid containing a quaternary amine or quaternary ammonium cation, i.e., R′, R′, or R′, by treatment with, for example, chloromethane (CHCl) in acetonitrile (CHCN) and chloroform (CHCl). 1 and R 2 are each C1-C6 alkyl (all contemplated in this disclosure). The quaternary ammonium cations in such lipids are permanently charged regardless of the pH of their solution.

[0151] 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, lipid 11, lipid 12, lipid 13, lipid 14, lipid 15, lipid 16, lipid 17, lipid 18, lipid 19, lipid 20, lipid 21, lipid 22, lipid 23, lipid 24, or lipid 25 is protonated when the lipid is present under physiological conditions, e.g., at a pH of about 7.4 or less, e.g., a pH of about 7.4.

[0152] 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, lipid 11, lipid 12, lipid 13, lipid 14, lipid 15, lipid 16, lipid 17, lipid 18, lipid 19, lipid 20, lipid 21, lipid 22, lipid 23, lipid 24, or lipid 25 is protonated when the lipid is in aqueous solution.

[0153] 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, lipid 11, lipid 12, lipid 13, lipid 14, lipid 15, lipid 16, lipid 17, lipid 18, lipid 19, lipid 20, lipid 21, lipid 22, lipid 23, lipid 24, or lipid 25 is protonated when the lipid is present at a pH of about 7.4 or less (e.g., a pH of about 7.4).

[0154] 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, lipid 11, lipid 12, lipid 13, lipid 14, lipid 15, lipid 16, lipid 17, lipid 18, lipid 19, lipid 20, lipid 21, lipid 22, lipid 23, lipid 24, or lipid 25 is protonated when the lipid is in solution and at a pH of about 7.4 or less (e.g., a pH of about 7.4).

[0155] III. Lipid Nanoparticles (LNPs) LNPs as delivery vehicles for nucleic acids Lipid nanoparticles (LNPs) comprising a cationic lipid described herein and a capsid-free, non-viral vector or a therapeutic nucleic acid (TNA) (e.g., ceDNA), or pharmaceutical compositions thereof, can be used to deliver the capsid-free, non-viral DNA vector to a desired target site (e.g., a cell, tissue, organ, etc.). Accordingly, another aspect of the present disclosure relates to lipid nanoparticles (LNPs) comprising one or more cationic lipids described herein or pharmaceutically acceptable salts thereof and a therapeutic nucleic acid (TNA).

[0156] Cationic lipids are typically used to condense nucleic acid cargoes, such as ceDNA, at low pH and to drive membrane association and fusogenicity. Generally, cationic lipids are lipids that contain at least one amino group that is positively charged or that is protonated under acidic conditions, e.g., pH 6.5 or below, to form quaternary amine-containing lipids.

[0157] In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the cationic lipid provided herein or a pharmaceutically acceptable salt thereof is present in an amount of 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 7 ... 0% 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 5 5% to about 70%, about 60% to about 70%, about 65% to about 70%, about 30% to about 65%, about 35% to about 65%, about 40% to about 65%, about 45% to about 65%, about 50% to about 65%, about 55% to about 65%, about 60% to about 65%, about 30% to about 60%, about 35% to about 60%, about 40% to about 60%, about 45% to about 60%, about 50% to about 60%, about 55% to about 60%, It is provided in a molar percentage of 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, the cationic lipid provided herein, or a pharmaceutically acceptable salt thereof, is present in the lipid nanoparticles at 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%.

[0158] sterols In one embodiment of any of the aspects or embodiments herein, in addition to the more cationic lipids described herein, or pharmaceutically 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).

[0159] Exemplary cholesterol derivatives are described in International Patent Application Publication No. 2009 / 127060 and US Patent Application Publication No. 2010 / 0130588, the contents of each of which are incorporated herein by reference in their entirety.

[0160] 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 can be used in the lipid particles is beta-sitosterol.

[0161] In one embodiment of any of the aspects or embodiments herein, the sterol is present in the lipid nanoparticle 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 at 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%.

[0162] 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, also known as structural lipids, can serve to increase membrane fusogenicity and stability of the LNP during formation to provide membrane integrity and lipid particle stability. Non-ionizable lipids include amphipathic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipids can be neutral, uncharged, zwitterionic, or anionic lipids.

[0163] 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 (DSPE), monomethyl-phosphatidylethanolamine (DOPE-mal), dioleoyl-sn-glycero-phospho ... amine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoyl dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE);It is to be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used.The acyl group in these lipids is preferably C; 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. 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).

[0164] 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, ceramide, sphingomyelin, and the like.

[0165] Additional exemplary non-cationic lipids are described in International Patent Application Publication No. 2017 / 099823 and U.S. Patent Application Publication No. 2018 / 0028664, the contents of each of which are incorporated herein by reference in their entirety.

[0166] In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the non-cationic lipid 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 20%, or about 3% to about 20%. % 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%, about It is present in a molar percentage of 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 herein, in the lipid nanoparticles, the non-cationic lipid 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% to about 15%, 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% molar percentage.

[0167] 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. Generally, 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.

[0168] 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, and examples thereof include, but are not limited to, PEG 2000, PEG 2500, PEG 3000, PEG 3350, PEG 3500, and PEG 4000.

[0169] The lipid to which one or more PEG chains are linked can 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-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol) PEGylated lipids include, but are not limited to, 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEGylated lipids are described, for example, in U.S. Patent Nos. 5,885,613 and 6,287,591, and U.S. Patent Application Publication Nos. 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, and 2017 / 0119904, the contents of each of which are incorporated herein by reference in their entirety.

[0170] In one embodiment of any of the aspects or embodiments herein, at least one PEGylated lipid in a 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 (DMG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-distearyloxypropyl. Terylglycerol; 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(ethylene glycol) ether (PEG-DMB), 1,2 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, mPEG-C18, or any combination thereof, e.g., a combination of two or three thereof. In one embodiment of any of the aspects or embodiments herein, the at least one PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, mPEG-C18, or any combination thereof, e.g., a combination of two or three thereof. In one embodiment of any of the aspects or embodiments herein, the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, or both.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, or mPEG-C18, or any combination thereof, for example, a combination of two or three thereof. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) provided herein comprise DMP-PEG2000 and DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles (LNPs) provided herein comprise DMG-PEG2000, DSPE-PEG2000, and DSPE-PEG2000-OH, or mPEG-C18, or any combination thereof, for example, a combination of two or three thereof. In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the at least one PEGylated lipid comprises, in total, about 1% to 10%, e.g., 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 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%, about It is present in a molar percentage of 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%.

[0171] 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 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 3. % to about 7%, about 3.5% to about 7%, about 4% to about 7%, about 4.5% to about 7%, about 5% to about 7%, about 5.5% to about 7%, about 6% to about 7%, about 6.5% to about 7%, 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%. In one embodiment of any of the aspects or embodiments herein, in the lipid nanoparticles, the at least one PEGylated lipid, in total, is 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% molar percentage.

[0172] 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 for the purpose of assisting, enhancing, and / or increasing delivery of the LNP to a desired target site. The ligand can be a peptide, protein, antibody, glycan, sugar, nucleic acid, lipid, glycan, sugar, nucleic acid, lipid, or conjugate comprising any of the above) that recognizes a receptor or surface antigen that is unique to particular cells and tissues.

[0173] 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 biological molecules (e.g., but not limited to, peptides, proteins, antibodies, glycans, sugars, nucleic acids, and lipids). The biological molecule 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 WO2017 / 084987 and WO2013 / 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).

[0174] In one embodiment of any of the aspects or embodiments herein, a 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-dimyristoylglycerol (DMG-PEG); PEG-dilaurylglycerol; 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-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether. (PEG-DMB), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG). 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-DSPE-PEG2000. In one embodiment of any of the aspects or embodiments herein, the PEGylated lipid conjugate is tetra-antennary GalNAc-DSPE-PEG2000.

[0175] 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%, e.g., 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%, or 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% to 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%, about 1 % to about 2%, about 1.5% to about 2%, about 0.1% to about 1.5%, 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%, 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%.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 0.1% to about 1.5%, 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%, 0.2% to about 1%, or about 0. 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% molar percentage.

[0176] Other components of lipid nanoparticles (LNPs) Additional components of LNPs, 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-polymeric lipid (CPL) conjugates, and mixtures thereof.

[0177] 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 within the LNP or by conjugation to a therapeutic nucleic acid or any one of the components of the LNP 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 LNPs described herein further comprise dexamethasone palmitate.

[0178] In some embodiments of any of the aspects and embodiments herein, in addition to the cationic lipid, the lipid nanoparticles include an agent for condensing and / or encapsulating nucleic acid cargo, such as ceDNA. Such agents are also referred to herein as condensing agents or encapsulating agents. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acids can be used as long as it is non-fusogenic. In other words, the agent can condense and / or encapsulate nucleic acid cargo, such as ceDNA, but has little or no 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 nanoparticles formed with the condensing agent may be non-fusogenic.

[0179] Total lipid to nucleic acid ratio Generally, lipid particles (e.g., lipid nanoparticles) have a final particle size 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 20:1 to about 60:1 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 to about 30:1, 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).

[0180] 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 greater. Generally, the total lipid content of a lipid particle formulation can range from about 5 mg / mL to about 30 mg / mL.

[0181] 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 110 nm, about 55 nm to about 110 nm, nm, approximately 60 nm to approximately 110 nm, approximately 65 nm to approximately 110 nm, approximately 70 nm to approximately 110 nm, approximately 75 nm to approximately 110 nm, approximately 80 nm to approximately 110 nm, approximately 85 nm to approximately 110nm, about 90nm to about 110nm, about 95nm to about 110nm, about 100nm to about 110nm, about 105nm to about 110nm, about 40nm to about 100nm, about 45 nm to about 100 nm, about 50 nm to about 100 nm, about 55 nm to about 100 nm, about 60 nm to about 100 nm, about 65 nm to about 100 nm, about 70 nm to about 100 nm, about 75 nm to about 100 nm, about 80 nm to about 100 nm, about 85 nm to about 100 nm, about 90 nm to about 100 nm, or about 95 nm to about 100 nm.

[0182] According to some embodiments of any of the aspects or embodiments herein, the LNPs are 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 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 about 65 nm to about 70 nm; for example, but not limited to, a diameter of 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.

[0183] 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.

[0184] 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 described herein. In one embodiment of any of the aspects or embodiments herein, the lipid nanoparticles provided herein consist essentially of at least one cationic lipid, at least one sterol, at least one non-cationic lipid, and at least one PEGylated lipid 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 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.

[0185] 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 consist essentially 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 essentially 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.

[0186] 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, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or nonviral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimally immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). Thus, aspects of the present disclosure generally provide ionizable lipid particles (e.g., lipid nanoparticles) containing TNAs.

[0187] siRNA or miRNA, which can downregulate the intracellular level of a specific protein through a process called RNA interference (RNAi), are also contemplated as nucleic acid therapeutics by the present invention. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of siRNA or miRNA is removed by the RISC complex. When the RISC complex binds to complementary mRNA, it cleaves the mRNA and releases the cleaved strand. RNAi works by inducing specific destruction of mRNA, which leads to the downregulation of the corresponding protein.

[0188] Antisense oligonucleotides (ASOs) and ribozymes, which inhibit mRNA translation into proteins, are potential nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxyribonucleotides have sequences complementary to the target protein mRNA and can bind to the mRNA through Watson-Crick base pairing. This binding prevents translation of the target mRNA and / or induces RNase H degradation of the mRNA transcript. As a result, antisense oligonucleotides have enhanced specificity of action (i.e., downregulation of specific disease-related proteins).

[0189] In any of the methods and compositions provided herein, the therapeutic nucleic acid (TNA) can be a therapeutic RNA. The therapeutic RNA can be an inhibitor of mRNA translation, an RNA interference (RNAi) agent, a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), or an RNA that binds to an mRNA transcript (ASO), a protein, or other molecular ligand (aptamer). In any of the methods provided herein, the RNAi agent can be double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, small hairpin RNA, or triple-helix-forming oligonucleotide.

[0190] In any of the method compositions provided herein, the therapeutic nucleic acid (TNA) is a therapeutic DNA such as a closed-ended double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministring"), doggybone™, protelomeric closed-end DNA, dumbbell linear DNA, plasmid, minicircle, etc.). Some embodiments of the present disclosure are based on methods and compositions comprising a closed-ended linear duplex (ceDNA) capable of expressing a transgene (e.g., a therapeutic nucleic acid). ceDNA vectors as described herein do not have the packaging constraints imposed by the limited space within a viral capsid. ceDNA vectors represent a versatile eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors.

[0191] The ceDNA vector preferably has a linear, continuous structure rather than a discontinuous structure. A linear, continuous structure is believed to be more stable against attack by cellular endonucleases and less likely to undergo recombination, leading to mutagenesis. Therefore, a ceDNA vector with a linear, continuous structure is a preferred embodiment. A continuous, linear, single-stranded, intramolecularly double-stranded ceDNA vector may have covalently linked termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. While the complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, a ceDNA vector, although having complementary strands, is a single DNA molecule and therefore remains a single molecule even when denatured. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector, unlike a plasmid, can be produced without prokaryotic cell-type DNA base methylation. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (in particular, linear versus circular) and the methods used to produce and purify these different entities, and also in terms of their DNA methylation, which is of prokaryotic cell type in the case of ceDNA-plasmids and eukaryotic cell type in the case of ceDNA-vectors.

[0192] 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) containing a heterologous gene (e.g., a transgene, particularly a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, where the ITRs are different from each other. In some embodiments of any of the aspects and embodiments herein, one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., an AAV ITR), and at least one of the ITRs contains 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) at 37°C for, for example, 1 hour or more.

[0193] 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 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 different three-dimensional spatial configurations. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a 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 have different sequences, different modifications, or are not identical modified ITRs and have different three-dimensional spatial configurations. In other words, ceDNA vectors with asymmetric ITRs may have ITRs in which any changes in one ITR relative to the WT-ITR are not reflected in the other ITR, or alternatively, the asymmetric ITRs may have modified asymmetric ITR pairs that have different sequences and different three-dimensional shapes relative to each other.

[0194] In one embodiment of any of the aspects and embodiments herein, the ceDNA vector comprises, from 5' to 3', a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical with respect to each other; i.e., the ceDNA vector may comprise ITR sequences with a symmetrical three-dimensional spatial organization, such that their structures are the same shape in geometric space or have the same A, C-C', and B-B' loops in three-dimensional space. In such embodiments, the symmetrical or substantially symmetrical ITR pair may be modified ITRs (e.g., mod-ITRs) that are not wild-type ITRs. A mod-ITR pair may have one or more modifications from the wild-type ITR and have the same sequence that is the reverse complement (inverted) of each other. In one embodiment of any of the aspects 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 or substantially symmetrical ITRs may be wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs of the same AAV serotype. In one embodiment of any of the aspects 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 embodiments, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they may have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial configuration.

[0195] The wild-type or mutant or otherwise modified ITR sequences provided herein represent DNA sequences included in expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses) for the production of ceDNA vectors. Thus, the ITR sequences actually contained in ceDNA vectors produced from ceDNA-plasmids or other expression constructs may or may not be identical to the ITR sequences provided herein as a result of naturally occurring variations (e.g., replication errors) that occur during the production process.

[0196] 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 enable or control 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.

[0197] In one embodiment of any of the aspects and embodiments herein, the expression cassette is located between two ITRs and comprises, in that order, a promoter operably linked to a transgene, a post-transcriptional regulatory element, and one or more polyadenylation and termination signals. In one embodiment of any of the aspects or embodiments herein, the promoter is regulatable—inducible or repressible. The promoter can be any sequence that promotes transcription of the transgene. In one embodiment of any of the aspects or embodiments herein, the promoter is a CAG promoter or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates expression of the transgene, and by way of non-limiting example, is any sequence that creates a tertiary structure that enhances expression of the transgene, which is a therapeutic nucleic acid sequence.

[0198] In one embodiment of any of the aspects or embodiments herein, the post-transcriptional regulatory element comprises a WPRE. In one embodiment of any of the aspects or embodiments herein, the polyadenylation and termination signal comprises a BGHpolyA. Any cis-regulatory element known in the art, or a combination thereof, may additionally be used, such as, for example, the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements (including, but not limited to, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV)). In one embodiment of any of the aspects or embodiments herein, the length of the expression cassette in the 5' to 3' direction exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment of any of the aspects or embodiments herein, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0199] In one embodiment of any of the aspects or embodiments herein, the expression cassette may comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range from about 4000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides.

