Cationic lipids and their compositions

Abstract

This article provides cationic lipids having formula I or Ia or pharmaceutically acceptable salts thereof, wherein R', ... 1 R 2 R 3 R 4 R 5 R 6a R 6b X and n are as defined herein. This document also provides lipid nanoparticle (LNP) compositions comprising a cationic lipid having formula I or Ia and a capsid-free nonviral vector (e.g., ceDNA). In any aspect of this document or any of the embodiments, these LNPs can be used to deliver a capsid-free nonviral DNA vector to a target site of interest (e.g., cells, tissues, organs, etc.). (I) or (Ia)

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202280049199.X, filed on June 14, 2022, entitled "Catonic Lipids and Compositions Thereof". Cross-references to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 210,204, filed June 14, 2021, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Gene therapy aims to improve clinical outcomes for patients suffering from genetic disorders or acquired diseases caused by abnormal gene expression profiles. To date, various types of gene therapies have been developed that deliver therapeutic nucleic acids as drugs to the patient's cells.

[0004] Corrective genes can be delivered and expressed in a patient's target cells using a variety of methods, including the use of engineered viral gene delivery vectors, as well as potential plasmids, small genes, oligonucleotides, small loops, or various terminally blocked DNAs. Among the many available viral-derived vectors (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated virus (rAAV) is recognized as a versatile and relatively reliable vector in gene therapy. However, viral vectors (such as adeno-associated vectors) can be highly immunogenic and induce humoral and cellular immunizations that can impair efficacy, particularly in re-administration.

[0005] Nonviral gene delivery circumvents some of the drawbacks associated with viral transduction, particularly the humoral and cellular immune responses to viral structural proteins that form the vector particles, as well as the disadvantages caused by any de novo viral gene expression. One advantage of nonviral delivery technologies is the use of lipid nanoparticles (LNPs) as carriers. LNPs offer a unique opportunity to allow the design of cationic lipids as LNP components, which can circumvent the humoral and cellular immune responses that cause significant toxicity associated with viral gene therapy.

[0006] Cationic lipids generally consist of a cationic amine moiety, a hydrophobic domain (i.e., a hydrophobic tail, which may be saturated or unsaturated) typically having one or two aliphatic hydrocarbon chains, and a linker or biodegradable group connecting the cationic amine moiety and the hydrophobic domain. The cationic amine moiety interacts electrostatically with the polyanionic nucleic acid to form a positively charged liposome or lipid membrane structure. This facilitates uptake into the cell and delivery of nucleic acids into the cell.

[0007] Some widely used cationic lipids are CLinDMA, DLinDMA (DODAP), and DOTAP. These lipids have been used for ribonucleic acid (siRNA or mRNA) delivery, but exhibit suboptimal delivery efficiency and toxicity at higher doses. Given the current limitations of cationic lipids, there is a need in the art to provide lipid scaffolds that not only demonstrate enhanced efficacy and reduced toxicity, but also improved pharmacokinetic and intracellular kinetics, such as cellular uptake and nucleic acid release from the lipid carrier. Summary of the Invention

[0008] The cationic lipids provided in this disclosure comprise a hydrophobic tail containing a biodegradable group and a hydrophobic tail not containing a biodegradable group. Some exemplary lipids provided in this disclosure comprise a hydrophobic tail that branches at the end to form two branched aliphatic hydrocarbon chains and a non-branched hydrophobic tail. The inventors have found that the cationic lipids of this disclosure can be synthesized with satisfactory yield and purity. The inventors have also found that, when formulated as lipid nanoparticles (LNPs) for carrying therapeutic nucleic acids, the cationic lipids of this disclosure provide sustained, excellent, and stable in vivo expression of transgenic inserts within nucleic acids, and are well tolerated. Furthermore, not wishing to be bound by theory, the inventors believe that the subtle interactions between the length of the terminal branched aliphatic hydrocarbon chain (i.e., the number of carbon atoms) in the branched hydrophobic tail, the length of the non-branched hydrophobic tail, and the distance between the biodegradable group and the branched hydrophobic tail are particularly important for achieving excellent encapsulation efficiency, expression levels, and in vivo tolerability of the LNP compositions.

[0009] Therefore, in one aspect, this paper provides cationic lipids represented by formula I or Ia: I or Ia And its pharmaceutically acceptable salts, of which R', R 1 R 2 R 3 R 4 R 5 R 6a R 6b X and n are defined in this paper for each of Equation I or Ia.

[0010] Also provided are pharmaceutical compositions comprising the cationic lipids described herein or pharmaceutically acceptable salts thereof; and pharmaceutically acceptable carriers.

[0011] Another aspect of this disclosure relates to a composition comprising lipid nanoparticles (LNPs) and a nucleic acid, the lipid nanoparticles (LNPs) comprising the cationic lipids described herein or pharmaceutically acceptable salts thereof. In any embodiment of any aspect or embodiment herein, the nucleic acid is encapsulated in the LNP. In a specific embodiment, the nucleic acid is terminally blocked DNA (ceDNA).

[0012] Another aspect of this disclosure relates to a method of treating a subject with a genetic condition using the cationic lipids or compositions disclosed herein. Attached Figure Description

[0013] The embodiments of this disclosure, which have been briefly summarized above and discussed in more detail below, can be understood by referring to the illustrative embodiments depicted in the accompanying drawings. However, the drawings only show typical embodiments of this disclosure and should not be considered as limiting the scope, as this disclosure may allow for other equivalent embodiments.

[0014] Figure 1 The results show that, as observed in the preclinical study (dose = 0.25 mg / kg), day 4 ceDNA-luciferase expression was achieved by using lipid nanoparticles LNP 2, LNP 3, and LNP 4, all formulated with lipid 6, as delivery media, compared with LNP 1 formulated with reference lipid A (positive control) and PBS (negative control).

[0015] Figure 2A This is a bar chart showing day 4 ceDNA-luciferase expression as measured by total throughput, as observed in preclinical studies (dose = 0.5 mg / kg), compared to LNP 5 (positive control) formulated with reference lipid A, LNP 6 formulated with MC3, and LNP 7 (positive control) formulated with reference lipid B, and PBS (negative control). This total throughput was achieved using lipid nanoparticles LNP 8, LNP 9, and LNP 10, formulated with lipids 7, 11, and 1, respectively, as delivery media. Figure 2B The longitudinal weight changes of mice in the same study from day 0 to day 4 are shown. Detailed Implementation

[0016] This disclosure provides lipid-based platforms for delivering therapeutic nucleic acids (TNAs), such as non-viral vectors (e.g., terminally blocked DNA) or synthetic viral vectors, which can be absorbed by cells and maintained at high levels of expression. For example, the immunogenicity associated with gene therapy based on viral vectors limits the number of patients that can be treated due to pre-existing background immunity and prevents re-administration to patients to titrate to an effective level for each patient or to maintain efficacy long-term. Furthermore, other nucleic acid modalities are greatly affected by immunogenicity due to innate DNA or RNA sensing mechanisms that trigger cascade immune responses. Due to the lack of pre-existing immunity, the TNA lipid particles (e.g., lipid nanoparticles) described herein allow for additional doses of TNA, such as mRNA, siRNA, synthetic viral vectors, or ceDNA, as needed, and further expand patient accessibility, including to pediatric populations that may require subsequent doses during tissue growth. Furthermore, this disclosure finds that TNA lipid particles (e.g., lipid nanoparticles), specifically comprising lipid compositions containing one or more tertiary amino groups and disulfide bonds, provide more efficient delivery, better tolerability, and improved safety of TNAs (e.g., ceDNA). Since the TNA lipid particles (e.g., lipid nanoparticles) described so far do not have the packaging constraints imposed by the space inside the viral capsid, theoretically, the only size limitation of TNA lipid particles (e.g., lipid nanoparticles) lies in the efficiency of expression (e.g., DNA replication or RNA translation) in the host cell.

[0017] One of the biggest hurdles in therapeutic development, particularly in rare diseases, is the sheer number of individual conditions. Approximately 350 million people worldwide live with rare diseases, though fewer than 200,000 are diagnosed with a condition or symptom, as defined by the National Institutes of Health (NIH). About 80% of these rare diseases are of genetic origin, and approximately 95% have not received FDA-approved treatment (rarediseases.info.nih.gov / diseases / pages / 31 / faqs-about-rare-diseases). One advantage of the TNA lipid particles (e.g., lipid nanoparticles) described in this article is that they provide a rapidly adaptable approach to a wide range of diseases (treatable with specific TNA pathways), particularly rare monogenic diseases, potentially transforming the treatment landscape for many genetic conditions or diseases.

[0018] I. Definition The term "alkyl" refers to a monovalent group in a saturated, straight-chain (i.e., unbranched) or branched hydrocarbon. Unless specifically described as unbranched, such as C1-C, alkyl groups are not explicitly defined. 16Non-branched alkyl groups; otherwise, as used herein, the term "alkyl" applies to both branched and non-branched alkyl groups. Exemplary alkyl groups include, but are not limited to, C1-C1 alkyl groups. 16 Non-branched alkyl, C7-C 12 Alkyl, C7-C 11 Alkyl, C8-C 10 Alkyl, C2-C 14 Non-branched alkyl, C2-C 12 Non-branched alkyl, C2-C 10 Non-branched alkyl, C2-C7 non-branched alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C7 non-branched alkyl, C8 non-branched alkyl, C9 non-branched alkyl, C 10 Non-branched alkyl, C 11 Non-branched alkyl, C8 alkyl, C 10 Alkyl, C 12 Alkyl, methyl, ethyl, propyl, isopropyl, 2-methyl-1-butyl, 3-methyl-2-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, etc.

[0019] The term "alkylene" refers to a divalent group in a saturated, straight-chain, or branched hydrocarbon. Unless specifically described as unbranched, such as C3-C, alkylene is not considered a branched group. 10 Unbranched alkylene and C1-C8 alkylene, otherwise the term "alkylene" as used herein applies to both branched and unbranched alkylene groups. Exemplary alkylene groups include, but are not limited to, C3-C9 alkylene, C3-C8 alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C2-C8 alkylene, C3-C7 alkylene, C5-C7 alkylene, C7 alkylene, C5 alkylene, and alkylene corresponding to any of the exemplary alkyl groups described above.

[0020] The term "alkenyl" refers to a monovalent group of a straight-chain or branched hydrocarbon having one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl group includes groups having "cis" and "trans" orientations, or, according to alternative nomenclature, "E" and "Z" orientations. Unless specifically described as unbranched, such as C2-C, the alkenyl group is not considered to be branched. 16Unbranched alkenyl groups; otherwise, as used herein, the term "alkenyl" applies to both branched and unbranched alkenyl groups. Exemplary alkenyl groups include, but are not limited to, C2-C. 16 Unbranched alkenyl, C7-C 16 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 Alkenyl groups, and alkenyl groups corresponding to the exemplary alkyl groups containing two or more carbon atoms described above.

[0021] The term "alkenyl" refers to a divalent group of a straight-chain or branched hydrocarbon having one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl group includes groups having "cis" and "trans" orientations, or, according to alternative nomenclature, "E" and "Z" orientations. Unless specifically described as unbranched, such as C3-C, the alkenyl group is not defined as such. 10 Non-branched alkylene groups, or otherwise the term "alkylene group" as used herein applies to both branched and unbranched alkylene groups. Exemplary alkylene groups include, but are not limited to, C3-C9 alkylene, C3-C8 alkylene, C2-C8 alkylene, C2-C6 alkylene, C3-C7 alkylene, C5-C7 alkylene, C2-C4 alkylene, C1-C8 alkylene, C2-C8 alkylene, C3-C7 alkylene, C5-C7 alkylene, C7 alkylene, C5 alkylene, and alkenyl groups corresponding to the exemplary alkyl groups containing two or more carbon atoms described above.

[0022] 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, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, sucrose salts, formates, benzoates, glutamates, methanesulfonates ("methanesulfonates"), ethylsulfonates, phenylsulfonates, p-toluenesulfonates, bis(hydroxynaphthyl)ates (i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthylcarbamate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve another molecule, such as an acetate ion, a succinate ion, or other counterions. This counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. The presence of multiple charged atoms as part of a pharmaceutically acceptable salt can result in multiple counterions. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0023] As used in this specification and the appended claims, the term “about” when referring to a measurable value such as a quantity, duration, etc., is intended to include a deviation from the specified value of ±20% or ±10%, or ±5%, or ±1%, or ±0.5%, and more preferably ±0.1%, because such deviation is suitable for performing the disclosed method.

[0024] As used herein, “comprise,” “comprising,” “comprises,” and “comprisedof” are intended to be synonymous with “include,” “including,” “includes,” or “contain,” “containing,” and are inclusive or open-ended terms that specify the presence of, for example, components, and do not exclude or preclude the presence of additional, unlisted components, features, elements, members, or steps known in the art or disclosed herein.

[0025] The term “composed of” means the composition, method, process and its corresponding components as described herein, excluding any elements not described in the description of the embodiments.

[0026] As used herein, the term "consistently of" refers to those elements required for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic and novel or functional features of that embodiment of the invention.

[0027] As used herein, the term "administration" and its variations refer to the introduction of a composition or agent (e.g., nucleic acid, especially ceDNA) into a subject and include the simultaneous and sequential introduction of one or more compositions or agents. The introduction of the composition or agent into the subject is by any suitable route, including oral, pulmonary, nasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or local administration. Administration includes self-administration and administration by another person. Administration can be performed by any suitable route. A suitable route of administration enables the composition or agent to perform its intended function. For example, if a suitable route is intravenous, the composition or agent is administered by introducing it into the subject's vein. In any aspect of this document or any of the embodiments, "administration" means therapeutic administration.

[0028] As used herein, the phrases “anti-therapeutic nucleic acid immune response,” “anti-transfer vector immune response,” “immune response against therapeutic nucleic acid,” and “immune response against transfer vector,” etc., refer to any undesired immune response to a therapeutic nucleic acid, virus, or non-virus of its origin. In some embodiments of any of the aspects and embodiments herein, the undesired immune response is an antigen-specific immune response against 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 sequence-specific to the transfer vector. In other embodiments, the immune response is CpG-specific to the transfer vector.

[0029] As used herein, the terms “carrier” and “excipient” are used interchangeably and are intended to include any and all solvents, dispersion media, mediators, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption-retarding agents, buffers, carrier solutions, suspensions, colloids, etc. Such media and agents are well known in the art for their use with pharmaceutically active substances. Complementary active ingredients may also be incorporated into the composition. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce toxicity, sensitization, or similar adverse reactions when administered to a host.

[0030] As used herein, the term "ceDNA" means linear double-stranded (ds) DNA without capsid ends for nonviral gene transfer, synthesis, or other forms. A detailed description of ceDNA is described in International Application PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for producing ceDNA comprising various inverted terminal repeat (ITR) sequences and conformations using cell-based methods are described in Example 1 of International Patent Application No. PCT / US2018 / 049996, filed September 7, 2018, and International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Some methods for generating synthetic ceDNA vectors including various ITR sequences and conformations are described, for example, in International Application 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 aspect or embodiment of the present invention, ceDNA is a terminally closed linear double-stranded (CELiD) CELiD DNA. According to some embodiments of any aspect or embodiment of the present invention, ceDNA is a small loop based on DNA. According to some embodiments of any aspect or embodiment of the present invention, ceDNA is a simplified immunologically defined gene expression (MIDGE) vector. According to some embodiments of any aspect or embodiment of the present invention, ceDNA is a mini-strand DNA. According to some embodiments of any aspect or embodiment of the present invention, ceDNA is a dumbbell-shaped, terminally closed linear double-stranded DNA including two hairpin structures of the ITR at the 5' and 3' ends of the expression cassette. According to some embodiments of any aspect or implementation of this document, ceDNA is doggybone ™ DNA.

[0031] As used herein, the term "ceDNA-baculosome" is intended to refer to an infectious baculovirus genome containing a ceDNA genome as an intermolecular double strand that can be propagated as a plasmid in E. coli and thus can be manipulated as a shuttle vector for baculoviruses.

[0032] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus whose genome includes ceDNA as an intermolecular double strand within its genome.

[0033] As used herein, the terms “ceDNA-baculovirus-infected insect cells” and “ceDNA-BIIC” are used interchangeably to refer to invertebrate host cells (including, but not limited to, insect cells (e.g., Sf9 cells)) infected with ceDNA-baculovirus.

[0034] As used herein, the term "ceDNA genome" refers to an expression cassette that also incorporates at least one inverted terminal repeat region. A ceDNA genome may also include one or more spacer regions. In some embodiments of any of the aspects and embodiments herein, the ceDNA genome is incorporated as an intermolecular double-stranded polynucleotide of DNA into a plasmid or viral genome.

[0035] As used herein, the terms “DNA regulatory sequence,” “control element,” and “regulatory element” are used interchangeably and refer to transcriptional and translational control sequences that provide and / or regulate the transcription of non-coding sequences (e.g., RNA targeting DNA) or coding sequences (e.g., site-modified peptides or Cas9 / Csn1 peptides) and / or regulate the translation of the encoded peptides, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, etc.

[0036] As used herein, the term “exogenous” is intended to refer to a substance present in a cell other than its native source. When used herein, the term “exogenous” can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced into a biological system such as a cell or organism by a process involving human intervention, where the nucleic acid or polypeptide is not normally found in that cell or organism, and it is desirable to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or polypeptide that has been introduced into a biological system such as a cell or organism by a process involving human intervention, where the amount of the nucleic acid or polypeptide found in that cell or organism is relatively low, and it is desirable to increase the amount of the nucleic acid or polypeptide in the cell or organism, for example, to produce ectopic expression or levels. In contrast, as used herein, the term “endogenous” refers to a substance native to a biological system or cell.

[0037] As used herein, the term “expression” refers to cellular processes involving the production of RNA and proteins, and, where appropriate, the secretion of proteins, including, where applicable, transcription, transcript processing, translation, and protein folding, modification, and manipulation. As used herein, the phrase “expression product” includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translating mRNA transcribed from a gene.

[0038] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is typically, but not necessarily, heterologous to the host cell. Expression vectors may include other elements; for example, an expression vector may have two replication systems, allowing it to be maintained in two organisms, such as for expression in human cells and for cloning and amplification in a prokaryotic host. Expression vectors may be recombinant vectors.

[0039] As used herein, the terms “expression cassette” and “expression unit” are used interchangeably to refer to a heterologous DNA sequence operatively linked to a promoter or other DNA regulatory sequence sufficient to direct transgenic transcription of a DNA vector (e.g., a synthetic AAV vector). Suitable promoters include, for example, tissue-specific promoters. Promoters may also be of AAV origin.

[0040] As used herein, the term "sidejoint" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Typically, in sequence ABC, B is flanked by A and C. The same applies to the arrangement of AxBxC. Therefore, the sidejoint sequence is before or after the sidejoined sequence, but does not necessarily have to be adjacent to or immediately next to the sidejoined sequence. In any embodiment of any aspect of this document or of any embodiment, the term sidejoint refers to a terminal repeat sequence at each end of a linear single-stranded synthetic AAV vector.

[0041] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid associated with the in vitro or in vivo expression of a given RNA or protein. Therefore, a gene includes the region encoding the expressed RNA (which typically includes a polypeptide-coding sequence) and the regulatory sequences usually required for its expression. Genes can be obtained from a variety of sources, including cloning from sources of interest or synthesis from known or predicted sequence information, and can include sequences designed to have the desired parameters.

[0042] As used herein, the phrase “genetic disease” or “genetic symptom” refers to a disease or defect that is partly or entirely caused, directly or indirectly, by one or more abnormalities in the genome, including, and especially, symptoms present from birth. Abnormalities can be mutations, insertions, or deletions in genes. Abnormalities may affect the coding sequence of a gene or its regulatory sequences.

[0043] As used herein, the term "heterologous" refers to a nucleotide or polypeptide sequence that is not found in native nucleic acids or proteins, respectively. Heterologous nucleic acid sequences can be linked (e.g., through genetic engineering) to generate chimeric nucleotide sequences encoding chimeric polypeptides. Heterologous nucleic acid sequences can be linked (e.g., through genetic engineering) to generate nucleotide sequences encoding fusion variant polypeptides.

[0044] As used herein, the term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., by the nucleic acid therapeutic agents of this disclosure. As a non-limiting example, the host cell can be isolated primary cells, pluripotent stem cells, CD34 cells, etc. + The host cell may be any of the following: a cell, an induced pluripotent stem cell, or a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be an in situ or in vivo cell in a tissue, organ, or organism. Furthermore, the host cell may be, for example, the target cell of a mammalian subject (e.g., a human patient requiring gene therapy).

[0045] As used herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity in the presence, influence of, or contact with an inducer or inducer. An "inducer" or "inducer" as used herein can be endogenous or typically exogenous compounds or proteins administered in a manner capable of inducing transcriptional activity from an inducible promoter. In some embodiments of any aspect and embodiment herein, the inducer or inducer, i.e., a chemical substance, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducible protein expressed by another component or module), the nucleic acid sequence itself being under the control of the inducible promoter. In some embodiments of any aspect and embodiment herein, the inducible promoter is induced in the absence of certain agents, such as repressors. Examples of inducible promoters include, but are not limited to, tetracyclines, metallothioneins, ecdysone, mammalian viruses (e.g., adenovirus late promoters; and mouse mammary tumor virus long terminal repeat sequences (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters, etc.

[0046] As used in this article, the term "in vitro" ” This refers to assays and methods that do not require the presence of cells with intact membranes (such as cell extracts), and can refer to the introduction of programmable synthetic biological circuits into non-cellular systems (such as media that do not contain cells) or cellular systems (such as cell extracts).

[0047] As used in this article, the term "in vivo" ” This refers to a measurement or process performed in or within an organism (such as a multicellular animal). In some aspects described herein, when using single-celled organisms (such as bacteria), it can be said that the method or use occurs “in vivo.” The term “ex vivo” refers to a method and use using living cells with intact membranes outside a multicellular animal or plant body, such as explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissues or cells, including blood cells, etc.

[0048] As used herein, the term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, characterized by poor water solubility but generally soluble in many organic solvents. They are generally classified into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include 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, distearylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, the glycosphingolipid family, diacylglycerols, and β-acyloxy acids, are also in the group referred to as amphiphilic lipids. Furthermore, the aforementioned amphiphilic lipids can be mixed with other lipids, including triglycerides and sterols.

[0049] As used herein, the term "encapsulated" is intended to refer to lipid particles that provide active agents or therapeutic agents (such as nucleic acids (e.g., ASO, mRNA, siRNA, ceDNA, viral vectors)) through complete encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is completely encapsulated within the lipid particle (e.g., to form a lipid particle containing the nucleic acid).

[0050] As used herein, the terms "lipid particle" or "lipid nanoparticle" refer to a lipid formulation (referred to as "TNA lipid particle," "TNA lipid nanoparticle," or "TNA LNP") that can be used to deliver therapeutic agents, such as nucleic acid therapeutic agents (TNA), to a target site of interest (e.g., cells, tissues, organs, etc.). 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, wherein the LNP typically consists of cationic lipids, sterols, non-cationic lipids, and optionally polyethylene glycol-modified lipids to prevent particle aggregation, and additionally optionally tissue-specific targeting ligands for delivering the LNP to the target site of interest. In other preferred embodiments, therapeutic agents, such as therapeutic nucleic acids, may be encapsulated in the lipid portion of the particle, thereby protecting them from enzymatic degradation. In one embodiment of any of the aspects or embodiments herein, the LNP comprises a nucleotide (e.g., ceDNA) and an LNP formulated with the cationic lipids described herein.

[0051] As used herein, the term "ionizable lipid" means a lipid having at least one protonable or deprotonable group, such as a cationic lipid, such that the lipid is positively charged at a pH equal to or below physiological pH (e.g., pH 7.4) and neutral at a second pH (preferably equal to or above physiological pH). Those skilled in the art will understand that the addition or removal of protons according to pH is a balancing process, and that references to charged or neutral lipids refer to the properties of the primary substance and do not require all lipids to be present in a charged or neutral form. Typically, the pKa of the protonable group of cationic lipids is in the range of about 4 to about 7. Therefore, as used herein, the term "cationic" encompasses both the ionized (or charged) and neutral forms of the lipids of the present invention.

[0052] As used herein, the term "neutral lipid" is intended to refer to any type of lipid that exists as an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0053] As used herein, the term "anionic lipid" refers to any lipid that carries a negative charge 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 neutral lipids with added anionic modifying groups.

[0054] As used herein, the term “non-cationic lipid” means any amphiphilic lipid as well as any other neutral or anionic lipid.

[0055] As used herein, the term "organic lipid solution" is intended to refer to a composition which comprises, in whole or in part, an organic solvent having lipids.

[0056] As used herein, the term "liposome" refers to a lipid molecule assembled into a spherical structure that encapsulates an internal aqueous volume isolated from an aqueous exterior. A liposome is a vesicle having at least one lipid bilayer. In the context of pharmaceutical research and development, liposomes are commonly used as carriers for drug / therapeutic agent delivery. They function by fusing with cell membranes and repositioning their lipid structure to deliver drugs or active pharmaceutical ingredients. Liposome compositions used for such delivery typically consist of phospholipids, particularly compounds having phosphatidylcholine groups; however, these compositions may also include other lipids.

[0057] As used herein, the term "local delivery" refers to the direct delivery of an active agent, such as interfering RNA (e.g., siRNA), to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site (such as a tumor or other target site, such as an inflamed area or a target organ, such as the liver, heart, pancreas, kidney, etc.).