[0200] 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 for 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 kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] In one embodiment of any of the aspects or embodiments herein, the sequences provided in the expression cassettes, expression constructs, or donor sequences of the ceDNA vectors described herein can be codon-optimized for the host cell. As used herein, the term "optimized codons" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a substantial number of codons of the native sequence (e.g., a prokaryotic sequence) with codons more frequently or most frequently used in the genes of a vertebrate of interest, such as a mouse or human, for enhanced expression in the cells of that vertebrate. Different species exhibit particular biases for certain codons for particular amino acids.

[0205] 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.

[0206] Many organisms exhibit a bias for using certain codons to encode the insertion of specific amino acids in growing peptide chains. Codon preference, or codon bias, is a difference in codon usage among organisms, resulting from the degeneracy of the genetic code and is well documented among many organisms. Codon bias is often correlated with the efficiency of messenger RNA (mRNA) translation and is thought to depend, in particular, on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The prevalence of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0207] Given the large number of gene sequences available in a wide variety of animal, plant, and microbial species, it is possible to calculate the relative frequencies of codon usage (Nakamura, Y., et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000)).

[0208] inverted terminal repeat (ITR) As described herein, a ceDNA vector is a non-encapsid-containing linear double-stranded DNA molecule formed from continuous strands of complementary DNA with covalently linked ends (linear, continuous, non-encapsid structure), and contains 5' inverted terminal repeat (ITR) and 3' ITR sequences that are different or asymmetric with respect to each other. At least one of the ITRs contains a functional terminal resolution site and a replication protein binding site (RPS) (sometimes called a replication protein binding site), e.g., a Rep binding site. Generally, a ceDNA vector contains at least one modified AAV inverted terminal repeat (ITR), i.e., a deletion, insertion, and / or substitution relative to the other ITR, and an expressible transgene.

[0209] In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is an AAV ITR, e.g., a wild-type AAV ITR. In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is a modified ITR relative to the other ITRs, i.e., the ceDNA comprises ITRs that are asymmetric with respect to each other. In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is a non-functional ITR.

[0210] 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, wherein the ceDNA vector is not associated with capsid proteins. 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 an AAV terminal resolution site (TRS) or a functional variant of the RBE, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments of any of the aspects or 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 may also comprise a regulatory switch that is a kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0211] In any one of the aspects or embodiments herein, the two self-complementary sequences can be ITR sequences from any known parvovirus, e.g., a dependovirus, such as AAV (e.g., AAV1-AAV12). Any AAV serotype can be used, including, but not limited to, a modified AAV2 ITR sequence that retains a Rep binding site (RBS) and a terminal resolution site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to a variable palindromic sequence that allows for hairpin secondary structure formation. In some embodiments of any of the aspects and embodiments herein, the ITRs can be synthetic. In one embodiment of any of the aspects 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 contain AAV base sequences. In yet another embodiment, the synthetic ITRs preserve the ITR structure described above but have little or no AAV-origin sequences. In some aspects, the synthetic ITRs may preferentially interact with wild-type Rep or Rep of a particular serotype, or in some cases, are not recognized by wild-type Rep but are recognized only by mutant Rep. In some embodiments of any of the aspects and embodiments herein, the ITRs are synthetic ITR sequences that retain a functional Rep binding site (RBS) and terminal resolution site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to a variable palindromic sequence that allows for hairpin secondary structure formation. In some examples, the modified ITR sequence retains the 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-26B of International Patent Application No. PCT / US2018 / 049996, filed September 7, 2018.In some embodiments of any of the aspects and embodiments herein, the ceDNA vector may comprise an ITR with a modification in the ITR that corresponds to any of the modifications in the ITR sequence or ITR subsequence set forth in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A and 10B of International Patent Application No. PCT / US2018 / 49996, filed September 7, 2018.

[0212] In one embodiment of any of the aspects and embodiments herein, the ceDNA vector can be produced from an expression construct that further comprises a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments of any of the aspects or embodiments herein, the ITRs can act as promoters for the transgene. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector comprises additional components for regulating expression of the transgene, such as a regulatory switch described in International Patent Application No. PCT / US2018 / 049996, filed September 7, 2018, to regulate expression of the transgene or a kill switch that can kill cells containing the ceDNA vector.

[0213] In one embodiment of any of the aspects or embodiments herein, the expression cassette may also include post-transcriptional elements to increase transgene expression. In one embodiment of any of the aspects or embodiments herein, the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase transgene expression. Other post-transcriptional processing elements, such as post-transcriptional elements from the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV), can be used. A secretory sequence may be linked to the transgene, e.g., VH-02 and VK-A26 sequences. The expression cassette may include a polyadenylation sequence known in the art, such as a natural sequence isolated from bovine BGHpA or viral SV40pA, or a synthetic sequence, or a variant thereof. Some expression cassettes may also include an SV40 late polyA signal upstream enhancer (USE) sequence. The USE may be used in combination with SV40pA or a heterologous polyA signal.

[0214] Figures 1A-1C of International Patent Application No. PCT / US2018 / 050042, filed September 7, 2018, and incorporated herein by reference in its entirety, show schematic diagrams of non-limiting exemplary ceDNA vectors, or corresponding sequences of ceDNA plasmids. The ceDNA vectors are capsid-free and can be obtained from plasmids encoding, in that order, a first ITR, an expressible transgene cassette, and a second ITR, where at least one of the first and / or second ITR sequences is mutated relative to the corresponding wild-type AAV2 ITR sequence. The expressible transgene cassette preferably comprises, in that order, one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA).

[0215] promoter Suitable promoters, including those described above, may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. 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)), human H1 promoter (H1), CAG promoter, human alpha 1-antitrypsin (HAAT) promoter (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 exogenous to the promoter DNA.

[0216] In one embodiment of any of the aspects or embodiments herein, the promoter can include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. The promoter can also include distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate the expression of genetic components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter, as well as the promoters listed below. Such promoters and / or enhancers can be used for expression of any gene of interest, e.g., a therapeutic protein. For example, a vector can include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein. In one embodiment of any of the aspects or embodiments herein, the promoter operably linked to the therapeutic protein coding sequence can be a promoter from Simian Virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, e.g., the long terminal repeat (LTR) promoter of bovine immunodeficiency virus (BIV), a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, e.g., a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.In one embodiment of any of the aspects or embodiments herein, the promoter may also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a natural or synthetic tissue-specific promoter, e.g., a liver-specific promoter, e.g., human alpha 1-antitrypsin (HAAT) or transthyretin (TTR). In one embodiment of any of the aspects or embodiments herein, delivery to the liver may be achieved using endogenous ApoE-specific targeting of a composition comprising a ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0217] 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).

[0218] 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.

[0219] Polyadenylation sequence A sequence encoding a polyadenylation sequence can be included in a ceDNA vector to stabilize mRNA expressed from the ceDNA vector and to aid in nuclear transport and translation. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector does not contain a polyadenylation sequence. In other embodiments, the vector contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments of any of the aspects or embodiments herein, the polyadenylation sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0220] In one embodiment of any of the aspects or embodiments herein, the ceDNA is obtainable from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two different inverted terminal repeats (ITRs) (e.g., AAV ITRs), at least one of the ITRs comprising a terminal resolution site and a replication protein binding site (RPS), e.g., a Rep binding site (e.g., a wt AAV ITR), and one of the ITRs comprising a deletion, insertion, and / or substitution relative to the other ITR, e.g., a functional ITR.

[0221] 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 AAV capsid genes, but also lacks capsid genes of other viruses). In one embodiment of any of the aspects or embodiments herein, the nucleic acid molecule also lacks AAV Rep protein-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.

[0222] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector does not have modified ITRs.

[0223] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector comprises a regulatory switch disclosed herein (or in International Patent Application No. PCT / US2018 / 049996, filed September 7, 2018).

[0224] Production of V.ce DNA vector Methods for producing the ceDNA vectors described herein, comprising asymmetric or symmetric ITR pairs as defined herein, are described in Section IV of International Application PCT / US18 / 49996, filed September 7, 2018, which is incorporated herein by reference in its entirety. As described herein, ceDNA vectors can be obtained, for example, by a process comprising: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., a ceDNA-plasmid, a ceDNA-bacmid, and / or a ceDNA-baculovirus), wherein the host cells lack viral capsid-coding sequences in the presence of Rep proteins, under conditions effective, and for a time sufficient to induce production of the ceDNA vector in the host cells; 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 a ceDNA vector in the host cell.

[0225] 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 virus-based vectors.

[0226] 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.

[0227] In one embodiment of any of the aspects or embodiments herein, the present disclosure provides for the use of host cell lines that have stably integrated a DNA vector polynucleotide expression template (ceDNA template) into their own genome in the production of non-viral DNA vectors, e.g., as described in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cells at an MOI of about 3. When the host cell line is a mammalian cell line, e.g., HEK293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector, such as a herpesvirus, may be used to introduce Rep proteins into the cells, allowing for excision and amplification of the ceDNA in the presence of Rep and a helper virus.

[0228] In one embodiment of any of the aspects or embodiments herein, the host cells used to generate the ceDNA vectors described herein are insect cells, and baculovirus is used to deliver both the polynucleotide encoding the Rep protein and the non-viral DNA vector polynucleotide expression construct template for the ceDNA. In some embodiments of any of the aspects and embodiments herein, the host cells are engineered to express Rep proteins.

[0229] 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 baculovirus toxicity. The DNA vector can be isolated using a plasmid purification kit, such as the Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation can also be adapted for DNA vectors. Generally, any nucleic acid purification method can be employed.

[0230] The DNA vector can be purified by any means known to those skilled in the art for purifying DNA. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is purified as a DNA molecule. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is purified as an exosome or microparticle. The presence of the ceDNA vector can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme that has a single recognition site on the DNA vector, and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0231] 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 a thermobarrel extruder, such as the Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration.

[0232] Generally, lipid particles (e.g., lipid nanoparticles) can be formed by any method known in the art. For example, lipid particles (e.g., lipid nanoparticles) can be prepared by the methods described in, for example, U.S. Patent Application Publication Nos. 2013 / 0037977, 2010 / 0015218, 2013 / 0156845, 2013 / 0164400, 2012 / 0225129, and 2010 / 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. A process and apparatus for preparing lipid nanoparticles using a serial dilution process is described in U.S. Patent Application Publication No. 2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0233] In one embodiment of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) can be prepared by an impinging jet process. Generally, the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer solution, such as citrate buffer, sodium acetate buffer, sodium acetate and magnesium chloride buffer, malic acid buffer, malic acid and sodium chloride buffer, or sodium citrate and sodium chloride buffer. The lipid to ceDNA ratio can be about 45-55% lipid and about 65-45% ceDNA.

[0234] 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 an alcohol, e.g., ethanol, at a total lipid concentration of 5-30 mg / mL, more preferably 5-15 mg / mL, and most preferably 9-12 mg / mL. In the lipid solution, the molar ratio of lipids can range from about 25-98%, e.g., about 35-65%, for cationic lipid; from about 0-15%, e.g., about 0-12%, for non-ionic lipid; from about 0-15%, e.g., about 1-6%, for PEGylated lipid; and from about 0-75%, e.g., about 30-50%, for sterol.

[0235] 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.

[0236] To form LNPs, in one exemplary but non-limiting embodiment, two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impingement jet mixer to instantly form 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, and the total flow rate can be 10-600 mL / min. The combination of flow rate and tube ID can have the effect of controlling the particle size of the LNPs to 30 nm-200 nm. The solution can then be mixed with a buffer solution at a higher pH in a mixing ratio of 1:1 to 1:3 volume:volume, preferably about 1:2 volume:volume. If necessary, this buffer solution can be at a temperature in the range of 15-40°C or 30-40°C. The mixed LNPs can then undergo an anion exchange filtration step. Prior to anion exchange, the mixed LNPs can be incubated for a period of time, for example, 30 minutes to 2 hours. The temperature during incubation can range from 15 to 40 °C or from 30 to 40 °C. After incubation, 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 of 10 to 2000 mL / min can be used for this process.

[0237] After formation, the LNPs can be concentrated and ultrafiltered via an ultrafiltration process in which the alcohol is removed and the buffer is exchanged for a final buffer solution, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0238] The ultrafiltration process can use a tangential flow filtration (TFF) format with membranes having a nominal molecular weight cutoff range of 30–500 kD. The membrane format can be hollow fiber or flat-sheet cassette. In a TFF process with an appropriate molecular weight cutoff, the LNPs can be retained in the retentate, while the filtrate or permeate contains alcohol, citrate buffer, and final buffer waste. The TFF process is a multi-step process that results in an initial ceDNA concentration of 1–3 mg / mL. After concentration, the LNP solution is ultrafiltered with 10–20 volumes of final buffer to remove alcohol and perform buffer exchange. The material can then be further concentrated 1–3 times. The concentrated LNP solution can be sterile filtered.

[0239] VII. Pharmaceutical Compositions and Formulations Also provided herein is a pharmaceutical composition comprising a TNA lipid particle and a pharmaceutically acceptable carrier or excipient. In one embodiment of any of the aspects or embodiments herein, the invention further relates to a pharmaceutical composition comprising a cationic lipid as described in any embodiment of any of the aspects or embodiments herein, or a lipid nanoparticle as described in any embodiment of any of the aspects or embodiments herein, and a pharmaceutically acceptable excipient.

[0240] Generally, the lipid particles (eg, lipid nanoparticles) of the present invention have an average diameter selected to provide the intended therapeutic effect.

[0241] Depending on the intended use of the lipid particle (e.g., lipid nanoparticle), the proportions of the components may vary, and the delivery efficiency of a particular formulation may be measured using, for example, an endosomal release parameter (ERP) assay.

[0242] In one embodiment of any of the aspects or embodiments herein, the ceDNA may be complexed with the lipid portion of the particle or encapsulated in the lipid portion of the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments herein, the ceDNA may be fully encapsulated in the lipid portion of the lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by 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 or embodiments herein, the ceDNA in the lipid particles (e.g., lipid nanoparticles) is not substantially degraded after incubation of the particles in serum at 37°C for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0243] In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) are substantially non-toxic to a subject, e.g., a mammal, such as a human.

[0244] In one embodiment of any of the aspects or embodiments herein, a pharmaceutical composition comprising a therapeutic nucleic acid of the present disclosure may be formulated into a lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects 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, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), minicircle DNA, minigene, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vector, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, doggybone™ DNA vector, minimal immunologically defined gene expression (MIDGE) vector, non-viral ministring DNA vector (linear covalently closed DNA vector), or dumbbell-shaped DNA minimal vector ("dumbbell DNA").

[0245] 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 particle aggregation.

[0246] 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.

[0247] 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.

[0248] In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to the subject's cells, tissues, or organs. Typically, the pharmaceutical composition comprises the TNA lipid particles (e.g., lipid nanoparticles) disclosed herein and a pharmaceutically acceptable carrier. In one embodiment of any of the aspects or embodiments herein, the TNA lipid particles (e.g., lipid nanoparticles) of the present disclosure can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high-pressure intravenous or intra-arterial infusion, as well as intracellular injections such as intranuclear microinjections or intracytoplasmic injections, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. A sterile injectable solution can be prepared by incorporating the required amount of the ceDNA vector compound in an appropriate buffer with one or a combination of the above-listed ingredients, as needed, followed by filter sterilization.

[0249] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intra-thecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intra-thecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrainterstitial, intracameral, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high-pressure intravenous or intra-arterial infusion is also contemplated, as are intracellular injections such as intranuclear microinjection or intracytoplasmic injection.

[0250] Pharmaceutically active compositions containing TNA lipid particles (e.g., lipid nanoparticles) can be formulated to deliver a transgene in a nucleic acid to recipient cells, resulting in therapeutic expression of the transgene therein. The composition can also include a pharmaceutically acceptable carrier.

[0251] Pharmaceutical compositions for therapeutic purposes are typically sterile and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high ceDNA vector concentration.A sterile injectable solution can be prepared by incorporating the required amount of ceDNA vector compound in an appropriate buffer with one or a combination of the above-listed ingredients as needed, and then filtering sterilization.

[0252] 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, non-bilayer morphologies can include, for example, three-dimensional tube, rod, cubic symmetry, etc. The non-lamellar morphology (i.e., non-bilayer structure) of a lipid particle (e.g., lipid nanoparticle) can be determined using analytical techniques known and used by those skilled in the art. Such techniques include, but are not limited to, cryo-transmission electron microscopy ("Cryo-TEM"), differential scanning calorimetry ("DSC"), X-ray diffraction, etc. For example, the morphology of the lipid particles (lamellar vs. non-lamellar) can be readily assessed and characterized using, for example, Cryo-TEM analysis as described in U.S. Patent Application Publication No. 2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0253] 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.