[0058] As used herein, the term “neDNA” or “gap DNA” refers to DNA with a closed end containing a 2-100 base pair gap or nick in the 5' of the stem or spacer region upstream of an open reading frame (e.g., the promoter and transgene to be expressed).

[0059] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form and including DNA, RNA, and hybrids thereof. DNA can take the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. DNA can take the form of small circular DNA, plasmids, rods, small genes, mini-string DNA (linearly covalently closed DNA vectors), terminally closed linear double-helix DNA (CELiD or ceDNA), doggybone, etc. ™ DNA, dumbbell-shaped DNA, a simplified 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), cleats-substrate dsRNA, small 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 bonds, which are synthetic, naturally occurring, or non-natural, and have binding properties similar to a reference nucleic acid. Examples of such analogs and / or modified residues include (but are not limited to): thiophosphates, phosphoryl diamine morpholino oligomers (morpholino), aminophosphates, methyl phosphonate, chiral methyl phosphonate, 2'-O-methylribonucleotides, locked nucleic acids (LNAs). ™ ) and peptide nucleic acids (PNA). Unless otherwise specified, the term covers nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid. Unless otherwise stated, a particular nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as explicitly stated sequences.

[0060] As used herein, the phrases “nucleic acid therapeutic,” “therapeutic nucleic acid,” and “TNA” are used interchangeably and refer to any modality of treatment that uses nucleic acid as the active component of a therapeutic agent for treating a disease or condition. As used herein, these phrases 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), clecase-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutic agents include small circular DNA, small genes, viral DNA (e.g., lentivirus or AAV genome) or non-viral DNA vectors, terminally closed linear double-stranded DNA (ceDNA / CELiD), plasmids, rod particles, and dog bone. ™ DNA vectors, simplified immunologically defined gene expression (MIDGE) vectors, nonviral mini-string DNA vectors (linearly-covalently blocked DNA vectors), and dumbbell-shaped DNA minimal vectors (“dumbbell DNA”). As used herein, the term “TNA LNP” refers to a lipid particle containing at least one of the aforementioned TNAs.

[0061] As used in this article, a "nucleotide" contains a sugar deoxyribonucleotide (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups.

[0062] As used herein, “operationally linked” means a juxtaposition in which the components described so far are in a relationship that allows them to function in the intended manner. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operationally linked to said coding sequence. A promoter can be described as driving the expression of the nucleic acid sequence it regulates or driving its transcription. The phrases “operationally linked,” “operationally positioned,” “operationally connected,” “under control,” and “under transcriptional control” indicate that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence it regulates to control the transcriptional initiation and / or expression of that sequence. As used herein, “reverse promoter” refers to a nucleic acid sequence in the opposite orientation, such that the coding strand is now the promoter of the non-coding strand, and vice versa. Reverse promoter sequences can be used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, promoters can be used in conjunction with enhancers.

[0063] 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 a nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inducible, repressive, tissue-specific, or any combination thereof. A promoter is the control region of a nucleic acid sequence in which the initiation and transcription rate of the remainder of the sequence are controlled. Promoters may also contain genetic elements that can bind regulatory proteins and molecules, such as RNA polymerases and other transcription factors. Within the promoter sequence will be a transcription start site and a protein-binding domain responsible for RNA polymerase binding. Eukaryotic promoters will often, but not always, contain both a "TATA" box and a "CAT" box. Various promoters, including inducible promoters, can be used to drive the expression of transgenes in the synthetic AAV vectors disclosed herein. The promoter sequence may extend upstream (in the 5' direction) from its 3' end, bounded by the transcription start site, to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above background.

[0064] A promoter can be a promoter that is naturally bound to a gene or sequence, such as one that can be obtained by isolating a 5' non-coding sequence located upstream of the coding region and / or an exon of a given gene or sequence. Such promoters may be referred to as "endogenous". Similarly, in some embodiments of any of the aspects and embodiments herein, an enhancer can be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. In some embodiments of any of the aspects and embodiments herein, the coding nucleic acid segment is located under the control of a "recombinant promoter" or a "heterologous promoter," both of which refer to a promoter that is not normally associated with a coding nucleic acid sequence that is operatively linked to it in its natural environment. Similarly, a "recombinant or heterologous enhancer" refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cells; and synthetic promoters or enhancers that are not "naturally present" (i.e., containing different elements of different transcriptional regulatory regions and / or mutations that alter expression through genetic engineering methods known in the art). In addition to synthesizing nucleic acid sequences that generate promoters and enhancers, promoter sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in conjunction with the synthetic biological circuits and modules disclosed herein (see, for example, U.S. Patent Nos. 4,683,202 and 5,928,906, each incorporated herein by reference in its entirety). Furthermore, it is anticipated that control sequences guiding transcription and / or expression in non-nuclear organelles such as mitochondria and chloroplasts can also be employed.

[0065] As used herein, the terms “Rep binding site” (“RBS”) and “Rep binding element” (“RBE”) are used interchangeably and refer to a binding site of a Rep protein (e.g., AAV Rep 78 or AAV Rep 68) that, upon binding, allows the Rep protein to exert its site-specific endonuclease activity on a sequence incorporating the RBS. The RBS sequence, together with its inverse complementary sequence, forms a single RBS. RBS sequences are known in the art and include, for example, the RBS sequence identified in AAV2, 5'-GCGCGCTCGCTCGCTC-3'.

[0066] As used herein, the phrase "recombinant vector" means a vector comprising a heterologous nucleic acid sequence or "transgenic" capable of being expressed in vivo. It should be understood that, in some embodiments of any of the aspects and embodiments herein, the vectors described herein may be combined with other suitable compositions and therapies. In some embodiments of any of the aspects and embodiments herein, the vector is free-form. The use of a suitable free-form vector provides a way to maintain the nucleotides of interest in a subject with high copy numbers of extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0067] As used herein, the term "reporter" refers to a protein that can be used to provide a detectable reading. Reporter proteins typically produce measurable signals, such as fluorescence, color, or luminescence. The reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observable.

[0068] As used in this article, the terms “sense” and “antense” refer to the orientation of structural elements on a polynucleotide. The sense and antisense forms of elements are mutually complementary and opposite.

[0069] As used herein, the term “sequence consistency” refers to the correlation between two nucleotide sequences. For the purposes of this disclosure, the degree of sequence consistency between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) performed in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.), preferably version 3.0.0 or later. Optional parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix. The Needle output labeled “Longest Consistency” (obtained using the -nobrief option) is used as the consistency percentage and is calculated as follows: (identical deoxyribonucleotides multiplied by 100) / (alignment length - total number of aligned vacancy). The length of the alignment is preferably at least 10 nucleotides, more preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0070] As used herein, the term "spacer region" refers to an intermediate sequence that isolates functional elements in a vector or genome. In some embodiments of any of the aspects and embodiments herein, the AAV spacer region maintains two functional elements at a distance desired for optimal functionality. In some embodiments of any of the aspects and embodiments herein, the spacer region provides or increases gene stability of the vector or genome. In some embodiments of any of the aspects and embodiments herein, the spacer region facilitates genome-ready gene manipulation by providing a suitable location for cloning sites and a designed number of base pairs of spacing. For example, in some aspects, oligonucleotide "multi-restriction linkers" or "polycloning sites" containing several restriction endonuclease sites, or non-open reading frame sequences designed not to have known protein (e.g., transcription factor) binding sites, can be located in the vector or genome to isolate cis-acting factors, such as inserting 6-mers, 12-mers, 18-mers, 24-mers, 48-mers, 86-mers, 176-mers, etc.

[0071] As used herein, the term "subject" refers to a person or animal to whom treatment, including preventative treatment, of the therapeutic nucleic acid according to the invention is administered. Typically, the animal is a vertebrate, such as, but not limited to, primates, rodents, domesticated animals, or hunted animals. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and hunted animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject can be male or female. Additionally, the subject can be an infant or a child. In some embodiments of any of the aspects and embodiments described herein, the subject can be a newborn or an unborn subject, such as a subject still in the womb. Preferably, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects in animal models representing diseases and symptoms. Additionally, the methods and compositions described herein can be used with domestic animals and / or pets. Human subjects can be of any age, sex, race, or ethnicity, such as Caucasian (white), Asian, African, Black, African American, African European, Hispanic, Middle Eastern, etc. In some embodiments of any aspect and implementation thereof, the subject can be a patient or other subject in a clinical setting. In some embodiments of any aspect and implementation thereof, the subject is already receiving treatment. In some embodiments of any aspect and implementation thereof, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments of any aspect and implementation thereof, the subject is a human fetus, human newborn, human infant, human child, human adolescent, or human adult. In some embodiments of any aspect and implementation thereof, 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 and implementation methods herein, the subject is a human embryo.

[0072] As used herein, the phrase “subject in need” means (i) a subject to be administered TNA liposomes (or a pharmaceutical composition containing TNA liposomes) according to the invention, (ii) a subject currently receiving TNA liposomes (or a pharmaceutical composition containing TNA liposomes) according to the invention, or (iii) a subject who has received TNA liposomes (or a pharmaceutical composition containing TNA liposomes) according to the invention, unless the context and usage of the phrase otherwise indicate.

[0073] As used herein, the terms “containment,” “reduction,” “interference,” “suppression,” and / or “reduction” (and similar terms) generally refer to actions that directly or indirectly reduce the concentration, level, function, activity, or behavior relative to natural, expected, or average conditions, or relative to control conditions.

[0074] As used herein, the terms “synthetic AAV vector” and “synthetic production of AAV vector” refer to AAV vectors and their synthetic production methods in a completely cell-free environment.

[0075] As used herein, the term "systemic delivery" refers to the delivery of lipid particles that result in the widespread biodistribution of active agents, such as interfering RNA (e.g., siRNA), within an organism. Some administration techniques can lead to systemic delivery of certain agents but not others. Systemic delivery means exposing a useful (preferably therapeutic) dose of an agent to most parts of the body. To achieve widespread biodistribution, blood lifetime is typically required so that the agent is not rapidly degraded or cleared before reaching the disease site distal to the administration site (e.g., via first-pass organs (liver, lungs, etc.) or via rapid, nonspecific cell binding). Systemic delivery of lipid particles (e.g., lipid nanoparticles) can be performed by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal delivery. In a preferred embodiment, systemic delivery of lipid particles (e.g., lipid nanoparticles) is performed via intravenous delivery.

[0076] As used herein, the terms “terminal dissociation site” and “TRS” are used interchangeably to refer to a region where Rep forms a tyrosine-phosphodiester bond with 5'-thymidine, producing a 3'-OH, which acts as a substrate for DNA elongation via cellular DNA polymerases, such as DNApol δ or DNApol ε. Alternatively, the Rep-thymidine complex can participate in coordination conjugation reactions.

[0077] As used herein, the terms “therapeutic dose,” “therapeutic effective dose,” “effective dose” or “pharmaceutical effective dose” of an active agent (such as TNA lipid particles described herein) are used interchangeably to refer to an amount sufficient to provide the expected benefit of treatment or effect, for example, inhibition of target sequence expression compared to expression levels detected in the absence of therapeutic nucleic acids. Suitable analyses for measuring the expression of a target gene or target sequence include, for example, examining protein or RNA levels using techniques known to those skilled in the art, such as dot blot, northern blot, in situ heterozygosity, ELISA, immunoprecipitation, enzyme function, and phenotypic analyses known to those skilled in the art. However, dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, the patient’s medical condition, the severity of the condition, the route of administration, and the specific active agent used. Therefore, dosage regimens can vary widely but can be routinely determined by a physician using standard methods. Additionally, the terms “therapeutic dose,” “effective dose,” “therapeutic effective dose,” and “pharmaceutical effective dose” include preventative or protective doses of the compositions of the present invention described herein. In the preventive or preventative applications of the invention described herein, a pharmaceutical composition or agent is administered to a patient susceptible to, or otherwise at risk of, a disease, symptom, or condition, in an amount sufficient to eliminate or reduce the risk, severity, or delay the onset of the disease, symptom, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, symptom, or condition, its complications, and intermediate pathological phenotypes present during the development of the disease, symptom, or condition. In one aspect, the terms “therapeutic amount,” “effective amount,” “therapeutic effective amount,” and “pharmaceutical effective amount” do not include preventive or preventative amounts of the compositions of the invention described herein. Generally, the maximum dose is preferred, i.e., the highest safe dose based on some medical judgment. The term “dose / dosage” is used interchangeably herein. In any aspect of this document or in any embodiment, “therapeutic amount,” “therapeutic effective amount,” and “pharmaceutical effective amount” refer to non-preventative or non-protective applications.