[0254] In one embodiment of any of the aspects or embodiments herein, the present disclosure provides lipid particles (e.g., lipid nanoparticles) that are either unilamellar or multilamellar in structure. In some aspects, the present disclosure provides lipid particle (e.g., lipid nanoparticle) formulations that include multivesicular particles and / or effervescent base particles. By controlling the composition and concentration of the lipid components, the rate at which conjugated lipids exchange out of the lipid particle (lipid nanoparticle), which in turn controls the rate at which the lipid nanoparticle becomes fusogenic, can be controlled. In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which lipid particles (e.g., lipid nanoparticles) become fusogenic. Other methods that can be used to control the rate at which lipid particles (e.g., lipid nanoparticles) become fusogenic will be apparent to those skilled in the art based on the present disclosure. It will also be apparent that lipid particle size can be controlled by controlling the composition and concentration of the conjugated lipids.

[0255] 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 the cationic lipid can be determined in lipid particles (e.g., lipid nanoparticles) using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0256] In one embodiment of any of the aspects or embodiments herein, encapsulation of ceDNA in lipid particles (e.g., lipid nanoparticles) can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or the PicoGreen® assay, which uses a dye that exhibits enhanced fluorescence upon association with nucleic acids. Generally, encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic surfactant. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ceDNA, allowing it to interact with the membrane-impermeable dye. Encapsulation of ceDNA can be calculated as E = (Io - I) / Io, where I and Io refer to the fluorescence intensity before and after the addition of surfactant.

[0257] 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 intracerebroventricular administration. In some embodiments of any of the aspects and embodiments herein, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient 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.

[0258] VIII. Treatment method The lipid nanoparticles and methods described herein (e.g., TNA lipid particles, such as lipid nanoparticles) can be used to introduce nucleic acid sequences (e.g., therapeutic nucleic acid sequences) into host cells. In one embodiment of any of the aspects and embodiments herein, introduction of a nucleic acid sequence into a host cell using a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) can be monitored with appropriate biomarkers from the treated patient to assess gene expression.

[0259] 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., 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.

[0260] Provided herein are methods for treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein), optionally with a pharmaceutically acceptable carrier, into target cells (e.g., liver cells, muscle cells, kidney cells, nerve cells, or other diseased cell types) of the subject in need of treatment. The implemented TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) comprise a nucleotide sequence of interest useful for treating the disease. In particular, the TNA may comprise a desired exogenous DNA sequence operably linked to a control element capable of directing transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into a subject. The TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be administered via any suitable route described herein and known in the art. In one embodiment of any of the aspects or embodiments herein, the target cells are within a human subject.

[0261] Provided herein are methods of providing a diagnostically or therapeutically effective amount of TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) to a subject in need thereof, the method comprising providing an amount of TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) to a tissue or organ of the subject in need thereof, and providing a diagnostically or therapeutically effective amount of a protein, peptide, or nucleic acid expressed by the TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) to the subject for a time effective to allow expression of a transgene from the TNA LNPs. In one embodiment of any of the aspects or embodiments herein, the subject is a human.

[0262] Provided herein are methods for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or trauma in a subject. Generally, the methods include at least the step of administering a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to a subject in need thereof in an amount and for a time sufficient to diagnose, prevent, treat, or ameliorate one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the subject. In one embodiment of any of the aspects or embodiments herein, the subject is a human.

[0263] Provided herein are methods for using TNA LNPs as a tool for treating one or more symptoms of a disease or disease state. There are several genetic diseases for which the defective gene is known, typically falling into two classes: enzyme deficiency states, which are generally 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 states, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be used to deliver transgenes to carry normal genes into 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 enable 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.

[0264] Generally, any transgene can be delivered using TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) as described above to treat, prevent, or ameliorate symptoms associated with any disorder related to gene expression. Exemplary disease states include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes, muscular dystrophies (e.g., Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondriopathy (e.g., Leber's hereditary optic neuropathy (LHON), Leigh's syndrome, and subacute sclerosing encephalitis), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like. In some embodiments of any of the aspects 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).

[0265] 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., the 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).

[0266] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to deliver heterologous nucleotide sequences in situations where it is desirable to regulate the expression level of a transgene (e.g., a transgene encoding a hormone or growth factor).

[0267] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to correct abnormal levels and / or function of gene products (e.g., absence or defect in a protein) that result in a disease or disorder. The TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can produce functional proteins and / or restore protein levels to alleviate or reduce symptoms or confer a benefit resulting from a particular disease or disorder caused by the absence or defect in the protein. For example, treatment of OTC deficiency can be achieved by producing a functional OTC enzyme; treatment of hemophilia A and B can be achieved by altering the levels of factor VIII, factor IX, and factor 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 conductance 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.

[0268] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceRNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver RNA-based therapeutics to cells in vitro or in vivo. Examples of RNA-based therapeutics include, but are not limited to, mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). For example, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver antisense nucleic acids to cells in vitro or in vivo. For example, if the transgene is an RNAi molecule, expression of the antisense nucleic acid or RNAi in the target cell reduces the expression of a specific protein by the cell. Thus, a transgene that is an RNAi molecule or an antisense nucleic acid can be administered to reduce the expression of a specific protein in a subject in need thereof. Antisense nucleic acids can also be administered to cells in vitro to regulate cellular physiology, for example, to optimize a cell or tissue culture system.

[0269] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver DNA-based therapeutics to cells in vitro or in vivo. Examples of DNA-based therapeutics include, but are not limited to, minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE)-vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). For example, in one embodiment of any of the aspects or embodiments herein, ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver minicircles to cells in vitro or in vivo. For example, if the transgene is minicircle DNA, expression of the minicircle DNA in target cells reduces the expression of a specific protein by the cells. Therefore, a transgene that is a minicircle DNA can be administered to reduce the expression of a particular protein in a subject in need thereof. Minicircle DNA can also be administered to cells in vitro to regulate cell physiology, for example, to optimize cell or tissue culture systems.

[0270] In one embodiment of any of the aspects or embodiments herein, exemplary transgenes encoded by the TNA vector comprising the expression cassette include a gene encoding a lysosomal enzyme (e.g., hexosaminidase A associated with Tay-Sachs disease, or a gene encoding ... II-related iduronate sulfatase), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factor-3 and 4, brain-derived neurotrophic factor (BDNF), glial-derived growth factor (GDNF), transforming growth factor-a and -b, etc.), receptors (e.g., tumor necrosis factor receptor). In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, two or more transgenes are encoded by a ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects 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 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.

[0271] 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, Da Non-onset muscular dystrophy (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),The genetic disorder is selected from the group consisting of dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt's macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular 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. 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.

[0272] 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 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), such as the exemplary genetic disorders 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 disease 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.

[0273] 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 described in any of the aspects or embodiments herein for use in treating a genetic disorder in a subject (e.g., a human), such as the exemplary genetic disorders 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 disease 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.

[0274] Administration In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles described herein) can be administered to an organism for transduction of cells in vivo. In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles) can be administered to an organism for transduction of cells ex vivo.

[0275] Generally, administration is by any of the routes normally used to ultimately bring molecules into contact with blood or tissue cells. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and although more than one route may be used to administer a particular composition, 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, intraendothelial, 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., and direct tissue or organ injection (e.g., into the liver, eye, skeletal muscle, myocardium, diaphragm, muscle, or brain).

[0276] Administration of a TNA LNP-like ceDNA vector (e.g., ceDNA LNP) can be to any site of a subject, including, but not limited to, a site selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. In one embodiment of any of the aspects or embodiments herein, administration of the ceDNA LNP can also be to a tumor (e.g., within or near a tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition to be treated, ameliorated, and / or prevented, as well as the properties of the particular ceDNA LNP being used. Additionally, ceDNA allows for the administration of two or more transgenes in a single vector or multiple ceDNA vectors (e.g., a ceDNA cocktail).

[0277] 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, intra-arterial administration, intraperitoneal administration, limb perfusion (optionally, isolated limb perfusion of the legs and / or arms, see, e.g., Arruda et al., (2005) Blood 105:3458-3464), and / or direct intramuscular injection. In certain embodiments, ceDNA LNPs are administered to a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-arterial administration). In one embodiment of any of the aspects or embodiments herein, the ceDNA LNPs can be administered without the use of "hydrodynamic" techniques.

[0278] 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.

[0279] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA LNPs) are administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (e.g., to treat, ameliorate, and / or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure).

[0280] TNA LNPs (e.g., ceDNA LNPs) can be administered to the CNS (e.g., the brain or eye). TNA LNPs (e.g., ceDNA LNPs) may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (cerebrum including striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. 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).

[0281] In one embodiment of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA LNPs) can be administered to the desired region of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intracerebroventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's area) delivery, and intramuscular delivery with retrograde delivery to motor neurons.

[0282] According to some embodiments of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA LNPs) are administered in a liquid formulation by direct injection (e.g., stereotactic injection) into a desired region or compartment in the CNS. According to other embodiments, TNA LNPs (e.g., ceDNA LNPs) can be provided by topical application to a desired region or by intranasal administration of an aerosol formulation. Administration to the eye can also be by topical application of liquid drops. As a further alternative, ceDNA vectors can be administered as solid sustained-release formulations (see, e.g., U.S. Patent No. 7,201,898, incorporated herein by reference in its entirety). In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA LNPs) can be used for retrograde transport to treat, ameliorate, and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, TNA LNPs (e.g., ceDNA LNPs) can be delivered to muscle tissue and from there translocate into neurons.

[0283] In one embodiment of any of the aspects or embodiments herein, repeated administration of a therapeutic product can be performed until an appropriate level of expression is achieved. Thus, in one embodiment of any of the aspects or embodiments herein, a therapeutic nucleic acid can be administered and re-administered multiple times. For example, a therapeutic nucleic acid can be administered on day 0. Following the initial treatment on day 0, the therapeutic nucleic acid can be administered at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, or about 18 years after the initial treatment with the therapeutic nucleic acid. A second administration (re-administration) can be administered after about 18 years, about 19 years, about 20 years, about 21 years, about 22 years, about 23 years, about 24 years, about 25 years, about 26 years, about 27 years, about 28 years, about 29 years, about 30 years, about 31 years, about 32 years, about 33 years, about 34 years, about 35 years, about 36 years, about 37 years, about 38 years, about 39 years, about 40 years, about 41 years, about 42 years, about 43 years, about 44 years, about 45 years, about 46 years, about 47 years, about 48 years, about 49 years, or about 50 years.

[0284] 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 different from the first one. Without limitation, 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 derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0285] In one embodiment of any of the aspects or embodiments herein, the one or more additional compounds may be therapeutic agents. The therapeutic agent may be selected from any class suitable for therapeutic purposes. Thus, the therapeutic agent may be selected from any class suitable for therapeutic purposes. The therapeutic agent may be selected according to the desired therapeutic purpose and biological effect. For example, in one embodiment of any of the aspects or embodiments herein, if the TNA in the LNP is useful for treating cancer, the additional compound may be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate)). In one embodiment of any of the aspects or embodiments herein, if the TNA-containing LNP is useful for treating an infectious disease, the additional compound may be an antimicrobial agent (e.g., an antibiotic or an antiviral compound). In one embodiment of any of the aspects or embodiments herein, if the TNA-containing LNP is useful for treating an immune disease or disorder, the additional compound may be an anti-cancer agent (e.g., an antibiotic or an anti-viral compound). In one embodiment of any of the aspects or embodiments herein, if the TNA-containing LNP is useful for treating an immune disease or disorder, the additional compound may be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate)). If so, the additional compound can be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In one embodiment of any of the aspects or embodiments herein, different cocktails of different lipid particles containing different compounds, such as TNAs encoding different proteins or different compounds (e.g., therapeutic agents), can be used in the compositions and methods of the invention. In one embodiment of any of the aspects or embodiments herein, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressant. In some embodiments of any of the aspects or embodiments herein, the additional compound is an immunostimulatory compound. [Example]

[0286] 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.

[0287] Example 1: General Synthesis Lipids of formula I were designed and synthesized according to the following scheme 1 (R5 does not exist) and Scheme 2 (R 5 All other variables in the compounds shown in Scheme 1 and Scheme 2, i.e., R 1 , R 2 , R 3 , R 4 , R 6a , R 6b , X 1 , X 2 , and n is as defined in formula I. 1’ is, as defined, X 1 but with an additional protecting group such as benzyl or pyridine.

[0288] [ka]

[0289] [ka] R x is R 5 It is an alkylene or alkenylene having one less carbon atom.

[0290] Diester lipids of formula III were designed and synthesized as follows in Scheme 3 (R 5 does not exist) and Scheme 4 (R 5 All other variables in the compounds shown in Scheme 3 and Scheme 4, i.e., R 1 , R 2 , R 3 , R 4 , R 6a , R 6b , and n is as defined in Formula III.

[0291] [ka]

[0292] [ka] R x is R 5 It is an alkylene or alkenylene having one less carbon atom.

[0293] Scheme 1 and Scheme 3 Referring to Scheme 1 and Scheme 3, in step 1, to a stirred solution of acid 1 and alcohol 2 (or 2a) in dichloromethane (DCM) was added 4-dimethylaminopyridine (DMAP) followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). The resulting mixture was stirred overnight at room temperature and then washed with hydrochloric acid (HCl) and water. The organic layer was dried over magnesium sulfate (MgSO), evaporated to dryness, and purified by silica gel column chromatography using 0-10% methanol (MeOH) in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give acid 3 as a white solid.

[0294] In step 2, to a solution of acid 3 (or 3a) in DCM, EDCI and triethylamine (TEA) were added, and the mixture was stirred at room temperature for 15 min. N,O-dimethylhydroxylamine hydrochloride and DMAP were then added, and the mixture was stirred at room temperature overnight. The next day, the reaction was quenched with aqueous ammonium chloride (NH4Cl(aq)) and diluted with DCM. The organic layer was washed with NH4Cl and brine and dried over anhydrous sodium sulfate (Na2SO4). The solvent was evaporated under vacuum. The product 4 (or 4a) was used in the next step without further purification.

[0295] In step 3, compound 4 (or 4a) was dissolved in anhydrous tetrahydrofuran (THF). Then, 5 was added dropwise to a solution of magnesium bromide in diethyl ether (EtO) at 0 °C. The resulting mixture was stirred at room temperature under nitrogen gas (N) for 16 h. The reaction was quenched with saturated aqueous NHCl and extracted with ether. The organic phase was washed with brine and dried over anhydrous NaSO. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0–10% ethyl acetate (EtOAc) in hexane as the eluent to give 6 (or 6a).

[0296] In step 4, to a solution of 6 (or 6a) in anhydrous THF was added sodium borohydride (NaBH) at 0 °C, and the mixture was stirred overnight under a N atmosphere. The reaction was quenched with saturated aqueous NH Cl and extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na SO . The solvent was evaporated under vacuum, and purification by column chromatography using 0–10% EtOAc in hexane as the eluent gave 7.

[0297] In step 5, N,N-diisopropylethylamine (DIPEA) was added to a solution of compound 7 (or 7a) and compound 8 (or 8a) in DCM. EDCI and DMAP (0.012 g, 0.1 mmol) were then added, and the mixture was stirred overnight at room temperature under a N2 atmosphere. The next day, the reaction was diluted with DCM. The organic layer was washed with aqueous sodium bicarbonate (NaHCO3 (aq)) and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0–5% MeOH in DCM as the eluent to give the final product 9 (or diester 9a).

[0298] Scheme 2 and Scheme 4 Referring to Scheme 2 and Scheme 4, in Step 1, to an ice-cold solution of 3 g (11.8 mmol) of ketone 10 in THF was added dropwise a solution of phosphoric anhydride 11. The reaction was stirred for 30 minutes, and then sodium hydride (NaH) was added. The reaction afforded 12.

[0299] In step 2, compound 2 in THF was reacted with lithium aluminum hydride solution (LiAlH4). After 48 h, the crude product was quenched with water and extracted with ether to give alcohol 13.

[0300] Subsequent steps 3 to 7 of Scheme 2 and Scheme 4 were carried out in a similar manner as described for steps 1 to 5 of Scheme 1 and Scheme 3, using alcohol 13 as the appropriate starting material, with other modifications within the knowledge of one skilled in the art.

[0301] Example 2: General Synthesis Lipids of formula I can be synthesized using alternative synthetic procedures such as those described in this example.

[0302] Another general synthesis (A) At least lipid 11, lipid 1, and lipid 2, and any lipid of formula I (wherein R' is absent and R 1 and R 2 methyl, n is 3, and R 3 is a C6 alkylene, and R 4 is a C7 alkyl, and R 5 does not exist, and X 1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b (wherein R is as defined in Formula I) was prepared or can be prepared using synthetic methods similar to those shown in Scheme 5, provided below. Minor modifications, such as, but not limited to, replacing compound 2b with compound 2a, replacing heptylmagnesium bromide with compound 5, and / or replacing 4-(dimethylamino)butanoic acid with compound 8a to produce lipids of Formula I, can be applied to the general synthesis shown in Scheme 5 to produce other lipids of Formula I.

[0303] [ka]

[0304] Step 1: Synthesis of 9-(methoxy(methyl)amino)-9-oxononanoic acid (25c) To a stirred mixture of nonanedioic acid (2b) (20 g, 106 mmol) and N,O-dimethylhydroxylamine hydrochloride (10.3 g, 106 mmol) in DCM (200 mL) was added triethylamine (20 mL), followed by EDCI (24.3 g, 127 mmol) and DMAP (0.5 g, 4 mmol). The resulting mixture was stirred overnight at room temperature and then washed with 150 mL of 1 N HCl and 150 mL of water. The organic layer was dried over MgSO, evaporated to dryness, and 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 25c (12.8 g, 52%). 1 H-NMR (300 MHz, d-chloroform): δ 3.67 (s, 3H), 3.17 (s, 3H), 2.39 (t, 2H), 2.32 (t, 2H), 1.55-1.70 (m, 4H), 1.24-1.36 (m, 6H).

[0305] Step 2: Synthesis of compound 4f To a stirred solution of acid 25c and alcohol 1 in DCM, DMAP was added followed by EDCI. The resulting mixture was stirred at room temperature overnight and then washed with 1N HCl and water. The organic layer was dried over MgSO4, evaporated to dryness, and 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 4f.

[0306] Step 3: Synthesis of compound 6g Compound 4f was dissolved in anhydrous THF. Then, a 1 M solution of heptylmagnesium bromide in EtO was added dropwise at 0 °C. The resulting mixture was stirred at room temperature under N for 16 h. The reaction was quenched with saturated aqueous NH4Cl and extracted with ether. The organic phase was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the residue was purified by column chromatography using 0-30% EtOAc in hexane as the eluent to give 6g.

[0307] Step 4: Synthesis of compound 7h To a solution of 6 g in anhydrous THF, NaBH was added at 0 °C and stirred overnight under a N atmosphere. The reaction was quenched with saturated aqueous NH Cl and extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na SO . The solvent was evaporated under vacuum, and purified by column chromatography using 0–50% EtOAc in hexane as the eluent to give 7 h.

[0308] Step 5: Synthesis of compound 9c To a solution of compound 7h and 4-(dimethylamino)butanoic acid in DCM, DIPEA was added. EDCI and DMAP were then added, and the mixture was stirred overnight at room temperature under a N2 atmosphere. The next day, the reaction was diluted with DCM. The organic layer was washed with NaHCO3 (aq) and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and purified by column chromatography using 0-5% MeOH in DCM as the eluent to give compound 9c (which is a lipid of formula I, where R' is absent and R 1 and R 2 methyl, n is 3, and R 3 is a C6 alkylene, and R 4 is a C7 alkyl, and R 5 does not exist, and X 1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b is as defined in formula I).

[0309] Alternative general synthesis (B) At least lipid 12, lipid 3, and lipid 4, and any lipid of formula I (wherein R' is absent and R 1 and R 2 is methyl, n is 3, and R 3 is a C3 alkylene, and R 4 is a C7 alkyl, and R 5 is a C4 alkylene, and X1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b are the same and are as defined in formula I, and R 6 (equal to R in Formula I) have been prepared or can be prepared using synthetic methods similar to those shown in Scheme 6, provided below. Minor modifications can be applied to the general synthesis shown in Scheme 6 to generate other lipids of Formula I, including, but not limited to, replacing compound 2d with compound 2a, replacing heptylmagnesium bromide with compound 5, replacing 4-(dimethylamino)butanoic acid with compound 8a, replacing compound 23 with R in Formula I, 6a and R 6b and / or substitution by equivalent diols having different numbers of carbon atoms in the two aliphatic chain tails, according to the scope and definition of R 6a and R 6b Examples include the use of two different species of compound 22, each with a different number of carbon atoms in the aliphatic chain, to generate lipids of formula I with different valence.

[0310] [ka]

[0311] Synthesis of compound 13b To a solution of alkylmagnesium bromide 22 in methyltetrahydrofuran (MeTHF) cooled to 0 °C under a nitrogen atmosphere, tetrahydro-2H-pyran-2-one (21) dissolved in anhydrous ethyl ether was added dropwise. After stirring for 5 min, the ice bath was removed and the reaction was stirred at room temperature overnight. The reaction was worked up as usual, and the crude product was purified by column chromatography using 0–25% ethyl acetate (EtOAc) in hexane as eluent to give compound 23.

[0312] Compound 23 was dissolved in 5 mL of toluene, p-toluenesulfonic acid monohydrate (pTSA) was added to the microwave tube, and the mixture was microwaved at 100° C. for 1 h. The solvent was evaporated under vacuum, and the mixture was purified by column chromatography using 0–5% EtOAc in hexane as the eluent to give compound 24.

[0313] To a solution of compound 24 in EtOAc, 200 mg of 10% palladium (Pd) on activated carbon, Pearlman (50-70% wet) was added, and the mixture was degassed. After that, a hydrogen balloon was placed over the mixture overnight. The mixture was filtered through Celite, and the crude material was purified using 0-10% EtOAc in hexane as the eluent to give alcohol compound 13b.

[0314] Synthesis of 5-(methoxy(methyl)amino)-5-oxopentanoic acid (25) To a stirred solution of glutaric acid 2d (5.4 g, 41 mmol) and N,O-dimethoxyamine hydrochloride (2 g, 20.5 mmol) in dichloromethane (DCM) (20 mL), 4-dimethylaminopyridine (DMAP) (0.25 g, 2 mmol) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and triethylamine (EtN) (5.7 mL, 41 mmol). The resulting mixture was stirred overnight at room temperature, then washed with 50 mL of aqueous NH4Cl and extracted with DCM (30 mL) and EtOAc (30 mL). The organic layer was dried over magnesium sulfate (MgSO4), evaporated to dryness, and purified by silica gel column chromatography using 0–25% EtOAc in hexane as the eluent. Fractions containing the desired compound were pooled and evaporated to give 5-(methoxy(methyl)amino)-5-oxopentanoic acid (25) (2.2 g, 60%) as an oil. 1 H-NMR (300 MHz, d-chloroform): δ 3.67 (s, 3H), 3.17 (s, 3H), 2.55-1.9 (m, 4H), 1.94-2.00 (m, 2H).

[0315] Step 1: Synthesis of compound 15c Alcohol 13b and amide 25 were dissolved in anhydrous DCM, followed by EDCI and DMAP. The reaction was stirred overnight under nitrogen and worked up by adding aqueous NH4Cl and extracting with DCM and EtOAc. The organic layer was dried over MgSO4. The solvent was evaporated in vacuo and purified by column chromatography using 0-25% EtOAc in hexane as eluent to give compound 15c.

[0316] Step 2: Synthesis of compound 16c To an ice-cold solution of 15c in anhydrous THF under nitrogen, heptylmagnesium bromide in ether was added dropwise. The reaction was stirred overnight, and the reaction mixture was cooled to 0 °C before being quenched with aqueous NH4Cl. The crude product was extracted with hexane. The organic layer was dried over MgSO4. The solvent was evaporated under vacuum, and purified by column chromatography using 0-10% EtOAc in hexane as the eluent to give 16c.

[0317] Step 3: Synthesis of compound 17c To an ice-cold solution of 16c in tetrahydrofuran / methanol (THF / MeOH) was added sodium borohydride (NaBH). The reduction reaction was monitored by thin layer chromatography (TLC). After 1 h, the starting material had completely disappeared, and the reaction was quenched with aqueous NH4Cl. The crude product was evaporated to dryness, then redissolved in DCM, washed once with water, and the organic layer was dried over MgSO4. Crude compound 17c was used in the next step without further purification.

[0318] Step 4: Synthesis of compound 18b To a solution of 17c and 4-dimethylaminobutyric acid HCl in DCM, EDCI, DMAP, and finally triethylamine were added. The reaction was stirred overnight, quenched with saturated NH4Cl solution, and extracted with DCM and EtOAc. The organic layer was dried over anhydrous MgSO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-5% MeOH in DCM as the eluent to give compound 18b (which is a lipid of formula I, where R' is absent and R 1 and R 2 methyl, n is 3, and R 3 is a C3 alkylene, and R 4 is a C7 alkyl, and R 5 is a C4 alkylene, and X1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b are the same and are as defined in formula I).

[0319] Alternative synthesis (C) At least lipid 13, lipid 5, and lipid 6, and any lipid of formula I (wherein R' is absent and R 1 and R 2 is methyl, n is 3, and R 3 is a C4 alkylene, and R 4 is a C7 alkyl, and R 5 is a C3 alkylene, and X 1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b are the same and are as defined in formula I, and R 6 (equal to R in Formula I) have been prepared or can be prepared using synthetic methods similar to those shown in Scheme 7, provided below. Minor modifications can be applied to the general synthesis shown in Scheme 7 to generate other lipids of Formula I, including, but not limited to, substituting heptylmagnesium bromide with compound 5, substituting 4-(dimethylamino)butanoic acid with compound 8a, substituting compound 23a for R in Formula I, and 6a and R 6b and / or substitution by equivalent diols having different numbers of carbon atoms in the two aliphatic chain tails, according to the scope and definition of R 6a and R 6b Examples include the use of two different species of compound 22, each with a different number of carbon atoms in the aliphatic chain, to generate lipids of formula I with different valence.

[0320] [ka]

[0321] Synthesis of compound 13c Alkyl magnesium bromide 22 was weighed into an oven-dried round-bottom flask under nitrogen gas and cooled to 0 °C. Then, a solution of γ-butyrolactone (21a) in diethyl ether (EtO) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature under nitrogen for 16 h. The reaction mixture was quenched with 3 M HCl solution and extracted with ether. The combined organic layers were washed with HO and dried over anhydrous NaSO. The solvent was evaporated under vacuum, and compound 23a was purified by column chromatography using 0–50% EtOAc in hexane as the eluent.

[0322] Diol 23a and PTSA were dissolved in toluene and microwaved for 2 h at 60° C. The solvent was then evaporated under vacuum and purified by column chromatography using 0-20% EtOAc in hexane as the eluent to give compound 24a.

[0323] Compound 24a was dissolved in EtOAc, degassed, 5% Pd / C was added, and degassed again. The reaction was maintained under hydrogen for 4 hours. The reaction mixture was then filtered through Celite. The solvent was evaporated under vacuum, and the mixture was purified by column chromatography using 0-20% EtOAc in hexane as the eluent to give compound 13c.

[0324] Synthesis of compound 29 Isopropylmagnesium chloride solution (2.0 M solution in THF; 10 mL, 20 mmol) was added dropwise to a mixture of 2-oxepanone (26) (0.34 g, 3 mmol) and N,O-dimethylhydroxylamine hydrochloride (0.88 g, 9 mmol) in THF (15 mL) at 0 °C and allowed to reach room temperature. After stirring at room temperature for 1 h, the mixture was cooled to 0 °C and saturated NH Cl solution was added. The layers were separated, and the aqueous layer was extracted with DMC. The organic phase was dried over anhydrous Na SO and concentrated under reduced pressure to give 6-hydroxy-N-methoxy-N-methylhexanamide (27), which was used in the next step without further purification.

[0325] To a solution of compound 27 (3.96 g, 22.6 mmol) in THF (138 mL) was added heptylmagnesium bromide (45.2 mL, 45.2 mmol) at 0 °C. The reaction mixture was stirred overnight under nitrogen. The next day, the reaction was cooled to 0 °C, quenched with saturated aqueous NH4Cl, and the product was extracted with DCM. The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 0-50% EtOAc and hexane as the eluent to give 1-hydroxytridecan-6-one (28) (3.53 g, 75%). 1 H NMR (300MHz, d-chloroform) δ 3.64(dd,J=11.8,6.3Hz,2H),2.39(dd,J=16.3,7.5Hz,4H),1.66-1.48(m,6H),1.32-1.26(m,10H),0.87(t,J=6.7Hz,3H).

[0326] To a stirred solution of compound 28 (0.17 g, 0.8 mmol) in acetone (4 mL) was added Jones reagent (i.e., a solution of chromium trioxide in aqueous sulfuric acid) until the color remained orange (3 mmol, 1.5 mL). The reaction mixture was allowed to reach room temperature and diluted with EtOAc. The organic layer was subsequently washed with HO and brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give compound 29, which was used in the next step as a reagent in the synthesis of compound 17i without further purification. 1 H NMR (300 MHz, d-chloroform) δ 2.50-2.28 (m, 5H), 1.67-1.48 (m, 6H), 1.26 (s, 8H), 0.87 (t, J = 6.7 Hz, 3H).

[0327] Step 1: Synthesis of compound 16i To a solution of compound 33, DCM, EDCI, and DMAP were added, and the mixture was stirred under a nitrogen atmosphere for 15 minutes. Compound 13c was then added to the reaction mixture and stirred overnight. The next day, the reaction was diluted with DCM. The combined organic layer was washed with HO and brine and dried over anhydrous NaSO. The solvent was evaporated under vacuum, and the residue was purified by column chromatography using 0-30% EtOAc in hexane as the eluent to give compound 16i.

[0328] Step 2: Synthesis of compound 17i To a solution of compound 16i in THF:MeOH (1:1) was added NaBH4 at 0 °C and stirred under a nitrogen atmosphere for 1 h. The reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the residue was purified by column chromatography using 0-20% EtOAc in hexane as the eluent to give compound 17i.

[0329] Step 3: Synthesis of compound 18c To a solution of compound 17i and 4-(dimethylamino)butanoic acid in DCM (2 mL) was added N,N-diisopropylethylamine (DIPEA). EDCI and DMAP were then added, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. The next day, the reaction was diluted with DCM. The organic layer was washed with aqueous sodium bicarbonate (NaHCO3) and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0-5% MeOH in DCM as the eluent to give compound 18c (which is a lipid of formula I, where R' is absent and R 1 and R 2 methyl, n is 3, and R 3 is a C4 alkylene, and R 4 is a C7 alkyl, and R 5 is a C3 alkylene, and X1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b are the same and are as defined in formula I, and R6s is equal to).

[0330] Alternative synthesis (D) At least lipid 14, lipid 7, and lipid 8, and any lipid of formula I (wherein R' is absent and R 1 and R 2 is methyl, n is 3, and R 3 is a C5 alkylene, and R 4 is a C7 alkyl, and R 5 is a C2 alkylene, and X 1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b (wherein R is the same or different and is as defined in Formula I) have been prepared or can be prepared using synthetic methods similar to those shown in Scheme 8, provided below. Minor modifications can be applied to the general synthesis shown in Scheme 8 to generate other lipids of Formula I, such as, but not limited to, replacing compound 2c with compound 2a, replacing heptylmagnesium bromide with compound 5, and / or replacing 4-(dimethylamino)butanoic acid with compound 8a.

[0331] [ka]

[0332] Synthesis of 7-(methoxy(methyl)amino)-7-oxoheptanoic acid (25a) Pimelic acid 2c (20.0 g, 0.125 mol) was dissolved in dichloromethane / DMF (120 mL / 15 mL), followed by the addition of EtN (58 mL, 0.50 mol) and N,O-dimethylhydroxylamine hydrochloride (10.1 g, 0.100 mol). The reaction mixture was cooled to 0 °C, and EDCI (28.8 g, 0.15 mol) was added, followed by DMAP (6.3 g, 0.050 mol). The ice bath was removed, and the reaction mixture was stirred at room temperature overnight. The thin suspension was diluted with water and extracted with dichloromethane. The organic phase was washed with 0.5 M HCl, water, dried over NaSO, and concentrated. The crude product was purified by column chromatography to give 2.8 g (11% yield) of pure compound 25a and 7.8 g of slightly impure material (containing the diamide as a by-product), which was set aside. 1 H NMR (300 MHz, d-chloroform) δ: 3.66 (s, 3H), 3.17 (s, 3H), 2.40-2.30 (m, 4H), 1.70-1.60 (m, 4H), 1.50-1.30 (m, 2H).