[0078] As used herein, the term "therapeutic effect" refers to the outcome of treatment that is deemed necessary and beneficial. Therapeutic effects can directly or indirectly include the containment, reduction, or elimination of disease manifestations. Therapeutic effects can also directly or indirectly include the containment, reduction, or elimination of the progression of disease manifestations.

[0079] For any of the therapeutic agents described herein, the therapeutically effective dose can be initially determined based on preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined based on human data. The administered dose can be adjusted based on relative bioavailability and the potency of the compound being administered. Adjusting the dose to achieve maximum efficacy based on the methods described above and other well-known methods is within the capabilities of a person of ordinary skill in the art. The general principles for determining therapeutic effectiveness are summarized below, which can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0080] Pharmacokinetic principles provide the basis for modifying dosing regimens to achieve the desired level of therapeutic efficacy with minimal unacceptable side effects. Further guidance for dose modification can be obtained when plasma drug concentrations can be measured and correlated with the therapeutic window.

[0081] As used herein, the term "treat / treating / treatment" includes eliminating, inhibiting, slowing, or reversing the progression of a condition, improving the clinical symptoms of the condition, or preventing the onset of clinical symptoms of the condition, or achieving a beneficial or desired clinical outcome. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the condition; (b) limiting the development of characteristic symptoms of the treated condition; (c) limiting the exacerbation of characteristic symptoms of the treated condition; (d) limiting the recurrence of the disorder in patients who previously had the condition; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic. In any aspect of this document or in any implementation thereof, the term "treat / treating / treatment" includes eliminating, inhibiting, slowing, or reversing the progression of a condition, or improving the clinical symptoms of the condition.

[0082] Beneficial or desired clinical outcomes, such as pharmacological and / or physiological effects, include (but are not limited to): prevention of the onset of a disease, condition, or symptom in subjects who may be susceptible to the disease, condition, or symptom but have not yet experienced or exhibited symptoms of the disease (preventive treatment); relief of symptoms of the disease, condition, or symptom; reduction of the severity of the disease, condition, or symptom; stabilization of the disease, condition, or symptom (i.e., no worsening); prevention of the spread of the disease, condition, or symptom; delay or slowing the progression of the disease, condition, or symptom; improvement or mitigation of the disease, condition, or symptom; and combinations thereof, and prolongation of survival compared to the expected survival without treatment.

[0083] As used herein, the term "vector" or "expression vector" means a replicon, such as a plasmid, rod, bacteriophage, virus, viral particle, or granule, which can attach another DNA segment, i.e., an "insertion," "transgenic," or "expression cassette," to enable 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 DNA vector. Thus, the term "vector" encompasses any genetic element that, when associated with a suitable control element, is capable of replication and can transfer a gene sequence into a cell. In some embodiments of any of the aspects and embodiments herein, the vector can be a recombinant vector or an expression vector.

[0084] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually, or in any combination with other members of that group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from a group. When any such inclusion or removal occurs, the specification is considered herein to contain a modified group, thereby satisfying the written description of all Markush groups as used in the appended claims.

[0085] In any of the embodiments described herein, the disclosure does not relate to methods of cloning humans, methods of modifying the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or methods of modifying the genetic identity of animals that may cause them suffering and have no substantial medical benefit to humans or animals, and animals produced by such methods.

[0086] Other terms are defined herein within the description of various aspects of the invention.

[0087] II. Lipids In the first embodiment, a cationic lipid represented by Formula I is provided: I Or its pharmaceutically acceptable salt, wherein: R' is absent, and is either hydrogen or a C1-C3 alkyl group; the prerequisite is that when R' is hydrogen or a C1-C3 alkyl group, R' and R... 1 and R 2 All the attached nitrogen atoms were protonated; R 1 and R 2Each is independently hydrogen or C1-C3 alkyl; R 3 For C3-C 10 Alkylene or C3-C 10 alkenyl; R 4 For C1-C 16 Non-branched alkyl, C2-C 16 non-branched alkenyl or ;in: R 4a and R 4b Each independently is C1-C 16 Non-branched alkyl or C2-C 16 Non-branched alkenyl groups; R 5 It does not exist; it is either a C1-C6 alkylene group or a C2-C6 alkenyl group. R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 alkenyl; X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-、-N=C(R a )-、-C(R a )=NO-、-ON=C(R a )-、-C(=O)NR a -、-NR a C(=O)-、-NR a C(=O)NR a -、-OC(=O)O-、-OSi(R a )2O-、-C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-; where: R a Each time it appears, it is independently either hydrogen or C. 1-6 Alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6.

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

[0089] In the third embodiment, the cationic lipid of this disclosure is represented by Formula II: II 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 of the foregoing embodiments. In an alternative third embodiment, n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula I or any of the foregoing embodiments.

[0090] In the fourth embodiment, the cationic lipid of this disclosure is represented by Formula III: III Or a pharmaceutically acceptable salt thereof; and all other remaining variables as described for any of Formula I, Formula II or the foregoing embodiments.

[0091] In the fifth embodiment, R is in the cationic lipid or a pharmaceutically acceptable salt thereof according to the first embodiment. 1 and R 2 Each is independently hydrogen or C1-C2 alkyl or C2-C3 alkenyl; or R', R 1 and R 2 Each is independently hydrogen, C1-C2 alkyl; and all other remaining variables are as described with respect to Formula I, Formula II, or any of the foregoing embodiments.

[0092] In the sixth embodiment, the cationic lipid of this disclosure is represented by formula IV: IV Or a pharmaceutically acceptable salt thereof; and all other remaining variables as described for any of Formula I, Formula II, Formula III or the foregoing embodiments.

[0093] In the seventh embodiment, R is a cationic lipid, or a pharmaceutically acceptable salt thereof, in accordance with Formula I, Formula II, Formula III, Formula IV, or any of the foregoing embodiments. 5 It does not exist, or is a C1-C8 alkylene group; or R 5 It does not exist; it is a C1-C6 alkylene group or a C2-C6 alkenyl group; or R 5 It does not exist; it is a C1-C4 alkylene group or a C2-C4 alkenyl group; or R 5 Does not exist; or R 5It is C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C6 alkenyl, C5 alkenyl, C4 alkenyl, C3 alkenyl, or 2 alkenyl; and all other remaining variables are as described for any of Formula I, Formula II, Formula III, Formula IV, or any of the foregoing embodiments.

[0094] In the eighth embodiment, the cationic lipid of this disclosure is represented by formula V: V Or a pharmaceutically acceptable salt thereof; and all other remaining variables as described for any of Formula I, Formula II, Formula III, Formula IV or any of the foregoing embodiments.

[0095] In the ninth embodiment, R is a cationic lipid, or a pharmaceutically acceptable salt thereof, in accordance with Formula I, Formula II, Formula III, Formula IV, Formula V, or any of the foregoing embodiments. 4 For C1-C 14 Non-branched alkyl, C2-C 14 non-branched alkenyl or , where R 4a and R 4b Each independently is C1-C 12 Non-branched alkyl or C2-C 12 Non-branched alkenyl; or R 4 For C2-C 12 Non-branched alkyl or C2-C 12 Non-branched alkenyl; or R 4 C5-C 12 Non-branched alkyl or C5-C 12 Non-branched alkenyl; or R 4 C 16 Non-branched alkyl, C 15 Non-branched alkyl, C 14 Non-branched alkyl, C 13 Non-branched alkyl, C 12 Non-branched alkyl, C 11 Non-branched 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 for , where R 4a and R 4b Each independently is C2-C 10 Non-branched alkyl or C2-C 10 Non-branched alkenyl; or R 4 for , where R 4a and R 4b Each independently is C 16 Non-branched alkyl, C 15 Non-branched alkyl, C 14 Non-branched alkyl, C 13 Non-branched alkyl, C 12 Non-branched alkyl, C 11 Non-branched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 Unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl or C2 alkenyl; and all other remaining variables as described with respect to Formula I, Formula II, Formula III, Formula IV, Formula V or any of the foregoing embodiments.

[0096] In the tenth embodiment, R is a cationic lipid, or a pharmaceutically acceptable salt thereof, in accordance with Formula I, Formula II, Formula III, Formula IV, Formula V, or any of the foregoing embodiments. 3 It is a C3-C8 alkylene or C3-C8 alkenylene, a C3-C7 alkylene or C3-C7 alkenylene, or a 3-C5 alkylene or C3-C5 alkenylene; or R 3It is a C8 alkylene, or a C7 alkylene, or a C6 alkylene, or a C5 alkylene, or a C4 alkylene, or a C3 alkylene, or a C1 alkylene, or a C8 alkenyl, or a C7 alkenyl, or a C6 alkenyl, or a C5 alkenyl, or a C4 alkenyl, or a C3 alkenyl; and all other remaining variables are as described for any of Formula I, Formula II, Formula III, Formula IV, Formula V or any of the foregoing embodiments.

[0097] In the eleventh embodiment, R is a cationic lipid according to Formula I, Formula II, Formula III, Formula IV, Formula V or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof. 6a and R 6b Each independently is C7-C 12 Alkyl or C7-C 12 alkenyl; or R 6a and R 6b Each independently is C8-C 10 Alkyl or C8-C 10 alkenyl; or R 6a and R 6b Each independently is C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 alkenyl, C 11 alkenyl, C 10 Alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; and all other remaining variables as described for any of Formula I, Formula II, Formula III, Formula IV, Formula V, or any of the foregoing embodiments.

[0098] In the twelfth embodiment, R is a cationic lipid according to Formula I, Formula II, Formula III, Formula IV, Formula V or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof. 6a and R 6b Contains an equal number of carbon atoms; R 6a and R 6b Same; or R 6a and R 6b All are C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 alkenyl, C 11 alkenyl, C 10 Alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; and all other remaining variables as described for any of Formula I, Formula II, Formula III, Formula IV, Formula V, or any of the foregoing embodiments.

[0099] In the thirteenth embodiment, in the cationic lipid according to Formula I, Formula II, Formula III, Formula V or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof, as defined in any of the foregoing embodiments, R 6a and R 6b Each contains a different number of carbon atoms; or carbon atom R 6a and R 6b The number differs by one or two carbon atoms; or carbon atom R 6a and R 6b The difference in quantity is one carbon atom; or R 6a It is a C7 alkyl group and R 6a It is a C8 alkyl group, R 6a It is a C8 alkyl group and R 6a It is a C7 alkyl group, R 6a It is a C8 alkyl group and R 6a It is a C9 alkyl group, R 6a It is a C9 alkyl group and R 6a It is a C8 alkyl group, R 6a It is a C9 alkyl group and R 6a C 10 Alkyl, R 6a C 10 Alkyl and R 6a It is a C9 alkyl group, R 6a C 10 Alkyl and R 6a C 11 Alkyl, R 6a C 11 Alkyl and R 6a C 10 Alkyl, R 6a C 11 Alkyl and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 11 Alkyl, R 6a It is a C7 alkyl group and R 6a It is a C9 alkyl group, R 6a It is a C9 alkyl group and R 6a It is a C7 alkyl group, R 6a It is a C8 alkyl group and R 6a C 10 Alkyl, R 6a C 10 Alkyl and R 6a It is a C8 alkyl group, R 6a It is a C9 alkyl group and R 6a C 11 Alkyl, R 6a C11 Alkyl and R 6a It is a C9 alkyl group, R 6a C 10 Alkyl and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 10 Alkyl groups, etc.; and all other remaining variables as described for any of the foregoing embodiments of formula I, II, III, IV, V, or the preceding embodiments.

[0100] In the fourteenth embodiment, R' is absent in the cationic lipid according to Formula I, Formula II, Formula III, Formula IV, Formula V, or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula I or any of the foregoing embodiments. In some embodiments, in the cationic lipid according to Formula I, Formula II, Formula III, Formula IV, Formula V, or any of the foregoing embodiments, wherein R' is hydrogen or C1-C6 alkyl, R', R 1 and R 2 All the attached nitrogen atoms are protonated because nitrogen atoms are positively charged.

[0101] In some embodiments, in the cationic lipid according to Formula I, Formula II, Formula III, Formula IV, Formula V or any of the foregoing embodiments, wherein R', R 1 and R 2 Each is a C1-C6 alkyl group, and wherein R' and R' are C1-C6 alkyl groups. 1 and R 2 Together with the nitrogen atom to which it is attached, it forms a quaternary ammonium cation or a quaternary amine.

[0102] In the fifteenth embodiment, a cationic lipid represented by formula Ia is provided: Ia Or its pharmaceutically acceptable salt, wherein: R' is absent, or is a C1-C3 alkyl group; R 1 and R 2 Each is independently hydrogen or C1-C3 alkyl; R 3 For C3-C 10 Alkylene or C3-C 10 alkenyl; R 4 For C1-C 16 Non-branched alkyl or C2-C 16 Non-branched alkenyl groups; R 5 It does not exist; it is either a C1-C6 alkylene group or a C2-C6 alkenyl group. R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 alkenyl; X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-、-N=C(R a )-、-C(R a )=NO-、-ON=C(R a )-、-C(=O)NR a -、-NR a C(=O)-、-NR a C(=O)NR a -、-OC(=O)O-、-OSi(R a )2O-、-C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-; where: R a Each time it appears, it is independently either hydrogen or C. 1-6 Alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0103] In the sixth embodiment, in the cationic lipid or its pharmaceutically acceptable salt according to the fifth embodiment, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-; and all other remaining variables are as described for formula Ia or the fifteenth embodiment.