[0333] Synthesis of compound 13d To an ice-cold solution of ketone 10 in THF (anhydrous) was added neat diethyl phosphate anhydride (11a) dropwise. The reaction was stirred for 30 min, followed by the portionwise addition of NaH 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 NaSO, and concentrated to give 7.5 g of crude material 13d, which was used directly in the next step.

[0334] Step 1: Synthesis of compound 15d Compound 13d and 7-(methoxy(methyl)amino)-7-oxoheptanoic acid (25a) were dissolved in DCM, and then DMAP and EDCI were added to the solution at room temperature. After stirring overnight, the reaction mixture was diluted with NH4Cl (sat. aq.) and extracted with DCM. The combined organic phase was washed with water, brine, dried over Na2SO4, and concentrated. Column chromatography purification (hexane-EtOAc) gave the title compound 15d.

[0335] Step 2: Synthesis of compound 16d Compound 15d was coevaporated with toluene several times and dried overnight over phosphorus pentoxide (PO) before reaction. The dried compound 15d was dissolved in THF in a flame-dried round-bottom flask, cooled to 0 °C, and heptylmagnesium bromide in ether was added dropwise. The reaction mixture was stirred at room temperature for 3.5 h, then cooled to 0 °C, quenched with NH4Cl (sat.), and extracted several times with hexane. The organic phase was dried over Na2SO4, concentrated, and purified by column chromatography (0–10% EtOAc in hexane) to give the title compound 16d.

[0336] Step 3: Synthesis of compound 17d To ice-cold compound 16d dissolved in THF:MeOH = 1:1 was added NaBH4 in one portion. After 5 min, the ice bath was removed and the reaction mixture was stirred at room temperature overnight. After confirming complete conversion by thin layer chromatography, the reaction mixture was quenched with NH4Cl (sat.) and concentrated to dryness. The residue was mixed with CHCl2, washed with water, dried over Na2SO4, concentrated, and purified by column chromatography (hexane-EtOAc) to give compound 17d in quantitative yield.

[0337] Step 4: Synthesis of compound 18d 4-(Dimethylamino)butanoic acid hydrochloride was dissolved in a CHCl / DMF mixture, followed by the addition of EtN, compound 17d, EDCI, and DMAP. The reaction mixture was stirred overnight at room temperature, quenched with NHCl (sat.), and extracted with CHCl. ​​The organic phase was dried over NaSO, concentrated, and purified by column chromatography (0-15% MeOH in CHCl) to give compound 18d, which is a lipid of formula I, where R' is absent and R 1 and R 2 methyl, n is 3, and R 3 is a C5 alkylene, and R 4 is a C7 alkyl, and R 5 is a C2 alkylene, and X1 and X 2 are each independently -C(=O)O-, and R6a and R 6b are the same or different and are as defined in formula I).

[0338] Alternative synthesis (E) At least lipid 15, lipid 9, and lipid 10, and any lipid of formula I (wherein R' is absent and R 1 and R 2 methyl, n is 3, and R 3 is a C6 alkylene, and R 4 is a C7 alkyl, and R 5 is a C1 alkylene, and X1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b are the same and are as defined in formula I, and R 6 (equivalent to) have been prepared or can be prepared using synthetic methods similar to those shown in Scheme 9, provided below. Minor modifications can be applied to the general synthesis shown in Scheme 9 to generate other lipids of Formula I, such as, but not limited to, replacing compound 2e with compound 2a, replacing heptylmagnesium bromide with compound 5, and / or replacing 4-(dimethylamino)butanoic acid with compound 8a.

[0339] [ka]

[0340] Synthesis of 8-(methoxy(methyl)amino)-8-oxooctanoic acid (25b) Suberic acid 2e (15.06 g, 86.45 mmol) was dissolved in dichloromethane / DMF (60 mL / 15 mL), followed by the addition of TEA (18.1 mL, 129.8 mmol) and N,O-dimethylhydroxylamine hydrochloride (4.22 g, 43.26 mol). The reaction mixture was cooled to 0 °C, and EDCI (10.36 g, 54.07 mol) was added, followed by DMAP (2.64 g, 21.63 mol). The ice bath was removed, and the reaction mixture was stirred overnight at room temperature. The thin suspension was diluted with water and extracted with dichloromethane. The organic phase was washed with 0.5 M HCl, water, dried over NaSO, and concentrated. The crude product was purified by column chromatography to give 2.4 g (26% yield) of pure compound 25b. 1 H NMR (300 MHz, d-chloroform) δ: 3.67 (s, 3H), 3.17 (s, 3H), 2.42-2.30 (m, 4H), 1.70-1.60 (m, 4H), 1.40-1.30 (m, 4H).

[0341] Step 1: Synthesis of Compound 1 To an ice-cold 0.5 M solution of alkylmagnesium bromide (22) in THF was added ethyl formate in THF. After stirring overnight at room temperature, the reaction mixture was diluted with approximately 60 mL of NH4Cl (saturated aqueous solution) and extracted with either ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by recrystallization from dichloromethane-hexane to give 2.82 g (82% yield) of pure compound 1.

[0342] Step 2: Synthesis of compound 10 Alcohol 1 was mixed with 18 mL of dichloromethane, cooled to 0° C., and Dess-Martin periodinane was added in one portion. The reaction mixture was stirred at room temperature overnight, then cooled to 0° C. and quenched with a 1:1 mixture (25:25 mL) of NaHCO (saturated) and Na-SO (15% aqueous solution) and stirred at room temperature for 20 min. The layers were separated, and the organic layer was washed with water, brine, dried over NaSO, and concentrated to give crude compound 10, which was used in the next step without purification.

[0343] Step 3: Synthesis of compound 31 To a suspension containing ketone 10 and (methoxymethyl)triphenylphosphonium chloride in THF was added a 1 M solution of KOtBu in THF dropwise over 15 min. The reaction mixture was stirred overnight at room temperature, diluted with EtO, washed with water, brine, dried over NaSO, and concentrated. The crude material was purified by column chromatography (0–2% EtOAc in hexanes) to give compound 31.

[0344] Step 4: Synthesis of Compound 32 To an ice-cooled cloudy solution of ketal 31 in dioxane / water, 4N HCl was added dropwise over 30 min. The reaction mixture was stirred at room temperature for 48 h. After confirming complete conversion by thin-layer chromatography, the reaction mixture was diluted with ether, cooled to 0 °C, and quenched by the slow addition of NaHCO (sat.) and 10% NaCO. The layers were separated, and the organic layer was washed with brine, dried over NaSO, and concentrated. The crude material was purified by column chromatography (0–5% EtOAc in hexanes) to give compound 32 in quantitative yield.

[0345] Step 5: Synthesis of compound 13e To ice-cold compound 32 dissolved in THF:MeOH=1:1 was added NaBH in one portion. The reaction mixture was stirred at room temperature overnight, quenched with NH4Cl (sat.) at 0 °C, and concentrated to dryness. The residue was mixed with CHCl, washed with water, dried over Na2SO4, concentrated, and purified by column chromatography (hexane-EtOAc) to give compound 13e in quantitative yield.

[0346] Step 6: Synthesis of compound 15e Compounds 25b and 13e were dissolved in dichloromethane, and then DMAP and EDCI were added to the solution at room temperature. After stirring overnight, the reaction mixture was diluted with NH4Cl (saturated aqueous solution) and extracted with dichloromethane. The combined organic phase was washed with water, brine, dried over Na2SO4, and concentrated. Column chromatography purification (hexane-EtOAc) gave pure compound 15e.

[0347] Step 7: Synthesis of compound 16e Compound 15e was coevaporated with toluene several times and dried overnight over PO before reaction. The dried compound 15e was dissolved in THF in a flame-dried round-bottom flask, cooled to 0 °C, and heptylmagnesium bromide (1 M in ether) was added dropwise. The reaction mixture was stirred at room temperature for 3.5 h, then cooled to 0 °C, quenched with NH Cl (sat.), and extracted several times with hexane. The organic phase was dried over Na SO , concentrated, and purified by column chromatography (0–10% EtOAc in hexane) to give compound 16e.

[0348] Step 8: Synthesis of compound 17e To ice-cold compound 16e dissolved in THF:MeOH=1:1 was added NaBH4 in one portion. The reaction mixture was stirred at room temperature for approximately 2 hours until complete conversion was confirmed by thin layer chromatography, then quenched with 2 mL of NH4Cl (sat.) and concentrated to dryness. The residue was mixed with CHCl2, washed with water, dried over Na2SO4, concentrated, and purified by column chromatography (hexane-EtOAc) to give compound 17e.

[0349] Step 9: Synthesis of Compound 18e 4-(Dimethylamino)butanoic acid hydrochloride was dissolved in a CHCl / DMF mixture, followed by the addition of TEA, compound 17e, EDCI, and DMAP. The reaction mixture was stirred overnight at room temperature, quenched with NHCl (sat.), and extracted with CHCl. ​​The organic phase was dried over NaSO, concentrated, and purified by column chromatography (0-15% MeOH in DCM) to give compound 18e, which is a lipid of formula I, where R' is absent and R 1 and R 2 methyl, n is 3, and R 3 is a C6 alkylene, and R 4 is a C7 alkyl, and R 5 is a C1 alkylene, and X1 and X 2 are each independently -C(=O)O-, and R 6a and R 6b are the same and are as defined in formula I, and R 6 is equal to).

[0350] Example 3: Synthesis of lipid 20, lipid 21, lipid 19, lipid 22, and lipid 11 Procedures for synthesizing lipid 20, lipid 21, lipid 19, lipid 22, and lipid 11 are described below with reference to Scheme 10 provided below.

[0351] [ka]

[0352] Step 1: Synthesis of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (3b) To a stirred solution of nonanedioic acid (2b, also known as azelaic acid) (7.34 g, 39 mmol) and heptadecan-9-ol (1a) (5 g, 19 mmol) in DCM (1000 mL) was added DMAP (2.37 g, 19 mmol), followed by EDCI (3 g, 19 mmol). The resulting mixture was stirred overnight at room temperature and then washed with 250 mL of 1 N HCl and 250 mL of water. The organic layer was dried over MgSO4, evaporated to dryness, and purified by silica gel column chromatography using 0–10% methanol in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 3b (6.2 g, 75%) as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.80-4.90 (m, 1H), 2.25-2.34 (m, 4H), 1.55-1.70 (m, 4H), 1.40-1.50 (m, 4H), 1.20-1.40 (m, 30H), 0.84-0.90 (t, 3H).

[0353] Step 2: Synthesis of heptadecan-9-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4b) To a solution of compound 3 (5.4 g, 12.7 mmol) in DCM (60 mL), EDCI (3.6 g, 19.7 mmol) and TEA (3.5 mL, 25.4 mmol) were added, and the mixture was stirred at room temperature for 15 min. N,O-dimethylhydroxylamine hydrochloride (1.36 g, 13.97 mmol) and DMAP (0.15 g, 1.27 mmol) were then added, and the mixture was stirred at room temperature overnight. The next day, the reaction was quenched with NH4Cl (aq) and diluted with DCM. The organic layer was washed with NH4Cl and brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum. Product 4b was used in the next step without further purification. 1H NMR(300MHz,d-chloroform)δ 4.85(t,J=6.2Hz,1H),3.67(s,3H),3.58(s,2H),3.17(s,3H),2.40(t,J=7.6Hz,2H),2.27(t,J=7.5Hz ,2H),1.63(dd,J=14.8,5.5Hz,6H),1.49(d,J=5.4Hz,4H),1.37-1.19(m,32H),0.86(d,J=6.8Hz,6H).

[0354] Step 3: Heptadecan-9-yl 9-oxohexadecanoate (R 4 is C7 alkyl 6b), heptadecan-9-yl 9-oxoheptadecanoate (R 4 is C8 alkyl 6b), heptadecan-9-yl 9-oxooctadecanoate (R 4 is C9 alkyl 6b), heptadecan-9-yl 9-oxononadecanoate (R 4 C 10 6b), or heptadecan-9-yl 9-oxoicosanoate (R 4 C 11 Synthesis of alkyl 6b) Heptadecan-9-yl 9-oxohexadecanoate (R 4 6b) when is C7 alkyl Compound 4b (1.0 g, 2.13 mmol) was dissolved in 10 mL of anhydrous THF. Then, 1 M heptylmagnesium bromide solution (R 4 A solution of compound 5a) in EtO (3.2 mL, 3.2 mmol) where is C7 alkyl was added dropwise at 0 °C. The mixture was stirred at room temperature under N for 16 h. The reaction was quenched with saturated aqueous NH4Cl and extracted with ether. The organic phase was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% EtOAc in hexane as the eluent to give R 4 In the case where is a C7 alkyl, 6b was obtained (0.3 g, 30%). 1H NMR (300 MHz, d-chloroform) δ 4.85 (t, J = 6.2 Hz, 1H), 2.37 (t, J = 7.4 Hz, 4H), 2.27 (t, J = 7.5 Hz, 2H), 1.64-1.43 (m, 12H), 1.27 (s, 36), 0.87 (t, J = 6.7 Hz, 9H).

[0355] Heptadecan-9-yl 9-oxoheptadecanoate (R 4 6b) when is C8 alkyl Compound 4b (1.0 g, 2.13 mmol) was dissolved in 10 mL of anhydrous THF. Then, 1 M octylmagnesium bromide solution (R 4 A solution of compound 5) in EtO (1.6 mL, 3.2 mmol) where is C8 alkyl was added dropwise at 0 °C. The mixture was stirred at room temperature under N2 for 16 h. The reaction was quenched with saturated aqueous NH4Cl and extracted with ether. The organic phase was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% EtOAc in hexane as the eluent to give R 4 When is C8 alkyl, 6b was obtained (0.41 g, 40%). 1 H NMR (300 MHz, d-chloroform) δ 4.85 (t, J = 6.2 Hz, 1H), 2.37 (t, J = 7.4 Hz, 4H), 2.26 (t, J = 7.5 Hz, 2H), 1.65-1.38 (m, 8H), 1.33-1.18 (m, 42H), 0.87 (t, J = 6.5 Hz, 9H).

[0356] Heptadecan-9-yl 9-oxooctadecanoate (R 4 6b) when is C9 alkyl Compound 4b (1.1 g, 2.3 mmol) was dissolved in 20 mL of anhydrous THF. Then, 1 M nonylmagnesium bromide solution (R 4A solution of compound 5 (wherein is C9 alkyl) in Et2O (6.13 mL, 3.2 mmol) was added dropwise at 0 °C. The resulting mixture was allowed to reach room temperature over 2 h. The reaction was quenched with saturated NH4Cl solution and extracted with ether. The organic phase was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-30% EtOAc in hexane as the eluent to give R 4 When is C9 alkyl, 6b was obtained (1.2 g, 96%). 1 H NMR (300 MHz, d-chloroform) δ 4.85 (t, J = 6.2 Hz, 1H), 2.37 (t, J = 7.4 Hz, 4H), 2.26 (t, J = 7.5 Hz, 2H), 1.65-1.38 (m, 8H), 1.33-1.18 (m, 44H), 0.87 (t, J = 6.5 Hz, 9H).

[0357] Heptadecan-9-yl 9-oxononadecanoate (R 4 C 10 6b) when alkyl Compound 4b (0.3 g, 0.64 mmol) was dissolved in 2 mL of anhydrous THF. Then, 1 M decylmagnesium bromide solution (R 4 C 10 A solution of compound 5 (in the alkyl form) in EtO (1.28 mL, 0.77 mmol) was added dropwise at 0°C. The resulting mixture was allowed to reach room temperature over 2 hours. The reaction was quenched with saturated NH4Cl solution and extracted with hexane. The organic phase was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% EtOAc in hexane as the eluent to give R 4 C 10 The alkyl form of 6b was obtained (0.2 g, 47%).

[0358] Heptadecan-9-yl 9-oxoicosanoate (R 4 C 11 6b) when alkyl To a solution of 1-bromoundecane (0.47 g, 2 mmol) in 2 mL of anhydrous ether, Mg (0.072 g, 3 mmol) and one drop of 1,2-dibromoethane were added. The resulting mixture was stirred for 1 hour, filtered, and dried. The product, undecylmagnesium bromide (R 4 C 11 Compound 5) when alkyl was used in the next step without further purification.