[0104] In the seventh embodiment, the cationic lipid of this disclosure is represented by formula IIa: IIa 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 of the foregoing embodiments. In an alternative third embodiment, n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula Ia or the sixteenth embodiment.

[0105] In the eighteenth embodiment, the cationic lipid of this disclosure is represented by formula IIIa: IIIa Or a pharmaceutically acceptable salt thereof; and all other remaining variables as described for Formula Ia, Formula IIa, or the fifteenth, sixteenth, or seventeenth embodiments.

[0106] In the nineteenth embodiment, R is present in the cationic lipid or a pharmaceutically acceptable salt thereof according to the first embodiment. 1 and R 2 Each is independently hydrogen or C1-C2 alkyl or C2-C3 alkenyl; or R', R 1 and R 2 Each is independently hydrogen, C1-C2 alkyl; and all other remaining variables are as described with respect to formula Ia, formula IIa or the foregoing embodiments.

[0107] In the twentieth embodiment, the cationic lipid of this disclosure is represented by formula IVa: IVa Or a pharmaceutically acceptable salt thereof; and all other remaining variables as described in any one of the following embodiments: Formula Ia, Formula IIa, Formula IIIa, or the fifteenth, sixteenth, seventeenth, eighteenth, or nineteenth embodiments.

[0108] In the twenty-first embodiment, R is a cationic lipid according to formula Ia, IIa, IIIa, IVa, or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof. 5 It does not exist, or is a C1-C8 alkylene group; or R 5 It does not exist; it is a C1-C6 alkylene group or a C2-C6 alkenyl group; or R 5 It does not exist; it is a C1-C4 alkylene group or a C2-C4 alkenyl group; or R 5 Does not exist; or R 5 It is a C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C6 alkenyl, C5 alkenyl, C4 alkenyl, C3 alkenyl, or 2 alkenyl; and all other remaining variables are as described with respect to any one of the following embodiments: Ia, IIa, IIIa, IVa, or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth embodiments.

[0109] In the twenty-second embodiment, the cationic lipid of this disclosure is represented by the formula Va: Va Or a pharmaceutically acceptable salt thereof; and all other remaining variables as described in any one of the following embodiments: Ia, IIa, IIIa, IVa, or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, or twenty-first embodiments.

[0110] In the twenty-third embodiment, R is a cationic lipid according to formula Ia, IIa, IIIa, IVa, Va, or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof. 4 For C1-C 14 Non-branched alkyl or C2-C 14 Non-branched alkenyl; or R 4 For C2-C 12 Non-branched alkyl or C2-C 12 Non-branched alkenyl; or R 4 C5-C 12 Non-branched alkyl or C5-C 12 Non-branched alkenyl; or R 4 C 16 Non-branched alkyl, C 15 Non-branched alkyl, C 14 Non-branched alkyl, C 13 Non-branched alkyl, C 12 Non-branched alkyl, C 11 Non-branched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 Unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl or C2 alkenyl; and all other remaining variables as described with respect to any one of the following embodiments: Ia, IIa, IIIa, IVa, Va or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first or twenty-second.

[0111] In the twenty-fourth embodiment, R is a cationic lipid according to formula Ia, IIa, IIIa, IVa, Va, or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof. 3 It is a C3-C8 alkylene or C3-C8 alkenylene, a C3-C7 alkylene or C3-C7 alkenylene, or a 3-C5 alkylene or C3-C5 alkenylene; or R 3 It is a C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C1 alkylene, or C8 alkenyl, or C7 alkenyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl; and all other remaining variables are as described with respect to any one of the following embodiments: Ia, IIa, IIIa, IVa, Va, or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, or twenty-third embodiments.

[0112] In the twenty-fifth embodiment, R is a cationic lipid according to formula Ia, IIa, IIIa, IVa, Va, or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof. 6a and R 6b Each independently is C7-C 12 Alkyl or C7-C 12 alkenyl; or R 6a and R 6b Each independently is C8-C 10 Alkyl or C8-C 10 alkenyl; or R 6a and R 6b Each independently is C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 alkenyl, C 11 alkenyl, C 10 Alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; and all other remaining variables as described in any one of the following embodiments: Ia, IIa, IIIa, IVa, Va, or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, or twenty-fourth embodiments.

[0113] In the twenty-sixth embodiment, R is a cationic lipid according to formula Ia, IIa, IIIa, IVa, Va, or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof.6a and R 6b Contains an equal number of carbon atoms; R 6a and R 6b Same; or R 6a and R 6b All are C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 alkenyl, C 11 alkenyl, C 10 Alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl; and all other remaining variables as described in any one of the following embodiments: Ia, IIa, IIIa, IVa, Va, or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, or twenty-fifth embodiments.

[0114] In the twenty-seventh embodiment, in the cationic lipid according to formula Ia, IIa, IIIa, IVa, Va or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof, as defined in any of the foregoing embodiments, R 6a and R 6b Each contains a different number of carbon atoms; or carbon atom R 6a and R 6b The number differs by one or two carbon atoms; or carbon atom R 6a and R 6b The difference in quantity is one carbon atom; or R 6a It is a C7 alkyl group and R 6a It is a C8 alkyl group, R 6a It is a C8 alkyl group and R 6a It is a C7 alkyl group, R 6a It is a C8 alkyl group and R 6a It is a C9 alkyl group, R 6a It is a C9 alkyl group and R 6a It is a C8 alkyl group, R 6a It is a C9 alkyl group and R 6a C 10 Alkyl, R 6a C 10 Alkyl and R 6a It is a C9 alkyl group, R 6a C 10 Alkyl and R 6a C 11 Alkyl, R 6a C 11 Alkyl and R 6a C10 Alkyl, R 6a C 11 Alkyl and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 11 Alkyl, R 6a It is a C7 alkyl group and R 6a It is a C9 alkyl group, R 6a It is a C9 alkyl group and R 6a It is a C7 alkyl group, R 6a It is a C8 alkyl group and R 6a C 10 Alkyl, R 6a C 10 Alkyl and R 6a It is a C8 alkyl group, R 6a It is a C9 alkyl group and R 6a C 11 Alkyl, R 6a C 11 Alkyl and R 6a It is a C9 alkyl group, R 6a C 10 Alkyl and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 10 Alkyl groups, etc.; and all other remaining variables as described in any one of the following embodiments: Ia, IIa, IIIa, IVa, Va, or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, or twenty-sixth embodiments.

[0115] In the twenty-eighth embodiment, R' is absent in the cationic lipid or pharmaceutically acceptable salt thereof according to formula Ia, IIa, IIIa, IVa, Va or any of the preceding embodiments; and all other remaining variables are as described in any of the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth or twenty-seventh embodiments of formula Ia.

[0116] In the twenty-ninth embodiment, R' is absent in the cationic lipid according to formula Ia, IIa, IIIa, IVa, Va or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof, and R', R 1 and R 2 All attached nitrogen atoms are protonated when the lipids are present at, for example, a pH of about 7.4 or lower, such as physiological conditions at a pH of about 7.4; and all other remaining variables are as described in any one of the following embodiments: Ia or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, or twenty-eighth.

[0117] In the thirtieth embodiment, R' is absent in the cationic lipid according to formula Ia, IIa, IIIa, IVa, Va or any of the foregoing embodiments, or in a pharmaceutically acceptable salt thereof, and R', R 1 and R 2 All attached nitrogen atoms are protonated when the lipid is present in an aqueous solution; and all other remaining variables are as described in any one of the following embodiments: Ia or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, or twenty-ninth embodiments.

[0118] In the thirty-first embodiment, in the cationic lipid according to formula Ia, IIa, IIIa, IVa, Va or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof, R' is absent, and R', R 1 and R 2 All attached nitrogen atoms are protonated in the presence of lipids at a pH of about 7.4 or lower; and all other remaining variables are as described in any one of the following embodiments: Ia or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, or thirtieth.

[0119] In the thirty-second embodiment, R' is absent in the cationic lipid according to formula Ia, IIa, IIIa, IVa, Va or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof, and R', R 1 and R 2 All attached nitrogen atoms are protonated in the presence of lipids in an aqueous solution and at a pH of about 7.4 or lower (e.g., a pH of about 7.4); and all other remaining variables are as described in any one of the following embodiments: Ia or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-eighth, twenty-ninth, thirtieth, or thirty-first embodiment.

[0120] In the thirty-third embodiment, in the cationic lipid according to formula Ia, IIa, IIIa, IVa, Va or any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof, wherein R', R 1 and R 2 Together with the nitrogen atom to which it is attached, it forms a quaternary ammonium cation or a quaternary amine; and all other remaining variables are as described in any one of the following embodiments: Ia or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirtieth, thirty-first, or thirty-second.

[0121] In some embodiments, in the cationic lipid according to any one of formula Ia, IIa, IIIa, IVa, Va, or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirtieth, thirty-first, thirty-second, or thirty-third embodiments, wherein R' is hydrogen or a C1-C6 alkyl group, R', R 1 and R 2 All the attached nitrogen atoms are protonated because the nitrogen atom carries a positive charge.

[0122] In some embodiments, in the cationic lipid according to any one of Formula Ia, Formula IIa, Formula IIIa, Formula IVa, Formula Va or the fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirtieth, thirty-first, thirty-second or thirty-third embodiments, wherein R', R 1 and R 2 Each is a C1-C6 alkyl group, and wherein R' and R' are C1-C6 alkyl groups. 1 and R 2 Together with the nitrogen atom to which it is attached, it forms a quaternary ammonium cation or a quaternary amine.

[0123] In one embodiment, the cationic lipid of this disclosure, or the cationic lipid of formula I or Ia, is: 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid, 11-yl dodecanoic acid ester (Lipid 1); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid tridecyl-12-yl ester (Lipid 2); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid nonadecanoyl ester (Lipid 3); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid pentadecane-13-yl ester (Lipid 4); 8-((2-(dimethylamino)ethyl)(heptyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 5); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 6); 6-((2-(dimethylamino)ethyl)(nonyl)amino)hexanoic acid 3-decyltridecyl ester (Lipid 7); 3-Octylundecyl ester of 6-((2-(dimethylamino)ethyl)(nonyl)amino)hexanoic acid (Lipid 8); 8-((2-(dimethylamino)ethyl)(undecyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 9); 8-(decyl(2-(dimethylamino)ethyl)amino)octanoic acid heptadecano-9-yl ester (Lipid 10); and 8-((2-(dimethylamino)ethyl)(octyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 11); Or its pharmaceutically acceptable salt.

[0124] Furthermore, lipids of Formula I, II, III, IV, V, Ia, IIa, IIIa, IVa, Va, or pharmaceutically acceptable salts thereof (e.g., quaternary ammonium salts), or any exemplary lipids disclosed herein, can be converted into corresponding lipids comprising quaternary ammonium or quaternary ammonium cations, namely R', ... 1 and R 2 Each is a C1-C6 alkyl group (all considered in this disclosure), for example by treatment with chloromethane (CH3Cl) in acetonitrile (CH3CN) and chloroform (CHCl3). The quaternary ammonium cations in these lipids are permanently charged, regardless of the pH of their solution.

[0125] In some implementations, when lipids are present under physiological conditions, such as a pH of about 7.4 or lower, the nitrogen atom of any one of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated.

[0126] In some embodiments, when the lipids are present in an aqueous solution, the nitrogen atom of any one of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated.

[0127] In some embodiments, when the lipids are present at a pH of about 7.4 or lower (e.g., a pH of about 7.4), the nitrogen atom of any one of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated.

[0128] In some embodiments, when the lipids are present in an aqueous solution and at a pH of about 7.4 or lower (e.g., a pH of about 7.4), the nitrogen atom of any one of lipid 1, lipid 2, lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 8, lipid 9, lipid 10, or lipid 11 is protonated.

[0129] III. Lipid nanoparticles (LNP) LNP As a delivery carrier for nucleic acids Lipid nanoparticles (LNPs) comprising the cationic lipids described herein and uncoated nonviral vectors or therapeutic nucleic acids (TNAs) (e.g., ceDNA) or pharmaceutical compositions thereof may be used to deliver uncoated nonviral DNA vectors to target sites of interest (e.g., cells, tissues, organs, etc.). Therefore, another aspect of this disclosure relates to lipid nanoparticles (LNPs) comprising one or more of the cationic lipids described herein or pharmaceutically acceptable salts thereof, and therapeutic nucleic acids (TNAs).