[0359] Compound 4b (0.47 g, 1 mmol) was dissolved in 3 mL of anhydrous THF. Then, a solution of undecylmagnesium bromide (1.1 mL, 1 mmol) in THF was added dropwise at 0 °C. The resulting mixture was allowed to reach room temperature over 2 h. The reaction was quenched with saturated NH4Cl solution and extracted with hexane. The organic phase was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0-10% EtOAc in hexane as the eluent to give the compound (R4 = C). 11 Compound 5) was obtained in the alkyl case (0.27 g, 48%). 1 H NMR (300 MHz, d-chloroform) δ 4.86 (t, J = 6.2 Hz, 1H), 2.37 (t, J = 7.4 Hz, 4H), 2.27 (t, J = 7.5 Hz, 2H), 1.70-1.45 (m, 8H), 1.29-1.25 (m, 48H), 0.87 (t, J = 6.6 Hz, 9H).

[0360] Step 4: Heptadecan-9-yl 9-oxohexadecanoate (R 4 7b) when R is C7 alkyl, heptadecan-9-yl 9-oxoheptadecanoate (R 4 7b) when R is C8 alkyl, heptadecan-9-yl 9-oxooctadecanoate (R 4 7b) where R is C9 alkyl, heptadecan-9-yl 9-oxononadecanoate (R 4 C 10 7b) when alkyl, or heptadecan-9-yl 9-oxoicosanoate (R 4 C 11 Synthesis of 7b) when alkyl Heptadecan-9-yl 9-hydroxyhexadecanoate (R 4 7b) when is C7 alkyl Heptadecan-9-yl 9-oxohexadecanoate (R) in 10 mL of anhydrous THF 4 To a solution of 6b) (0.3 g, 0.6 mmol) where R is a C7 alkyl, NaBH4 (0.09 g, 2.4 mmol) was added at 0 °C and stirred overnight under N2 atmosphere. The reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% EtOAc in hexane as eluent to give R 4 In the case where is a C7 alkyl, 7b was obtained (0.25 g, 82%). 1 H NMR (300 MHz, d-chloroform) δ 4.92-4.78 (m, 1H), 3.57 (m, 1H), 2.27 (t, J = 7.5 Hz, 2H), 1.66-1.36 (m, 12H), 1.31-1.25 (m, 40H), 0.87 (t, J = 6.1 Hz, 9H).

[0361] Heptadecan-9-yl 9-hydroxyheptadecanoate (R 4 7b) when is C8 alkyl Heptadecan-9-yl 9-oxoheptadecanoate (R 4 To a solution of 6b) (0.4 g, 0.77 mmol) where R is a C8 alkyl, NaBH4 (0.04 g, 1.15 mmol) was added at 0 °C and stirred overnight under N2 atmosphere. The reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 0-10% EtOAc in hexane as eluent to give R 4 When is C8 alkyl, 7b was obtained (0.21 g, 52%). 1H NMR (300 MHz, d-chloroform) δ 4.92-4.80 (m, 1H), 3.57 (m, 1H), 2.27 (t, J = 7.5 Hz, 2H), 1.64-1.40 (m, 12H), 1.36-1.18 (m, 42H), 0.87 (t, J = 6.5 Hz, 9H).

[0362] Heptadecan-9-yl 9-hydroxyoctadecanoate (R 4 7b) when is C9 alkyl Heptadecan-9-yl 9-oxooctadecanoate (R) in 40 mL of a DCM:MeOH (1:1) mixture 4 To a solution of 6b) (1.1 g, 2.05 mmol) where is C9 alkyl, NaBH4 (0.3 g, 8 mmol) was added at 0 °C and stirred under N2 atmosphere for 2 h. The reaction mixture was quenched with 1 M HCl solution (aq) and extracted with DCM. The organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using 5-40% EtOAc in hexane as eluent to give R 4 When is C9 alkyl, 7b was obtained (0.9 g, 83%). 1 H NMR (300 MHz, d-chloroform) δ 4.88-4.83 (m, 1H), 3.57 (m, 1H), 2.27 (t, J = 7.5 Hz, 2H), 1.61 (t, J = 7.5 Hz, 2H), 1.48-1.41 (m, 8H), 1.36-1.18 (m, 44H), 0.87 (t, J = 6.5 Hz, 9H).

[0363] Heptadecan-9-yl 9-hydroxynonadecanoate (R 4 C 10 7b) when alkyl Heptadecan-9-yl 9-oxononadecanoate (R) in 3 mL of a THF:DCM:MeOH (1:1:1) mixture 4 C 10To a solution of 6b) (0.2 g, 0.36 mmol) in alkyl form was added NaBH (0.03 g, 0.8 mmol) at 0 °C and stirred under N atmosphere for 3 h. The reaction was quenched with 0.5 mL of HO and extracted with DCM. The organic layer was washed with brine and dried over anhydrous MgSO. The solvent was evaporated under vacuum and purified by column chromatography using 5-40% EtOAc in hexane as eluent to give R 4 C 10 The alkyl form of 7b was obtained (0.16 g, 80%). 1 H NMR (300 MHz, d-chloroform) δ 4.86 (t, J = 6.2 Hz, 1H), 3.58 (m, 1H), 2.27 (t, J = 7.5 Hz, 2H), 1.61-1.37 (m, 12H), 1.32-1.18 (m, 46H), 0.87 (t, J = 6.6 Hz, 9H).

[0364] Heptadecan-9-yl 9-hydroxyicosanoate (R 4 C 11 7b) when alkyl Heptadecan-9-yl 9-oxoicosanoate (R) in 3 mL of a THF:DCM:MeOH (1:1:1) mixture 4 C 11 To a solution of 6b) (0.27 g, 0.48 mmol) in alkyl form was added NaBH4 (0.05 g, 1.35 mmol) at 0 °C and stirred under N2 atmosphere for 3 h. The reaction was quenched with 0.5 mL of HO and extracted with DCM. The organic layer was washed with brine and dried over anhydrous MgSO4. The solvent was evaporated under vacuum and purified by column chromatography using 5-40% EtOAc in hexane as eluent to give R 4 C 11 The alkyl form of 7b was obtained (0.25 g, 92%). 1 H NMR (301 MHz, d-chloroform) δ 4.86 (t, J = 6.2 Hz, 1H), 3.57 (s, 1H), 2.27 (t, J = 7.5 Hz, 2H), 1.69-1.37 (m, 12H), 1.29-1.17 (m, 48H), 0.87 (t, J = 6.5 Hz, 9H).

[0365] Step 5: Synthesis of heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate (lipid 11), heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)heptadecanoate (lipid 19), heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)octadecenoate (lipid 20), heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)nonadecanoate (lipid 21), or heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)icosanoate (lipid 22) Heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate (Lipid 11) Heptadecan-9-yl 9-hydroxyhexadecanoate (R 4 To a solution of 7b) (0.25 g, 0.49 mmol) where is C7 alkyl) and 4-(dimethylamino)butanoic acid (0.125 g, 0.75 mmol), 0.27 mL of DIPEA was added. EDCI (0.143 g, 0.75 mmol) and DMAP (0.012 g, 0.1 mmol) were then added, and the mixture was stirred overnight at room temperature under a N2 atmosphere. The next day, the reaction mixture was diluted with DCM. The organic layer was washed with NaHCO3 (aq) and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0–5% MeOH in DCM as the eluent to give lipid 11 (0.14 g, 45%). 1 H NMR (300 MHz, d-chloroform) δ 4.93-4.77 (m, 2H), 2.37-2.23 (m, 5H), 2.21 (s, 6H), 1.83-1.73 (m, 2H), 1.70-1.40 (m, 10H), 1.25 (s, 43H), 0.87 (t, J = 6.6 Hz, 9H). [C 39 H 77 NO4] MS found 624.5 [M+H] + , calculated value 623.59.

[0366] Heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)heptadecanoate (Lipid 19) Heptadecan-9-yl 9-hydroxyheptadecanoate (R 4 To a solution of 7b) (0.21 g, 0.4 mmol) and 4-(dimethylamino)butanoic acid (0.08 g, 0.45 mmol), where is C8 alkyl, was added 0.16 mL of DIPEA. EDCI (0.09 g, 0.45 mmol) and DMAP (0.008 g, 0.06 mmol) were then added, and the mixture was stirred overnight at room temperature under a N2 atmosphere. The next day, the reaction mixture was diluted with DCM. The organic layer was washed with NaHCO3 (aq) and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0–5% MeOH in DCM as the eluent to give lipid 19 (0.112 g, 44%). 1 H NMR(300MHz,d-chloroform)δ 4.86(m,2H),2.34-2.24(m,5H),2.21(s,6H),1.78(p,J=7.6Hz,2H),1.6 8-1.56 (m, 2H), 1.54-1.40 (m, 8H), 1.25 (s, 45H), 0.87 (t, J=6.7Hz, 9H). [C 40 H 79 NO4] MS found 638.5 [M+H] + , calculated value 637.60.

[0367] Heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)octadecenoate (Lipid 20) Heptadecan-9-yl 9-hydroxyoctadecanoate (R 4 To a solution of 7b) (0.3 g, 0.56 mmol) where is a C alkyl, EDCI (0.21 g, 1.12 mmol) and DMAP (0.07 g, 0.56 mmol) were added and stirred under a N atmosphere for 15 minutes. 4-(Dimethylamino)butanoic acid (0.25 g, 1.5 mmol) was added to the reaction mixture and stirred overnight. The next day, the solvent was evaporated and redissolved in EtOAc (300 mL). The organic layer was washed with HO (300 mL), NaHCO (aq) (200 mL), and brine (200 mL) and dried over anhydrous NaSO. The organic layer was evaporated under vacuum and purified by column chromatography using 5-40% EtOAc in hexane as the eluent to give lipid 20 (0.124 g, 34%). 1 H NMR(300MHz,d-chloroform)δ 4.86(m,2H),2.38-2.23(m,6H),2.21(s,6H),1.85-1.71(m,2H),1.67-1.55(m,2H),1.50-1.44(m,8H),1.24(s,46H),0.86(t,J=6.5Hz,9H). [C 41 H 81 MS found for NO4: 652.7 [M+H] + , calculated value 651.62.

[0368] Heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)nonadecanoate (Lipid 21) Heptadecan-9-yl 9-hydroxynonadecanoate (R 4 C 10 To a solution of alkyl 7b) (0.16 g, 0.29 mmol), EDCI (0.052 g, 0.27 mmol) and DMAP (0.04 g, 0.033 mmol) were added and stirred under a N atmosphere for 15 minutes. 4-(Dimethylamino)butanoic acid (0.056 g, 0.33 mmol) was added to the reaction mixture and stirred overnight. The next day, the reaction was diluted with DCM. The organic layer was washed with NaHCO3 (aq) and dried over anhydrous MgSO4. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0-5% MeOH in DCM as the eluent to give lipid 21 (0.07 g, 36%). 1H NMR (300 MHz, d-chloroform) δ 4.93-4.81 (m, 2H), 2.34-2.24 (m, 5H), 2.22 (s, 6H), 1.85-1.67 (m, 4H), 1.63-1.57 (m, 2H), 1.48 (s, 7H), 1.24 (s, 47H), 0.87 (t, J = 6.6 Hz, 9H). [C 42 H 83 MS found for NO4: 665.63 [M+H] + , calculated value 666.5.

[0369] Heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)icosanoate (Lipid 22) Compound heptadecan-9-yl 9-hydroxyicosanoate (R 4 C 11 To a solution of 7b) (0.25 g, 0.44 mmol), EDCI (0.068 g, 0.36 mmol) and DMAP (0.054 g, 0.044 mmol) were added and stirred under a N atmosphere for 15 min. 4-(dimethylamino)butanoic acid (0.074 g, 0.44 mmol) was then added to the reaction mixture and stirred overnight. The next day, the reaction was diluted with DCM. The organic layer was washed with NaHCO (aq) and dried over anhydrous MgSO. The solvent was evaporated under vacuum, and the product was purified by column chromatography using 0-5% MeOH in DCM as the eluent to give lipid 22 (0.134 g, 45%). 1 H NMR(300MHz,d-chloroform)δ 4.87-4.81(m,2H),2.34-2.24(m,5H),2.23(d,J=7.2Hz,6H),1.87-1.76(m,2H),1.7 4-1.70(m,2H),1.65-1.57(m,2H),1.48(s,7H),1.24(s,50H),0.87(t,J=6.6Hz,9H). [C 43 H 85 NO4] MS found 680.6 [M+H] + , calculated value 679.65.

[0370] Example 4: Synthesis of lipid 23 The procedure for synthesizing lipid 23 is described below with reference to Scheme 11, which is also provided.

[0371] [ka]

[0372] Step 1 and Step 2: Synthesis of 3-octylundecan-1-ol (13a) To an ice-cold solution of 3 g (11.8 mmol) of heptadecan-9-one (10a) in 120 mL of THF was added 18 mL of (ethylcarboxylic)(diethylphosphoric)anhydride (11a). The reaction was stirred for 30 minutes, and then 3.2 g (80 mmol) of NaH was added. The reaction mixture was refluxed for 18 hours. The crude product was quenched with water and extracted with ether to give 2. One gram of ethyl 3-octylundec-2-enoate (12a) in 5 mL of THF was reacted with 1 mL of LiAlH4 (2 M solution). After 48 hours, the crude product was quenched with water and extracted with ether to give 0.6 g of 3-octylundecan-1-ol (13). 1 H-NMR (300 MHz, d-chloroform): δ 0.86 (t, 6H), 1.20-1.40 (m, 27H), 1.50-1.60 (m, 2H), 1.81-1.87 (m, 1H), 1.88-2.00 (m, 1H), 3.50-3.68 (m, 2H), 3.70-3.77 (m, 1H).

[0373] Step 3: Synthesis of 7-((3-octylundecyl)oxy)-7-oxoheptanoic acid (14b) 0.6 g (2.1 mmol) of 3-octylundecan-1-ol (13) was dissolved in 30 mL of methylene chloride, followed by 0.9 g (4.3 mmol) of pimelic acid. To this mixture, 0.87 g (4.3 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) was added, followed by 0.69 g (4.3 mmol) of 4-dimethylaminopyridine (DMAP). The reaction was stirred overnight. The reaction was quenched with 1 N aqueous HCl and purified on a silica gel column to give 0.45 g (50% yield) of 7-((3-octylundecyl)oxy)-7-oxoheptanoic acid (14b). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 6H), 1.20-1.55 (m, 27H), 1.56-1.75 (m, 5H), 2.30 (dd, 2H), 2.36 (dd, 2H), 4.07 (dd, 2H).

[0374] Step 4: Synthesis of 3-octylundecyl 7-(methoxy(methyl)amino)-7-oxoheptanoate (15b) To a solution of 0.45 g (1.1 mmol) of 7-((3-octylundecyl)oxy)-7-oxoheptanoic acid (14) in 10 mL of dichloromethane was added 0.25 g (1.6 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), followed by 0.16 g (1.6 mmol) of N,O-dimethylhydroxylamine hydrochloride, 0.16 g (1.6 mmol) of trimethylamine, and finally 0.19 g (1.6 mmol) of 4-dimethylaminopyridine (DMAP). The reaction mixture was stirred overnight and then quenched with 1 N HCl solution. The organic phase was dried over magnesium sulfate to give 0.45 g (90% yield) of 3-octylundecyl 7-(methoxy(methyl)amino)-7-oxoheptanoate (15b). The crude product was used in the next step without further purification. 1H-NMR (300MHz, d-chloroform): δ 0.87(t,6H),1.20-1.55(m,32H),1.56-1.80(m,4H),2.30(dd,2H),2.36(dd,2H),3.17(s,3H),3.67(s,3H),4.07(dd,2H). C 28 H 55 MS observed value for NO4: 470.3 [M+H] + , calculated value 469.4.

[0375] Step 5: Synthesis of 3-octylundecyl 7-oxohexadecanoate (16b) 1 mL of nonylmagnesium bromide (1 M in ether) was added dropwise to an ice-cold solution of 0.45 g (0.9 mmol) of 3-octylundecyl 7-(methoxy(methyl)amino)-7-oxoheptanoate (15b) in 3 mL of anhydrous THF. After stirring at 0 °C for 1 h, the reaction mixture was warmed to room temperature and stirred for 2 h. The reaction was cooled again and quenched with aqueous ammonium chloride solution. The crude product was extracted with hexane, dried over magnesium sulfate, and purified on silica gel (0–10% ethyl acetate / hexane) to give 0.3 g (58% yield) of 3-octylundecyl 7-oxohexadecanoate (16b). 1 H-NMR (300MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.40 (m, 38H), 1.45-1.70 (m, 7H), 2.27 (t, 2H), 2.32-2.39 (q, 4H), 4.07 (dd, 2H).