[0130] Generally, cationic lipids are commonly used to condense nucleic acid cargoes, such as ceDNA, under low pH conditions and to drive membrane association and fusion. Typically, cationic lipids are lipids comprising at least one amino group that is positively charged or protonated under acidic conditions (e.g., at pH 6.5 or lower) to form lipids containing quaternary ammonium.

[0131] In any embodiment of any aspect or embodiment herein, the lipid nanoparticles contain, for example, cationic lipids or pharmaceutically acceptable salts thereof as provided herein, at about 30% to about 80%, such as about 35% to about 80%, about 40% to about 80%, about 45% to about 80%, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, 30% to about 75%, about 35% to about 75%, about 40% to about 75%, about 45% to about 75%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, 30% to about 70%, about 35% to about 70%, about 40% to about 70%, about 45% to about 70%, about 50% to about 70%, about 55% to about 70%, about 55% to about 80%, about 55% to about 80%, about 45% to about 70%, about 50% to about 70%, about 55% to about 80%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%, about 55% to about 80%, about 30% to about 70%, about 35% to about 70%, about 40% to about 70%, about 45% to about 70%, about 50% to about 70%, about 55% to about 80%, about 50% to about 70%, about 55% to about 80%, about 45% to about 70%, about The molar percentages of 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%, 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% are present. In any embodiment of any aspect or implementation thereof, the lipid nanoparticles contain, in molar percentages such as, the cationic lipids or pharmaceutically acceptable salts thereof provided herein, at about 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%; such as, 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%.

[0132] Sterols In one embodiment of any of the aspects or embodiments herein, in addition to the more cationic lipids or their pharmaceutically acceptable salts and TNA described herein, the LNP described herein also comprises at least one sterol to provide membrane integrity and lipid particle stability. In one embodiment of any of the aspects or embodiments herein, an exemplary sterol that can be used for the lipid particles is cholesterol or a derivative thereof. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholesterol, 5β-codosterol, cholesterolyl-(2'-hydroxy)-ethyl ether, cholesterolyl-(4'-hydroxy)-butyl ether, and 6-ketocholesterol; nonpolar analogs such as 5α-cholestane, cholesterolenone, 5α-cholestanone, 5β-cholestanone, cholesterol decanoate, etc.; and mixtures thereof. In some embodiments of any of the aspects and embodiments herein, the cholesterol derivative is a polar analog such as cholesterolyl-(4'-hydroxy)-butyl ether. In some embodiments of any of the aspects and embodiments herein, the cholesterol derivative is hemisuccinate cholesterol ester (CHEMS).

[0133] Exemplary cholesterol derivatives are described in International Patent Application Publication No. WO2009 / 127060 and U.S. Patent Application Publication No. US2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0134] Other exemplary sterols include β-sitosterol, campesterol, stigmasterol, ergosterol, rapeseed sterol, lupeol, cycloartenol, and derivatives thereof. In one embodiment of any aspect or embodiment herein, an exemplary sterol that can be used for lipid particles is β-sitosterol.

[0135] In any embodiment of any aspect or embodiment herein, the sterol is present in the lipid nanoparticles in a molar percentage of about 20% to about 50%, such as 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 any embodiment of any aspect or implementation thereof, the sterol is present in the lipid nanoparticles in a molar percentage of about 35% to about 45%, or about 40% to about 45%, or about 35% to about 40%; such as, 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%.

[0136] non-cationic lipids In any embodiment of any aspect or embodiment described herein, the lipid nanoparticles (LNPs) further comprise at least one non-cationic lipid. Non-cationic lipids, also known as structural lipids, can be used to increase fusion and also enhance the stability of the LNPs during formation to provide membrane integrity and lipid particle stability. Non-cationic lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Therefore, non-cationic lipids can be neutral, zwitterionic, or anionic lipids.

[0137] Exemplary non-cationic lipids include, but are not limited to, phospholipids such as distearyl-sn-glycerol-phosphoethanolamine, distearyl-phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), and palmitoylphosphatidylethanolamine (P... OPE), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (such as 16-O-monomethylPE), dimethylphosphatidylethanolamine (such as 16-O-dimethylPE), 18-1-transPE, 1-stearoyl- 2-Oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), lecithin choline (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), disorhoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), ditransoleoylphosphatidylethanolamine (SOPE) DEPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE); 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphatidylphosphate, lysophosphatidylcholine, dilinoleylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably derived from a group having a C 10 -C 24 The acyl group of the fatty acid in the carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In any embodiment of any aspect or embodiment herein, the noncationic lipid is selected from one or more of dioleoylphosphatidylcholine (DOPC), distearylphosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

[0138] Other examples of non-cationic lipids suitable for lipid particles (e.g., lipid nanoparticles) include non-phospholipids such as stearamide, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramides, sphingomyelin, etc.

[0139] Additional exemplary descriptions of noncationic lipids are found in International Patent Application Publication No. WO2017 / 099823 and U.S. Patent Application Publication No. US2018 / 0028664, the contents of which are incorporated herein by reference in their entirety.

[0140] In any embodiment of any aspect or embodiment herein, in the lipid nanoparticles, non-cationic lipids are present in amounts of about 2% to about 20%, for example, about 3% to about 20%, about 5% to about 20%, about 7% to about 20%, about 8% to about 20%, about 10% to about 20%, about 12% to about 20%, about 13% to about 20%, about 15% to about 20%, about 17% to about 20%, about 18% to about 20%, about 2% to about 18%, about 3% to about 18%. %, about 5% to about 18%, about 7% to about 18%, about 8% to about 18%, about 10% to about 18%, about 12% to about 18%, about 13% to about 18%, about 15% to about 18%, about 17% to about 18%, about 2% to about 17%, about 3% to about 17%, about 5% to about 17%, about 7% to about 17%, about 8% to about 17%, about 10% to about 17%, about 12% to about 17%, about 13% to about 17%, about 15% to about 17%. About 2% to about 15%, about 3% to about 15%, about 5% to about 15%, about 7% to about 15%, about 8% to about 15%, about 10% to about 15%, about 12% to about 15%, about 13% to about 15%, about 2% to about 13%, about 3% to about 13%, about 5% to about 13%, about 7% to about 13%, about 8% to about 13%, about 10% to about 13%, about 12% to about 13%, about 2% to about 12%, about 3% to about 12%, about 5% to about 1 The molar percentages of 2%, about 7% to about 12%, about 8% to about 12%, about 10% to about 12%, about 2% to about 10%, about 3% to about 10%, about 5% to about 10%, about 7% to about 10%, about 8% to about 10%, about 2% to about 8%, about 3% to about 8%, about 5% to about 8%, about 7% to about 8%, about 2% to about 7%, about 3% to about 7%, about 5% to about 7%, about 2% to about 5%, about 3% to about 5%, or about 2% to about 3% are present. In any embodiment of any aspect or embodiment herein, the non-cationic lipids in the lipid nanoparticles are present in a molar percentage of 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 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%, such as, 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%.

[0141] PEGylated lipids In any embodiment of any aspect or embodiment described herein, the lipid nanoparticles (LNPs) further comprise at least one polyethylene glycol-modified lipid (e.g., one, two, or three). A polyethylene glycol-modified lipid is a lipid as defined herein, covalently or non-covalently linked to one or more polyethylene glycol (PEG) polymer chains, and is therefore a class of conjugated lipids. Typically, polyethylene glycol-modified lipids are incorporated into LNPs to inhibit particle aggregation and / or provide steric stability. In any embodiment of any aspect or embodiment described herein, the lipid is covalently linked to one or more PEG polymer chains.

[0142] Suitable PEG molecules for polyethylene glycol-modified lipids include, but are not limited to, those with molecular weights between about 500 and about 10,000, or between about 1,000 and about 7,500, or between about 1,000 and about 5,000, or between about 2,000 and about 5,000, or between about 2,000 and about 4,000, or between about 2,000 and about 3,500, or between about 2,000 and about 3,000; for example, PEG2000, PEG2500, PEG3000, PEG3350, PEG3500 and PEG4000.

[0143] The lipids linked to one or more PEG chains can be sterols, noncationic lipids, or phospholipids. Exemplary PEGylated lipids include (but are not limited to): PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramides (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG-dialkoxypropylcarbamate, N-(carbonyl-methoxy-polyethylene glycol) Sodium 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (2000) or mixtures thereof. Additional exemplary PEGylated lipids are described in, for example, U.S. Patent Nos. 5,885,613 and 6,287,591, and U.S. Patent Application Publications Nos. 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, and 2017 / 0119904, the entire contents of which are incorporated herein by reference.

[0144] In any embodiment of any aspect or embodiment of this document, at least one polyethylene glycol-modified lipid in the lipid nanoparticles (LNPs) provided herein is selected from the group consisting of: PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dispalmityloxypropyl; PEG-distearyloxypropyl; l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol-PEG (DMG-PEG); distearyl-racemic-glycerol-PEG (DSG-PEG); PEG-dilaurylglycerol; PEG-dispalmitoylglycerol; PEG-distearylglycerol ; PEG-dilauroyl saccharamide; PEG-dimyristoyl saccharamide; PEG-dipalmitoyl saccharamide; PEG-distearyl saccharamide; (l-[8'-(cholesterol-5-en-3[β]-oxy)formamido-3',6'-dioxooctanoyl]carbamoyl-[ω]-methyl-poly(ethylene glycol) (PEG-cholesterol); 3,4-bistetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N[methoxy(poly(ethylene glycol)) (DSPE-PEG), and 1,2-dimyristoyl- sn-glycerol-3-phosphate ethanolamine-N-poly(ethylene glycol)-hydroxy (DSPE-PEG-OH). In any embodiment of any aspect or embodiment herein, the at least one PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, DSG-PEG, or a combination thereof. In any embodiment of any aspect or embodiment herein, the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSG-PEG2000, or... Combinations of these. In any embodiment of any aspect or embodiment herein, the lipid nanoparticles (LNPs) provided herein comprise DMG-PEG2000 and DSPE-PEG2000. In any embodiment of any aspect or embodiment herein, the lipid nanoparticles (LNPs) provided herein comprise DMG-PEG2000 and DSG-PEG2000. In any embodiment of any aspect or embodiment herein, the lipid nanoparticles (LNPs) provided herein comprise DSPE-PEG2000 and DSPE-PEG2000-OH.

[0145] In any embodiment of any aspect or embodiment herein, in the lipid nanoparticles, at least one polyethylene glycol-modified lipid is present in amounts of about 1% to 10%, such as 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 ... The molar percentages of about 0.5% 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 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% are present. In any embodiment of any aspect or embodiment herein, in the lipid nanoparticles, at least one polyethylene glycol-modified lipid is present in total at a molar percentage of about 1% to about 2%, about 1.5% to about 2%, or about 1% to about 1.5%; such as, 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%.

[0146] In any embodiment of any aspect or embodiment herein, in the lipid nanoparticles, at least one polyethylene glycol-modified lipid is present in an amount of about 2.1% to 10%, such as 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 10%, etc. The molar percentages of 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% are present. In any embodiment of any aspect or embodiment herein, in the lipid nanoparticles, at least one polyethylene glycol-modified lipid comprises a total amount of 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 It is present in molar percentages such as, 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%.

[0147] Tissue-specific targeting ligands and PEGylated lipid conjugates In any embodiment of any aspect or implementation thereof, the lipid nanoparticles (LNPs) described herein further comprise at least one tissue-specific targeting ligand for the purpose of aiding, enhancing, and / or increasing the delivery of the LNP to the target site of interest. The ligand may be any biomolecule, such as a peptide, protein, antibody, glycan, sugar, nucleic acid, lipid, or conjugate comprising any of the foregoing substances, that recognizes a receptor or surface antigen specific to a particular cell and tissue.

[0148] In any embodiment of any aspect or embodiment of this document, 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, wherein one or more GalNAc molecules (natural or modified) are covalently linked to one or more functional groups or one or more classes of exemplary biomolecules, such as, but not limited to, peptides, proteins, antibodies, glycans, sugars, nucleic acids, and lipids. The biomolecules to which one or more GalNAc molecules can be conjugated generally contribute to increased stability and / or inhibition of aggregation. In any embodiment of any aspect or embodiment herein, the molar ratio between the tissue-specific target ligand (such as GalNAc) and the biomolecule conjugated to the ligand 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 any embodiment of any aspect or embodiment herein, the molar ratio between the tissue-specific target ligand (such as GalNAc) and the biomolecule conjugated to the ligand is 1:1 (e.g., monoanthalamic GalNAc), 2:1 (dianthalamic GalNAc), 3:1 (trianthalamic GalNAc), and 4:1 (tetraanthalamic GalNAc). Conjugated GalNAcs such as trianthal GalNAc (GalNAc3) or tetraanthal GalNAc (GalNAc4) can be synthesized as is known in the art (see WO2017 / 084987 and WO2013 / 166121) and chemically conjugated to lipids or PEG as is well known in the art (see Resen et al., "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and inVivo" (2001), Vol. 276, pp. 375577-37584).

[0149] In any embodiment of any aspect or embodiment herein, a tissue-specific targeting ligand is covalently linked to a polyethylene glycol-modified lipid as defined and described herein to form a polyethylene glycol-modified lipid conjugate. Exemplary polyethylene glycol-modified lipids are as described above and include PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dispalmityloxypropyl; PEG-distearyloxypropyl; l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG); PEG-dilaurylglycerol; PEG-dispalmitoylglycerol; PEG-distearylglycerol; PEG-dilauryl glycoamide; PEG-dimyristoyl glycoamide; PEG-dispalmitoyl glycoamide; PEG-distearyl glycoamide; (l-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxanoic acid) [Acyl]carbamoyl-[ω]-methyl-poly(ethylene glycol) (PEG-cholesterol); 3,4-bistetradecylbenzyl-[ω]-methyl-poly(ethylene glycol) ether (PEG-DMB); 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG); and 1,2-distearateyl-sn-glycerol-3-phosphate ethanolamine-N-poly(ethylene glycol)-hydroxy (DSPE-PEG-OH). In any embodiment of any aspect or embodiment herein, the lipid nanoparticles (LNPs) provided herein comprise DMG-PEG2000 and DSPE-PEG. EG2000. In any embodiment of any aspect or embodiment herein, the tissue-specific targeting ligand is covalently linked to GalNAc or a GalNAc derivative. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is a single-antenna, double-antenna, triple-antenna, or tetra-antenna GalNAc-DSPE-PEG. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is a single-antenna, double-antenna, triple-antenna, or tetra-antenna GalNAc-DSG-PEG. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate... The PEGylated lipid conjugate is a single-antenna, double-antenna, triple-antenna, or quadruple-antenna GalNAc-DSPE-PEG2000. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is a single-antenna, double-antenna, triple-antenna, or quadruple-antenna GalNAc-DSG-PEG2000. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is a triple-antenna GalNAc-DSPE-PEG2000. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is a triple-antenna GalNAc-DSG-PEG2000.In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is tetra-antenna GalNAc-DSPE-PEG2000. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is tetra-antenna GalNAc-DSG-PEG2000.

[0150] In any embodiment of any aspect or embodiment herein, the polyethylene glycol-modified lipid conjugate in the lipid nanoparticles comprises, for example, about 0.1% to about 10%, 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%, and about 5% to about 10%. Approximately 5.5% to approximately 10%, approximately 6% to approximately 10%, approximately 6.5% to approximately 10%, approximately 7% to approximately 10%, approximately 7.5% to approximately 10%, approximately 8% to approximately 10%, approximately 8.5% to approximately 10%, approximately 9% to approximately 10%, approximately 9.5% to approximately 10%, approximately 0.1% to approximately 5%, approximately 0.2% to approximately 5%, approximately 0.3% to approximately 5%, approximately 0.4% to approximately 5%, approximately 0.5% to approximately 5%, approximately 0.6% to approximately 5%, approximately 0.7% to approximately 5%, approximately 0.8% to approximately 5%, approximately 0.9% to approximately 10%, approximately 1% to approximately 5%, approximately 1.5% to approximately 5%, approximately 2% to approximately 5%, approximately 2.5% to approximately 5%, approximately 3% to approximately 5%, approximately 3.5% to approximately 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 The molar percentages of 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% are present.In any embodiment of any aspect or embodiment herein, the polyethylene glycol-modified lipid conjugate in the lipid nanoparticles comprises approximately 0.1% to approximately 1.5%, approximately 0.2% to approximately 1.5%, approximately 0.3% to approximately 1.5%, approximately 0.4% to approximately 1.5%, approximately 0.5% to approximately 1.5%, approximately 0.6% to approximately 1.5%, approximately 0.7% to approximately 1.5%, approximately 0.8% to approximately 1.5%, approximately 0.9% to approximately 1.5%, approximately 1% to approximately 1.5%, approximately 0.1% to approximately 1%, and approximately 0.2%. It is present in molar percentages of 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%; such as, 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%.

[0151] Lipid nanoparticles ( LNP Other components Additional components of LNPs, such as conjugated lipids, are also considered in this disclosure. Exemplary conjugated lipids include, but are not limited to, poly(LNPs). Azoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof.

[0152] Furthermore, in any embodiment of any aspect or implementation thereof, the lipid nanoparticles (LNPs) described herein also comprise immunomodulatory compounds, for example, by co-encapsulation within the LNP or by conjugation with a therapeutic nucleic acid or any component of the LNP described above. Immunomodulatory compounds, such as dexamethasone or modified dexamethasone, can help minimize immune responses. In any embodiment of any aspect or implementation thereof, the lipid nanoparticles (LNPs) described herein also comprise dexamethasone palmitate.

[0153] In some embodiments of any of the aspects and embodiments herein, in addition to cationic lipids, the lipid nanoparticles also contain a reagent, such as ceDNA, for concentrating and / or encapsulating nucleic acid cargo. Such a reagent is also referred to herein as a condensing agent or encapsulating agent. There are no limitations; any compound known in the art for condensing and / or encapsulating nucleic acids may be used, provided it is non-fusion. In other words, a reagent capable of condensing and / or encapsulating nucleic acid cargo (e.g., ceDNA) but with little or no fusion activity. Without wishing to be bound by theory, a condensing agent may have some fusion activity when not condensing / encapsulating nucleic acids (e.g., ceDNA), but the lipid nanoparticles encapsulating nucleic acids formed with said condensing agent may be non-fusion.

[0154] Total lipids to nucleic acid ratio Typically, lipid particles (e.g., lipid nanoparticles) are prepared such that the total lipid to therapeutic nucleic acid (mass or weight) ratio of the final particles is 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. Approximately 15:1 to approximately 55:1, Approximately 20:1 to approximately 55:1, Approximately 25:1 to approximately 55:1, Approximately 30:1 to approximately 55:1, Approximately 35:1 to approximately 55:1, Approximately 40:1 to approximately 55:1, Approximately 45:1 to approximately 55:1, Approximately 50:1 to approximately 55:1, Approximately 10:1 to approximately 50:1, Approximately 15:1 to approximately 50:1, Approximately 20:1 to approximately 50:1, Approximately 25:1 to approximately 50:1, Approximately 30:1 to approximately 50:1, Approximately 35:1 to approximately 50:1, Approximately 40:1 to Approximately 50:1, approximately 45:1 to approximately 50:1, approximately 10:1 to approximately 45:1, approximately 15:1 to approximately 45:1, approximately 20:1 to approximately 45:1, approximately 25:1 to approximately 45:1, approximately 30:1 to approximately 45:1, approximately 35:1 to approximately 45:1, approximately 40:1 to approximately 45:1, approximately 10:1 to approximately 40:1, approximately 15:1 to approximately 40:1, approximately 20:1 to approximately 40:1, approximately 25:1 to approximately 40:1, approximately 30:1 to approximately 40:1, approximately 35:1 to approximately 40:1, approximately 10 :1 to about 35:1, about 15:1 to about 35:1, about 20:1 to about 35:1, about 25:1 to about 35:1, about 30:1 to about 35:1, about 10:1 to about 30:1, about 15:1 to about 30:1, about 20:1 to about 30:1, about 25:1 to about 30:1, about 10:1 to about 25:1, about 15:1 to about 25:1, about 20:1 to about 25:1, about 10:1 to about 20:1, about 15:1 to about 20:1 or about 10:1 to about 15:1.

[0155] The amounts of lipids and nucleic acids are adjusted to provide the desired N / P ratio (i.e., the ratio of positively charged polymeric amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups), for example, N / P ratios of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or higher. Typically, the total lipid content of lipid particle formulations can range from about 5 mg / mL to about 30 mg / mL.

[0156] Lipid nanoparticles ( LNP ) size According to some embodiments of any aspect or implementation thereof, the diameter of the LNP ranges from about 40 nm to about 120 nm, for example, 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 120 nm, about 55 nm to about 120 nm, about 40 nm to about 110 nm, about 45 nm to about 110 nm, about 50 nm to about 1 ... 110nm, approximately 60nm to approximately 110nm, approximately 65nm to approximately 110nm, approximately 70nm to approximately 110nm, approximately 75nm to approximately 110nm, approximately 80nm to approximately 110nm, approximately 85nm to approximately 110nm, approximately 90nm to approximately 110nm, approximately 95nm to approximately 110nm, approximately 100nm to approximately 110nm, approximately 105nm to approximately 110nm, approximately 40nm to approximately 100nm, approximately 45nm to approximately 100nm, approximately 50nm to approximately 100nm, approximately 55nm to approximately 100nm, approximately 60nm to approximately 100nm, approximately 65nm to approximately 100nm, approximately 70nm to approximately 100nm, approximately 75nm to approximately 100nm, approximately 80nm to approximately 100nm, approximately 85nm to approximately 100nm, approximately 90nm to approximately 100nm, or approximately 95nm to approximately 100nm.

[0157] According to some embodiments of any aspect or implementation thereof, the diameter of the LNP is less than about 100 nm, for example, 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 80 ...80 nm to about 85 nm, about 80 nm to about 85 nm, about 80 nm to about 85 nm, about 85 nm to about 85 nm, about 80 nm to about 85 nm, about 80 nm to about 85 nm, about 85 nm to about 85 nm, about 80 nm to about 85 nm, about 80 nm to about 85 nm, about 85 nm to about 85 nm, about 80 nm to about 85 nm, about 85 nm to about 85 nm, about 80 nm to about 85 nm, about 85 nm to about 85 nm, about nm to 85nm, about 40nm to 80nm, about 45nm to 80nm, about 50nm to 80nm, about 55nm to 80nm, about 60nm to 80nm, about 65nm to 80nm, about 70nm to 80nm, about 75nm to 80nm, about 40nm to 75nm, about 45nm to 75nm, about 50nm to 75nm, about 55nm to 75nm, about 60nm to 75nm, about 65nm to 75nm, about 70nm to 75nm, about 40nm to 70nm, about 45nm to 70nm, about 50nm to 70nm, about 55nm to 70nm, about 60nm to 70nm or about 65nm to 70nm. In any embodiment of any aspect or implementation thereof, the diameter of the LNP is approximately 60 nm to approximately 85 nm, approximately 65 nm to approximately 85 nm, approximately 70 nm to approximately 85 nm, approximately 75 nm to approximately 85 nm, approximately 80 nm to approximately 85 nm, approximately 60 nm to approximately 80 nm, approximately 65 nm to approximately 80 nm, approximately 70 nm to approximately 80 nm, approximately 75 nm to approximately 80 nm, approximately 60 nm to approximately 75 nm, approximately 65 nm to approximately 75 nm, approximately 70 nm to approximately 75 nm, approximately 60 nm From to about 70nm or from about 65nm to about 70nm; such as, but not limited to, about 60mm, about 61mm, about 62mm, about 63mm, about 64mm, about 65mm, about 66mm, about 67mm, about 68mm, about 69mm, about 70mm, about 71mm, about 72mm, about 73mm, about 74mm, about 75mm, about 76mm, about 77mm, about 78mm, about 79mm, about 80mm, about 81mm, about 82mm, about 83mm, about 84mm or about 85mm.

[0158] In any embodiment of any aspect or implementation thereof, the size of the lipid particles (e.g., lipid nanoparticles) may be determined by quasi-elastic light scattering using, for example, the Malvern Zetasizer Nano ZS system (Malvern, UK).

[0159] Includes cationic lipids, sterols, non-cationic lipids, PEGylated lipids, and optional tissue-specific targeting compounds. body LNP According to some embodiments of any aspect or embodiment of this document, 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 polyethylene glycol-modified lipid as described herein. In one embodiment of any aspect or embodiment of this document, the lipid nanoparticles provided herein consist substantially of at least one cationic lipid, at least one sterol, at least one non-cationic lipid, and at least one polyethylene glycol-modified lipid. In one embodiment of any aspect or embodiment of this document, 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 polyethylene glycol-modified lipid. In one embodiment of any aspect or embodiment of this document, the molar ratio of cationic lipid:sterol:non-cationic lipid:polyethylene glycol-modified lipid is about 48 (±5):10 (±3):41 (±5):2 (±2), for example, about 47.5:10.0:40.7:1.8 or about 47.5:10.0:40.7:3.0.