[0376] Step 6: Synthesis of 3-octylundecyl 7-hydroxyhexadecanoate (17b) To an ice-cold solution of 0.3 g (0.56 mmol) of 3-octylundecyl 7-oxohexadecanoate (16b) in 3 mL of a 1 / 1 / 1 mixture of THF / MeOH / methylene chloride was added 50 mg (1.3 mmol) of sodium borohydride. The reaction was warmed to room temperature and stirred for 3 h before being quenched with water and evaporated. The crude product was redissolved in methylene chloride and washed with water. The organic phase was dried over magnesium sulfate, and 220 mg of the crude product was used in the next step without further purification. 1 H-NMR (300 MHz, d-chloroform): δ 0.88 (t, 9H), 1.10-1.45 (m, 46H), 1.50-1.70 (m, 2H), 2.31 (t, 2H), 3.65 (broad s, 1H), 4.07 (dd, 2H).

[0377] Step 7: Synthesis of 3-octylundecyl 7-((4-(dimethylamino)butanoyl)oxy)hexadecanoate (lipid 23) To a solution of 0.22 g (0.4 mmol) of 3-octylundecyl 7-hydroxyhexadecanoate (17b) in 1 mL of methylene chloride, 0.068 g (0.43 mmol) of EDCI, 0.074 g (0.44 mmol) of 4-(dimethylamino)butanoic acid hydrochloride, and 0.054 g (0.44 mmol) of DMAP were added, along with 0.054 g (0.5 mmol) of triethylamine, for 16 h. The reaction mixture was evaporated. After flash chromatography using 0-5% methanol / methylene chloride, pure 3-octylundecyl 7-((4-(dimethylamino)butanoyl)oxy)hexadecanoate (lipid 23) was obtained in an amount of 0.16 g (60% yield). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.40 (m, 48H), 1.41-1.70 (m, 6H), 1.72-1.85 (dd, 2H), 2.21 (s, 6H), 2.22-2.40 (m, 6H), 4.07 (dd, 2H), 4.80-4.90 (m, 1H). 41 H 81 MS observed value for NO4: 652.5 [M+H] + , calculated value 651.6.

[0378] Example 5: Synthesis of lipid 16 The procedure for synthesizing lipid 16 is described below with reference to Scheme 12, also provided below.

[0379] [ka]

[0380] Step 1: Synthesis of 9-(henicosan-11-yloxy)-9-oxononanoic acid (3c) In a round-bottom flask, 1.5 g (4.8 mmol) of henicosan-11-ol (1b) was dissolved in 60 mL of methylene chloride, followed by 1.8 g (9.6 mmol) of azelaic acid (2b). To this mixture, 1.48 g (9.6 mmol) of EDCI was added, followed by 1.2 g (9.6 mmol) of DMAP. The reaction was stirred overnight. The reaction was quenched with 1 N HCl (100 mL). The organic phase was dried over magnesium sulfate and purified on a silica gel column (0-25% ethyl acetate-hexane) to give 1.1 g (47% yield) of 9-(henicosan-11-yloxy)-9-oxononanoic acid (3c). 1 H-NMR (300 MHz, d-chloroform): δ 0.86 (t, 6H), 1.20-1.80 (m, 62H), 2.20-2.40 (m, 4H), 4.82-4.94 (m, 1H). 30 H 58 MS found for O4: 481.4 [M+H] + , calculated value 482.4.

[0381] Step 2: Synthesis of henicosan-11-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4c) To a solution of 1.1 g (2.3 mmol) of 9-(henicosan-11-yloxy)-9-oxononanoic acid (3c) in 20 mL of DCM, 0.53 g (3.4 mmol) of EDCI was added, followed by 0.35 g (3.4 mmol) of N,O-dimethylhydroxylamine hydrochloride, and finally, 0.42 g (3.4 mmol) of 4-dimethylaminopyridine (DMAP). The reaction mixture was stirred for 16 h and then quenched with 1 N HCl solution (2 × 5 mL). The organic phase was dried over magnesium sulfate and evaporated to give 1 g (80% yield) of henicosan-11-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4c). 1 H-NMR (300MHz, d-chloroform):δ 0.87(t,6H),1.20-1.40(m,35H),1.41-1.50(m,4H),1.55-1.68(m,4H),2 .26(dd,2H),2.37(dd,2H),3.17(s,3H),3.67(s,3H),4.80-4.87(m,1H).

[0382] Step 3: Synthesis of henicosan-11-yl 9-oxooctadecanoate (6c) 2.3 mL of nonylmagnesium bromide (1 M in ether) was added dropwise to an ice-cooled solution of 1 g (1.9 mmol) of henicosan-11-yl 9-(methoxy(methyl)amino)-9-oxoheptanoate (4c) in 6 mL of anhydrous THF. After stirring at 0 °C for 1 h, the reaction mixture was warmed to room temperature and stirred for 2 h. The reaction was cooled again and quenched with aqueous ammonium chloride solution. The crude product was extracted with hexane, dried over magnesium sulfate, and purified on a silica gel column (0–10% ethyl acetate / hexane) to give 0.7 g (62% yield) of henicosan-11-yl 9-oxooctadecanoate (6c). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.40 (m, 45H), 1.45-1.61 (m, 11H), 2.26 (t, 2H), 2.37 (t, 4H), 4.83-4.87 (m, 1H).

[0383] Step 4: Synthesis of henicosan-11-yl 9-hydroxyoctadecanoate (7c) To an ice-cold solution of 0.7 g (1.2 mmol) of henicosan-11-yl 9-oxooctadecanoate (6c) in 2 mL of THF / 2 mL of MeOH / 2 mL of DCM was added 0.2 g (5.3 mmol) of sodium borohydride. The reaction was warmed to room temperature and stirred for 3 h, then quenched with water and evaporated to dryness. The crude product was redissolved in DCM and washed with water. After evaporation and column purification, 0.6 g (86% yield) of henicosan-11-yl 9-hydroxyoctadecanoate (7c) was obtained. 1 H-NMR (300 MHz, d-chloroform): δ 0.88 (t, 9H), 1.10-159 (m, 59H), 2.27 (t, 2H), 3.51-3.62 (m, 1H), 4.83-4.87 (m, 1H).

[0384] Step 5: Synthesis of henicosan-11-yl 9-((4-(dimethylamino)butanoyl)oxy)octadecanoate (lipid 16) To a solution of 0.24 g (0.41 mmol) of henicosan-11-yl 9-hydroxyoctadecanoate (7c) in 1 mL of methylene chloride, 0.07 g (0.44 mmol) of EDCI, 0.074 g (0.44 mmol) of 4-(dimethylamino)butanoic acid hydrochloride, 0.054 g (0.44 mmol) of DMAP, and 0.054 g (0.53 mmol) of triethylamine were added. After 16 h, the reaction mixture was evaporated and purified by flash chromatography using 0-5% methanol / methylene chloride to afford pure henicosan-11-yl 9-((4-(dimethylamino)butanoyl)oxy)octadecanoate (lipid 16) in an amount of 0.160 g (55% yield). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.35 (m, 55H), 1.40-1.54 (m, 7H), 1.55-1.70 (m, 2H), 1.77-1.85 (m, 2H), 2.21 (s, 6H), 2.22-2.40 (m, 6H), 4.82-4.87 (m, 2H). 45 H 89MS observed value for NO4: 708.6 [M+H] + , calculated value 707.7.

[0385] Example 6: Synthesis of lipid 18 The procedure for synthesizing lipid 18 is described below with reference to Scheme 13, also provided below.

[0386] [ka]

[0387] Step 1: Synthesis of 7-(henicosan-11-yloxy)-7-oxoheptanoic acid (3d) In a round-bottom flask under a nitrogen atmosphere, 2 g (6.3 mmol) of henicosan-11-ol (1b) was dissolved in 30 mL of anhydrous methylene chloride, followed by 2.1 g (12.7 mmol) of pimelic acid (2c). To this mixture, 1.4 g (7.3 mmol) of EDCI was added, followed by 1.6 g (13 mmol) of DMAP. The reaction was stirred for 48 h. The reaction was quenched with aqueous ammonium chloride (100 mL) and extracted with methylene chloride. The organic phase was washed with brine (120 mL), dried over magnesium sulfate, and purified on a silica gel column (0–20% ethyl acetate–hexane) to give 1.33 g (45% yield) of 7-(henicosan-11-yloxy)-7-oxoheptanoic acid (3d). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 6H), 1.18-1.42 (m, 32H), 1.42-1.55 (m, 3H), 1.60-1.69 (m, 4H), 2.29 (t, 2H), 2.36 (t, 2H) 4.84-4.88 (m, 1H). 28 H 54 MS found for O4: 455.3 [M+H] + , calculated value 454.4.

[0388] Step 2: Synthesis of henicosan-11-yl 7-(methoxy(methyl)amino)-7-oxoheptanoate (4d) To a solution of 1.33 g (2.9 mmol) of 7-(henicosan-11-yloxy)-7-oxoheptanoic acid (3d) in 10 mL of anhydrous DCM under a nitrogen atmosphere, 0.84 g (4.4 mmol) of EDCI was added, followed by 0.82 mL (5.85 mmol) of triethylamine, 0.34 g (3.5 mmol) of N,O-dimethylhydroxylamine hydrochloride, and finally 36 mg (0.29 mmol) of DMAP. The reaction mixture was stirred for 16 h, then quenched with aqueous ammonium chloride (100 mL) and extracted with methylene chloride (3 × 40 mL). The organic phase was washed with brine (100 mL), dried over magnesium sulfate, and purified on a silica gel column (0–10% ethyl acetate–hexane) to give 0.91 g (64% yield) of henicosan-11-yl 7-(methoxy(methyl)amino)-7-oxoheptanoate (4d). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 6H), 1.24-1.29 (m, 38H), 1.30-1.52 (m, 8H), 1.55-1.75 (m, 5H), 2.29 (dd, 2H), 2.42 (dd, 2H), 3.17 (s, 3H), 3.67 (s, 3H), 4.83-4.87 (m, 1H).

[0389] Step 3: Synthesis of henicosan-11-yl 7-oxohexadecanoate (6d) 2.2 mL of nonylmagnesium bromide (1 M in ether) was added dropwise to an ice-cooled solution of 0.86 g (0.17 mmol) of henicosan-11-yl 7-(methoxy(methyl)amino)-7-oxoheptanoate (4d) in 5 mL of anhydrous THF. After stirring at 0 °C for 1 h, the reaction mixture was warmed to room temperature and stirred for 4 h. The reaction was cooled again and quenched with aqueous ammonium chloride (5 mL). The crude product was extracted with hexane (2 × 10 mL) and methylene chloride (2 × 15 mL), dried over magnesium sulfate, and purified on a silica gel column (0–10% ethyl acetate / hexane) to give 0.640 g (65% yield) of henicosan-11-yl 7-oxooctadecanoate (6d). 1H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.35 (m, 44H), 1.40-1.60 (m, 12H), 2.28 (t, 2H), 2.31-2.38 (q, 4H), 4.83-4.87 (m, 1H). 37 H 72 MS observed for O3: 565.5 [M+H] + , calculated value 564.6.

[0390] Step 4: Synthesis of henicosan-11-yl 7-hydroxyhexadecanoate (7d) To an ice-cold solution of 0.64 g (1.1 mmol) of henicosan-11-yl 7-oxohexadecanoate (7d) in 4 mL of 50% THF / MeOH was added 64 mg (1.7 mmol) of sodium borohydride. The reaction was warmed to room temperature and stirred for 1 h. After that, it was quenched with aqueous ammonium chloride (1 mL) and the crude product was extracted with methylene chloride (10 mL) and ethyl acetate (5 mL). The organic phase was dried over magnesium sulfate and used in the next step without further purification. 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-154 (m, 63H), 1.54-1.55 (m, 2H), 2.28 (t, 2H), 3.50-3.60 (m, 1H), 4.83-4.88 (m, 1H). 37 H 74 MS observed for O3: 567.5 [M+H] + , calculated value 566.56.

[0391] Step 5: Synthesis of henicosan-11-yl 7-((4-(dimethylamino)butanoyl)oxy)hexadecanoate (lipid 18) To a solution of 0.57 g (1 mmol) of henicosan-11-yl 7-hydroxyhexadecanoate (7d) in 4 mL of anhydrous methylene chloride was added 0.29 g (1.5 mmol) of EDCI, 0.20 g (1.2 mmol) of 4-(dimethylamino)butanoic acid hydrochloride, and 0.186 g (1.5 mmol) of DMAP under a nitrogen atmosphere. After 5 min, 0.21 mL (1.5 mmol) of triethylamine was injected into the reaction mixture. After 16 h, the reaction was quenched with aqueous ammonium chloride (5 mL), extracted with methylene chloride (2 × 10 mL) and ethyl acetate (20 mL), and dried over magnesium sulfate. After flash chromatography column 0–5% methanol / methylene chloride, pure henicosan-11-yl 7-((4-(dimethylamino)butanoyl)oxy)hexadecanoate (lipid 18) was obtained in an amount of 0.296 g (28% yield). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.30 (m, 47H), 1.40-1.84 (m, 15H), 2.23 (s, 6H), 2.24-2.4 (m, 6H), 4.81-4.88 (m, 2H). 43 H 85 MS observed value for NO4: 680.6 [M+H] + , calculated value 679.7.

[0392] Example 7: Synthesis of lipid 17 The procedure for synthesizing lipid 17 is described below with reference to Scheme 14, also provided below.

[0393] [ka]

[0394] Step 1: Synthesis of 9-oxo-9-(pentacosan-13-yloxy)nonanoic acid (3e) In a round-bottom flask under a nitrogen atmosphere, 0.9 g (2.4 mmol) of pentacosan-13-ol (1c) was dissolved in 50 mL of anhydrous methylene chloride, followed by 0.9 g (4.3 mmol) of pimelic acid (2b). To this mixture, 0.46 g (2.4 mmol) of EDCI was added, followed by 0.6 g (4.6 mmol) of DMAP. The reaction was stirred overnight. The reaction was quenched with aqueous ammonium chloride (100 mL) and extracted with methylene chloride. The organic phase was washed with brine (120 mL), dried over magnesium sulfate, and purified on a silica gel column (0–20% ethyl acetate–hexane) to give 0.45 g (34% yield) of 9-oxo-9-(pentacosan-13-yloxy)nonanoic acid (3e). 1 H-NMR (300 MHz, d-chloroform): δ 0.86 (t, 6H), 1.20-1.80 (m, 70H), 2.20-2.40 (m, 4H), 4.82-4.94 (m, 1H). C 34 H 66 MS found for O4: 539.4 [M+H] + , calculated value 538.5.

[0395] Step 2: Synthesis of pentacosan-13-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4e) To a solution of 0.45 g (0.8 mmol) of 9-oxo-9-(pentacosan-13-yloxy)nonanoic acid (3d) in 30 mL of anhydrous DCM, 0.24 g (1.25 mmol) of EDCI was added, followed by 0.2 mL (1.7 mmol) of trimethylamine, 0.09 g (0.9 mmol) of N,O-dimethylhydroxylamine hydrochloride, and finally 10 mg (0.08 mmol) of DMAP. The reaction mixture was stirred for 16 h, then quenched with aqueous ammonium chloride (60 mL) and extracted with methylene chloride. The organic phase was washed with brine (100 mL), dried over magnesium sulfate, and purified on a silica gel column (0–10% ethyl acetate–hexane) to give 0.35 g (70% yield) of pentacosan-13-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4e). 1H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 6H), 1.20-1.30 (m, 52H), 1.48-1.62 (m, 8H), 2.27 (dd, 2H), 2.29 (dd, 2H), 3.17 (s, 3H), 3.67 (s, 3H), 4.83-4.87 (m, 1H). 36 H 71 MS observed value for NO4: 582.4 [M+H] + , calculated value 581.54.

[0396] Step 3: Synthesis of pentacosan-13-yl 9-oxooctadecanoate (6e) 0.9 mL of nonylmagnesium bromide (1 M in ether) was added dropwise to an ice-cooled solution of 0.35 g (0.6 mmol) of pentacosan-13-yl 9-(methoxy(methyl)amino)-9-oxoheptanoate (4e) in 3 mL of anhydrous THF. After stirring at 0 °C for 1 h, the reaction mixture was warmed to room temperature and stirred for 4 h. The reaction was cooled again and quenched with aqueous ammonium chloride (3 mL). The crude product was extracted with hexane (3 × 20 mL) and methylene chloride (1 × 10 mL), dried over magnesium sulfate, and purified on silica gel (0–10% ethyl acetate / hexane) to give 0.321 g (82% yield) of pentacosan-13-yl 9-oxooctadecanoate (6e). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.30 (m, 54H), 1.40-1.65 (m, 9H), 2.27 (t, 2H), 2.37 (t, 4H), 4.83-4.87 (m, 1H). 43 H 84 MS observed for O3: 649.5 [M+H] + , calculated value 648.64.

[0397] Step 4: Synthesis of pentacosan-13-yl 9-hydroxyoctadecanoate (7e) To an ice-cold solution of 0.3 g (0.46 mmol) of pentasan-13-yl 9-oxooctadecanoate (6e) in 2 mL of 50% THF / MeOH was added 26 mg (0.69 mmol) of sodium borohydride. The reaction was warmed to room temperature and stirred for 1 h, then quenched with aqueous ammonium chloride (1 mL) and extracted with methylene chloride (10 mL) and ethyl acetate (5 mL). The organic phase was dried over magnesium sulfate and used in the next step without further purification. 1 H-NMR (300 MHz, d-chloroform): δ 0.88 (t, 9H), 1.10-154 (m, 72H), 1.55-1.70 (m, 2H), 2.27 (t, 2H), 3.51-3.62 (m, 1H), 4.83-4.87 (m, 1H). 43 H 86 MS found for O3: 651.6 [M+H] + , calculated value 650.66.

[0398] Step 5: Synthesis of pentacosan-13-yl 9-((4-(dimethylamino)butanoyl)oxy)octadecanoate (lipid 17) To a solution of 0.29 g (0.45 mmol) of pentacosan-13-yl 9-hydroxyoctadecanoate (6e) in 3 mL of anhydrous methylene chloride was added 0.128 g (0.67 mmol) of EDCI, 0.09 g (0.53 mmol) of 4-(dimethylamino)butanoic acid hydrochloride, and 0.082 g (0.67 mmol) of DMAP under a nitrogen atmosphere. After 5 min, 0.09 mL (0.67 mmol) of triethylamine was injected into the reaction mixture. After 16 h, the reaction was quenched with aqueous ammonium chloride (3 mL) and extracted with methylene chloride (2 × 15 mL) and ethyl acetate (15 mL). The organic phase was dried over magnesium sulfate and concentrated. After flash chromatography column 0–5% methanol / methylene chloride, pure pentacosan-13-yl 9-((4-(dimethylamino)butanoyl)oxy)octadecanoate (lipid 17) was obtained in an amount of 0.135 g (34% yield). 1H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 9H), 1.10-1.35 (m, 64H), 1.40-1.76 (m, 11H), 1.77-1.85 (m, 2H), 2.23 (s, 6H), 2.24-2.32 (m, 6H), 4.82-4.87 (m, 2H). 49 H 97 MS for NO4 found 764.7 [M+H]+, calculated 763.74.

[0399] Example 8: Synthesis of lipid 24 The procedure for synthesizing lipid 24 is described below with reference to Scheme 15, also provided below.

[0400] [ka]

[0401] Step 1: Synthesis of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (3b) In a round-bottom flask under a nitrogen atmosphere, 10 g (39 mmol) of heptadecan-9-ol (1a) was dissolved in 100 mL of anhydrous methylene chloride, followed by 14.7 g (78 mmol) of azelaic acid (2b). To this mixture, 9.7 g (50.7 mmol) of EDCI was added, followed by 9.5 g (78 mmol) of DMAP. The reaction was stirred overnight. The reaction was quenched with aqueous ammonium chloride (100 mL) and extracted with methylene chloride (100 mL). The organic phase was washed with brine (120 mL), dried over magnesium sulfate, and purified on a silica gel column (0–20% ethyl acetate–hexane) to give 7 g (42% yield) of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (3b). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 6H), 1.20-1.33 (m, 30H), 1.40-1.50 (m, 4H), 1.50-1.64 (m, 4H), 2.27 (t, 2H), 2.32 (t, 2H), 4.82-4.88 (m, 1H). 26 H 50 MS found for O4: 427.3 [M+H]+ , calculated value 426.37.

[0402] Step 2: Synthesis of heptadecan-9-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4b) To a solution of 5 g (11.7 mmol) of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (3b) in 40 mL of anhydrous dichloromethane was added 3.37 g (17.6 mmol) of EDCI, followed by 3.3 mL (23.5 mmol) of trimethylamine, 1.48 g (15.2 mmol) of N,O-dimethylhydroxylamine hydrochloride, and finally 143 mg (1.17 mmol) of DMAP. The reaction mixture was stirred for 16 h, then quenched with aqueous ammonium chloride (100 mL) and extracted with methylene chloride (150 mL). The organic phase was washed with brine (100 mL), dried over magnesium sulfate, and purified on a silica gel column (0–20% ethyl acetate–hexane) to give 3.9 g (70% yield) of heptadecan-9-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4b). 1 H-NMR (300 MHz, d-chloroform): δ 0.87 (t, 6H), 1.10-1.40 (m, 29H), 1.41-1.57 (m, 10H), 2.27 (dd, 2H), 2.29 (dd, 2H), 3.17 (s, 3H), 3.67 (s, 3H), 4.83-4.88 (m, 1H). 28 H 55 MS observed value for NO4: 470.4 [M+H] + , calculated value 469.41.

[0403] Step 3: Synthesis of heptadecan-9-yl 9-oxooctadecanoate (6f) 14.9 mL of nonylmagnesium bromide (1 M in ether) was added dropwise to an ice-cooled solution of 5 g (10.6 mmol) of heptadecan-9-yl 9-(methoxy(methyl)amino)-9-oxononanoate (4b) in 24 mL of anhydrous THF. After stirring at 0 °C for 1 h, the reaction mixture was warmed to room temperature and stirred for 4 h. The reaction was cooled again and quenched with aqueous ammonium chloride (20 mL). The crude product was extracted with ethyl acetate (3 × 30 mL), w...

Claims

1. A lipid of formula I, 【Chemistry 85】 or a pharma- ceutically acceptable salt thereof, wherein: R' is absent, hydrogen or C １ ~C ６ alkyl, where R′ is hydrogen or C １ ~C ６ When R′, R １ , and R ２ The nitrogen atoms to which all are attached are protonated, R １ and R ２ are each independently hydrogen, C １ ~C ６ Alkyl, or C ２ ~C ６ alkenyl, R ３ But, C １ ~C １２ Alkylene or C ２ ~C １２ alkenylene, R ４ But, C １ ~C １６ Unbranched alkyl, C ２ ~C １６ unbranched alkenyl, or 【Chemistry 86】 where: R ４ａ and R ４ｂ However, each independently, C １ ~C １６ Unbranched alkyl or C ２ ~C １６ is an unbranched alkenyl; R ５ But it doesn't exist, or C １ ~C ８ Alkylene, or C ２ ~C ８ alkenylene, R ６ａ and R ６ｂ However, each independently, C ７ ~C １６ Alkyl or C ７ ~C １６ alkenyl, where R ６ａ and R ６ｂ the total number of carbon atoms combined is greater than 15, X １ and X ２ each independently represents -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)-, wherein R ａ is, for each occurrence, independently, hydrogen or C １ ~C ６ is alkyl, A lipid, or a pharma- ceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, 4, 5, and 6.

2. X １ and X ２ The lipid according to claim 1 , or a pharma- ceutically acceptable salt thereof, wherein:

3. X １ and X ２ each independently represents -OC(=O)-, -SC(=O)-, -OC(=S)-, The lipid according to claim 1, which is -C(=O)O-, -C(=O)S-, or -S-S-, or a pharma- ceutically acceptable salt thereof.

4. The lipid is represented by formula II: 【Chemistry 87】 2. The lipid of claim 1, wherein n is an integer selected from 1, 2, 3, and 4, or a pharma- ceutically acceptable salt thereof.

5. The lipid is represented by formula III: 【Chemistry 88】 2. The lipid of claim 1, wherein n is an integer selected from 1, 2, and 3, or a pharma- ceutically acceptable salt thereof.

6. The lipid is represented by formula IV: 【Chemistry 89】 The lipid of claim 1, or a pharma- ceutically acceptable salt thereof.

7. R １ and R ２ are each independently hydrogen, C １ ~C ３ Alkyl, or C ２ ~C ３ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkenyl.

8. R １ and R ２ are each independently hydrogen, C １ ~C ２ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkyl.

9. The lipid is represented by formula V: 【Chemistry 90】 The lipid of claim 1, or a pharma- ceutically acceptable salt thereof.

10. R ３ But, C １ ~C ９ Alkylene or C ２ ~C ９ 2. The lipid of claim 1, which is an alkenylene, or a pharma- ceutically acceptable salt thereof.

11. R ３ But, C １ ~C ７ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkylene.

12. R ３ But, C ７ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the lipid is selected from the group consisting of alkylene, C6 alkylene, C5 alkylene, C4 alkylene, and C3 alkylene.

13. R ５ But it doesn't exist, or C １ ~C ４ Alkylene, or C ２ ~C ４ 2. The lipid of claim 1, which is an alkenylene, or a pharma- ceutically acceptable salt thereof.

14. R ５ The lipid of claim 1 , or a pharma- ceutically acceptable salt thereof, wherein:

15. R ５ But, C １ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the lipid is selected from the group consisting of alkylene, C2 alkylene, C3 alkylene, and C4 alkylene.

16. R ４ But, C １ ~C １４ Unbranched alkyl, C ２ ~C １４ unbranched alkenyl, or 【Chemistry 91】 Wherein R ４ａ and R ４ｂ However, each independently, C １ ~C １２ Unbranched alkyl or C ２ ~C １２ The lipid of claim 1 , or a pharma- ceutically acceptable salt thereof, which is an unbranched alkenyl.

17. R ４ But, C ２ ~C １２ Unbranched alkyl or C ２ ~C １２ The lipid of claim 1 , or a pharma- ceutically acceptable salt thereof, which is an unbranched alkenyl.

18. R ４ But, C ５ ~C １２ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is an unbranched alkyl.

19. R ４ But, C ６ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the lipid is selected from the group consisting of unbranched alkyl, C7 unbranched alkyl, C8 unbranched alkyl, and C9 unbranched alkyl.

20. R ４ But, C １０ ~C １２ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is an unbranched alkyl.

21. R ４ But, C ２ ~C ７ Unbranched alkyl or C ２ ~C ７ The lipid of claim 1 , or a pharma- ceutically acceptable salt thereof, which is an unbranched alkenyl.

22. R ４ but, 【Chemistry 92】 Wherein R ４ａ and R ４ｂ However, each independently, C ５ ~C ９ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is an unbranched alkyl.

23. R ６ａ and R ６ｂ However, each independently, C ７ ~C １４ Alkyl or C ７ ~C １４ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkenyl.

24. R ６ａ and R ６ｂ However, 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 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkenyl.

25. R ６ａ and R ６ｂ However, each independently, C ７ Alkyl, C ８ Alkyl, C ９ Alkyl, C １０ Alkyl, C １１ Alkyl, or C １２ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkyl.

26. R ６ａ and R ６ｂ However, each independently, C ９ Alkyl, C １０ Alkyl, C １１ Alkyl, C １２ Alkyl, C １３ Alkyl, C １４ Alkyl, C １５ Alkyl, C １６ Alkyl, C ９ Alkenyl, C １０ Alkenyl, C １１ Alkenyl, C １２ Alkenyl, C １３ Alkenyl, C １４ Alkenyl, C １５ Alkenyl, or C １６ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkenyl.

27. R ６ａ and R ６ｂ However, each independently, C ９ Alkyl, C １０ Alkyl, C １１ Alkyl, C １２ Alkyl, C １３ Alkyl, C １４ Alkyl, C １５ Alkyl, and C １６ 27. The lipid of claim 26, or a pharma- ceutically acceptable salt thereof, which is alkyl.

28. R ６ａ and R ６ｂ each contain the same number of carbon atoms, or a pharma- ceutically acceptable salt thereof.

29. R ６ａ and R ６ｂ The lipid according to claim 1 , or a pharma- ceutically acceptable salt thereof, wherein:

30. R ６ａ and R ６ｂ But both are C ７ 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl or C12 alkyl.

31. R ６ａ and R ６ｂ Each of the lipids contains a different number of carbon atoms, or a pharma- ceutically acceptable salt thereof.

32. R ６ａ But, C ９ alkyl, R ６ｂ But, C ８ 2. The lipid of claim 1, wherein R 6a is a C 8 alkyl and R 6b is a C 9 alkyl; R 6a is a C 9 alkyl and R 6b is a C 10 alkyl; or R 6a is a C 10 alkyl and R 6b is a C 9 alkyl, or a pharma- ceutically acceptable salt thereof.

33. 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, wherein R' is absent.

34. The lipid is 【Chemistry 93】 【Chemistry 94】 【Chemistry 95】 【Chemistry 96】 【Chemistry 97】 2. The lipid of claim 1, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of:

35. The lipid, 【Chemistry 101】 35. The lipid of claim 34, which is: or a pharma- ceutically acceptable salt thereof.

36. A lipid nanoparticle (LNP) comprising the lipid according to any one of claims 1 to 35, or a pharma- ceutically acceptable salt thereof, and a therapeutic nucleic acid.

37. The lipid nanoparticle of claim 36, wherein the therapeutic nucleic acid is encapsulated within the lipid.

38. The lipid nanoparticle of claim 36, wherein the therapeutic nucleic acid is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), an antisense oligonucleotide (ASO), a ribozyme, a ceDNA, a ministring, a doggybone™, a protelomeric closed-end DNA, or a dumbbell linear DNA, a dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), an mRNA, a tRNA, a rRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and any combination thereof.

39. The lipid nanoparticle of claim 36, wherein the therapeutic nucleic acid is closed-ended DNA (ceDNA), mRNA or siRNA.

40. The lipid nanoparticle of claim 36, further comprising a sterol.

41. The lipid nanoparticle of claim 40, wherein the sterol is cholesterol or beta-sitosterol.

42. The lipid nanoparticle of claim 36, further comprising a non-cationic lipid.

43. The non-cationic lipid may be selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine (DSPE), 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 (DSPE), monomethyl-phosphatidylethanolamine (DM ... amines (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamines (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylserine (DOPS), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE);43. The lipid nanoparticle of claim 42, selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

44. The lipid nanoparticle of claim 42, wherein the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).

45. The lipid nanoparticle of claim 36, further comprising at least one PEGylated lipid.

46. The at least one PEGylated lipid may be selected from the group consisting of 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 glycamide; PEG-dipalmitoyl glycamide; PEG-disteryl glycamide.

46. ​​The lipid nanoparticle of claim 45, wherein the lipid nanoparticle is selected from the group consisting of: (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol); 3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether (PEG-DMB); and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG).

47. The lipid nanoparticle of claim 36, further comprising a tissue-specific targeting ligand.

48. The lipid nanoparticle of claim 47, wherein the tissue-specific targeting ligand is N-acetylgalactosamine (GalNAc), a GalNAc derivative, or an antibody.

49. The cationic lipid is present in a molar percentage of about 30% to about 80%; the sterol is present in a molar percentage of about 20% to about 50%; said non-cationic lipid is present in a molar percentage of about 2% to about 20%; the at least one PEGylated lipid is present in a molar percentage of about 2.1% to about 10%; and / or The lipid nanoparticle of claim 36, wherein the PEGylated lipid conjugate is present in a molar percentage of about 0.1% to about 10%.

50. A pharmaceutical composition comprising a lipid according to any one of claims 1 to 35 and a pharma- ceutically acceptable excipient.

51. 37. A composition for use in a method for treating a genetic disorder in a subject, comprising the lipid nanoparticle described in claim 36.

52. 52. The composition for use according to claim 51, wherein the subject is a human.

53. The genetic disorder may be melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR deficiency), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, 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 H-S), Hunter syndrome (MPS II), Sanfilippo syndrome type A (MPS IIIA), Sanfilippo syndrome type B (MPS IIIB), Sanfilippo syndrome type B (MPS IIIC), Sanfilippo syndrome type C (MPS IIID), Sanfilippo syndrome type D (MPS IIID), Sanfilippo syndrome type E (MPS IIID), Sanfilippo syndrome type F ... 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 type A / B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis type I, mucolipidosis type II / III, mucolipidosis type IV, sialidosis type I, sialidosis type II, glycogen storage disease type I, glycogen storage disease type II (Pompe disease) 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), sphingolipidoses, 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's congenital amaurosis, Stargardt macular dystrophy (ABCA4 deficiency), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AM) 53. The composition for use according to claim 51 or 52, wherein the deficiency is selected from the group consisting of 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 deficiency), and cathepsin A deficiency.

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