[0160] According to some embodiments of any aspect or embodiment of this document, the lipid nanoparticles provided herein comprise at least one cationic lipid as described herein, at least one sterol, at least one non-cationic lipid, at least one polyethylene glycol-modified lipid, and a tissue-specific targeting ligand. In one embodiment of any aspect or embodiment of this document, the tissue-specific targeting ligand is GalNAc. In one embodiment of any aspect or embodiment of this document, the lipid nanoparticles provided herein are substantially composed of at least one cationic lipid as described herein, at least one sterol, at least one non-cationic lipid, at least one polyethylene glycol-modified lipid, and a tissue-specific targeting ligand. In one embodiment of any aspect or embodiment of this document, the lipid nanoparticles provided herein consist of at least one cationic lipid as described herein, at least one sterol, at least one non-cationic lipid, at least one polyethylene glycol-modified lipid, and a tissue-specific targeting ligand. In one embodiment of any aspect or embodiment of this document, the tissue-specific targeting ligand is conjugated with the polyethylene glycol-modified lipid to form a polyethylene glycol-modified lipid conjugate. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is a single-antenna, dual-antenna, tri-antenna, or tetra-antenna GalNAc-DSPE-PEG2000. In any embodiment of any aspect or embodiment herein, the PEGylated lipid conjugate is a tetra-antenna GalNAc-DSPE-PEG2000. In any embodiment of any aspect or embodiment 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), for example 47.5:10.0:40.2:1.8:0.5 or 47.5:10.0:39.5:2.5:0.5.

[0161] IV. Therapeutic Nucleic Acids (TNAs) This disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs). Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), clecase-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapies include small circular DNA, small genes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, terminally closed linear double-stranded DNA (ceDNA / CELiD), plasmids, rod particles, and canine bone. ™DNA vectors, simplified immunologically defined gene expression (MIDGE) vectors, nonviral mini-string DNA vectors (linearly-covalently blocked DNA vectors), or dumbbell-shaped DNA minimal vectors (“dumbbell DNA”). Therefore, aspects of this disclosure generally provide ionizable lipid particles (e.g., lipid nanoparticles) comprising TNA.

[0162] This invention also anticipates that siRNAs or miRNAs, which downregulate intracellular levels of specific proteins through a process called RNA interference (RNAi), can serve as nucleic acid therapeutics. After siRNAs or miRNAs are introduced into the cytoplasm of host cells, these double-stranded RNA constructs can bind to a protein called a RISC. The sense strand of the siRNA or miRNA is removed by the RISC complex. When the RISC complex combines with complementary mRNA, it cleaves the mRNA and releases the cleaved strand. RNAi leads to the downregulation of the corresponding protein by inducing specific disruption of the mRNA.

[0163] Antisense oligonucleotides (ASOs) and ribozymes that inhibit the translation of mRNA into proteins can be nucleic acid therapeutics. For antisense constructs, these single-stranded deoxyribonucleic acids have sequences complementary to the target protein mRNA sequence and are able to bind to mRNA via Watson-Crick base pairing. This binding prevents the translation of the target mRNA and / or triggers RNase H degradation of the mRNA transcript. Therefore, antisense oligonucleotides exhibit increased specificity (i.e., downregulation of specific disease-related proteins).

[0164] In any of the methods and compositions provided herein, the therapeutic nucleic acid (TNA) can be a therapeutic RNA. This therapeutic RNA can be an inhibitor of mRNA translation, an RNA interference agent (RNAi), a catalytically active RNA molecule (ribozyme), transfer RNA (tRNA), or an RNA, protein, or other molecular ligand (aptamer) that binds to mRNA transcripts (ASO). In any of the methods provided herein, the RNAi agent can be double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, short hairpin RNA, or triplet-forming oligonucleotides.

[0165] In any method composition provided herein, the therapeutic nucleic acid (TNA) is a therapeutic DNA, such as terminally closed double-stranded DNA (e.g., ceDNA, CELiD, linearly covalently closed DNA (“mini-string”), doggybone) ™This disclosure includes methods and compositions based on terminally blocked linear double-stranded DNA (ceDNA), such as terminally blocked anterior-particle-bound DNA, dumbbell-shaped linear DNA, plasmids, small circular DNA, etc. Some embodiments of this disclosure are based on methods and compositions comprising terminally blocked linear double-stranded DNA (ceDNA) capable of expressing transgenic molecules (e.g., therapeutic nucleic acids). As described herein, ceDNA vectors do not exhibit the encapsulation limitations imposed by the limited space within the viral capsid. ceDNA vectors are produced by living eukaryotes and represent an alternative to prokaryotically produced plasmid DNA vectors.

[0166] ceDNA vectors preferably have a linear and continuous structure rather than a discontinuous structure. Linear and continuous structures are believed to be more stable under attack by cellular endonucleases and less likely to recombine and induce mutagenesis. Therefore, linear and continuous ceDNA vectors are preferred embodiments. Continuous, linear, single-stranded intramolecular duplex ceDNA vectors may have covalently bound ends without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are bacterial-derived circular duplex nucleic acid molecules. The complementary strand of a plasmid can separate upon denaturation, resulting in two nucleic acid molecules, whereas a ceDNA vector, although having a complementary strand, is a single DNA molecule and therefore remains a single molecule even after denaturation. In some embodiments of any of the aspects and embodiments herein, unlike plasmids, the production of ceDNA vectors may be without prokaryotic DNA base methylation. Therefore, ceDNA vectors and ceDNA plasmids are different in terms of structure (especially linear versus circular), as well as in terms of the methods used to produce and purify these different objects, and also in terms of their DNA methylation, i.e., ceDNA-plasmids are prokaryotic while ceDNA vectors are eukaryotic.

[0167] This document provides nonviral capsid-free ceDNA molecules (ceDNA) with covalently closed ends. These nonviral capsid-free ceDNA molecules can be generated in licensed host cells from expression constructs (e.g., ceDNA plasmids, ceDNA-baculosomes, ceDNA-baculoviruses, or integrative cell lines) containing a heterologous gene (e.g., a transgene, especially a therapeutic transgene) located between two distinct inverted terminal repeat (ITR) sequences, wherein 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 a wild-type ITR sequence (e.g., an AAV ITR); and at least one of the ITRs contains a functional end resolution site (TRS) and a Rep binding site. The ceDNA vector is preferably double-stranded, for example, self-complementary to 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 vector has covalently closed ends and is therefore resistant to digestion by exonucleases (e.g., exonuclease I or exonuclease III), for example, by maintaining it at 37°C for more than one hour.

[0168] In any aspect of this document or any of the embodiments thereof, the ceDNA vector comprises, in the 5' to 3' direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR. In any embodiment of this document or any of the embodiments thereof, the first ITR (5' ITR) and the second ITR (3' ITR) are asymmetrical relative to each other, that is, they have different 3D spatial conformations relative to each other. As an exemplary embodiment, the first ITR may be a wild-type ITR, and the second ITR may be a mutant or modified ITR, or vice versa, wherein the first ITR may be a mutant or modified ITR and the second ITR may be a wild-type ITR. In any embodiment of this document or any of the embodiments thereof, the first ITR and the second ITR are both modified but are different sequences, or have different modifications, or are not the same modified ITR and have different 3D spatial conformations. In other words, a ceDNA vector with asymmetric ITRs has ITRs such that any change in one ITR relative to the WT-ITR is not reflected in the other ITR; or alternatively, the asymmetric ITRs with modified asymmetric ITR pairs may have sequences and different three-dimensional shapes relative to each other.

[0169] In any embodiment of any aspect or implementation thereof, the ceDNA vector comprises, in the 5' to 3' orientation: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, wherein the first ITR (5' ITR) and the second ITR (3' ITR) are symmetrical or substantially symmetrical with respect to each other; that is, the ceDNA vector is capable of including ITR sequences having a symmetrical three-dimensional spatial configuration such that their structures have the same shape in geometric space, or have the same A, C-C', and B-B' loops in 3D space. In such an embodiment, the symmetrical or substantially symmetrical ITR pair may be a modified ITR (e.g., a mod-ITR), which is not a wild-type ITR. A mod-ITR pair may have the same sequence with one or more modifications relative to the wild-type ITR and be inverted complementary (inverted) to each other. In any embodiment of any aspect or embodiment herein, the modified ITR pair is substantially symmetrical as defined herein, meaning that the modified ITR pair may have different sequences but have corresponding or identical symmetrical three-dimensional shapes. In some embodiments of any aspect or embodiment herein, the symmetrical or substantially symmetrical ITR may be wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs from the same AAV serotype. In any embodiment of any aspect or embodiment herein, one WT-ITR may be from one AAV serotype, while the other WT-ITR may be 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 conserved nucleotide modifications while still retaining a symmetrical three-dimensional spatial organization.

[0170] The wild-type, mutant, or otherwise modified ITR sequences provided herein represent the DNA sequences included in the expression constructs (e.g., ceDNA-plasmids, ceDNA-baculosomes, ceDNA-baculoviruses) used to generate ceDNA vectors. Therefore, the actual ITR sequences contained in ceDNA vectors generated from ceDNA-plasmids or other expression constructs may be the same as or different from the ITR sequences provided herein due to naturally occurring changes (e.g., replication errors) during the generation process.

[0171] In any embodiment of any aspect or implementation thereof, the ceDNA vector comprising an expression cassette having a transgene as a therapeutic nucleic acid sequence described herein can be operatively linked to one or more regulatory sequences that allow or control the expression of the transgene. In any embodiment of any aspect or implementation thereof, the polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is side-joined to the first and second ITR sequences, and the first and second ITR sequences are asymmetric to each other or symmetric to each other.

[0172] In any embodiment of any aspect or embodiment herein, the expression cassette is located between two ITRs and comprises, in order, one or more of the following: a promoter operatively linked to the transgene, a post-transcriptional regulatory element, and polyadenylation and termination signals. In any embodiment of any aspect or embodiment herein, the promoter is tunable-inducible or repressible. The promoter can be any sequence that promotes transgene transcription. In any embodiment of any aspect or embodiment herein, the promoter is a CAG promoter or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates transgene expression; by way of non-limiting example, it is any sequence that produces a tertiary structure that enhances the expression of the transgene as a therapeutic nucleic acid sequence.

[0173] In any embodiment of any aspect or embodiment herein, the posttranscriptional regulatory element comprises WPRE. In any embodiment of any aspect or embodiment herein, the polyadenylation and termination signal comprises BGHpolyA. Any cis-regulatory element or combination thereof known in the art may also be used, such as the upstream enhancer sequence (USE) of the SV40 late polyadenylation signal or other posttranscriptional processing elements, including but not limited to the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV). In any embodiment of any aspect or embodiment herein, the expression cassette is longer than the maximum known capsidation length in AAV viral particles in the 5' to 3' direction. In any embodiment of any aspect or embodiment 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 illustrated herein.

[0174] In any embodiment of any aspect or implementation thereof, the expression cassette may comprise more than 4,000 nucleotides, such as about 5,000 nucleotides, about 10,000 nucleotides, or about 20,000 nucleotides, or about 30,000 nucleotides, or about 40,000 nucleotides, or about 50,000 nucleotides, or any range between about 4,000 and 10,000 nucleotides or between 10,000 and 50,000 nucleotides, or more than 50,000 nucleotides.

[0175] In any embodiment of any aspect or embodiment herein, the expression cassette may further include an internal ribosome entry site (IRES) and / or 2A elements. Cis-regulatory elements include (but are not limited to): promoters, riboswitch, isolators, mir-regulatory elements, post-transcriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments of any aspect or embodiment herein, the ITR may act as a promoter for the transgene. In some embodiments of any aspect or embodiment herein, the ceDNA vector includes additional components for regulating transgene expression, such as regulatory switches for controlling and regulating transgene expression, and may, if desired, include regulatory switches as kill switches, thereby enabling controlled cell death in cells containing the ceDNA vector.

[0176] In any embodiment of any aspect or implementation thereof, the ceDNA vector is uncapped and may be obtained from a plasmid that sequentially encodes a first ITR, an expressible transgenic cassette, and a second ITR, wherein at least one of the first and / or second ITR sequences is mutated relative to the corresponding wild-type AAV2 ITR sequence.

[0177] In any embodiment of any aspect or implementation thereof, the ceDNA vector disclosed herein is used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary use) or for immunogenic peptides.

[0178] The expression cassette may include any transgene as a therapeutic nucleic acid sequence. In some embodiments, the ceDNA vector includes any gene of interest to the subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0179] In any embodiment of any aspect or implementation thereof, the sequence provided in the expression cassette, expression construct, or donor sequence of the ceDNA vector described herein may be codon-optimized for host cells. As used herein, the term "codon optimization" or "codon optimization" refers to the process of modifying a nucleic acid sequence to enhance its expression in the cells of a vertebrate species of interest by replacing at least one, more than one, or a large number of codons in a natural sequence (e.g., a prokaryotic sequence) with the most frequently or most frequently used codons in the genes of such vertebrates as mice or humans. Various species exhibit specific preferences for certain codons of specific amino acids.

[0180] Typically, codon optimization does not alter the amino acid sequence of the original translated protein. This can be achieved using, for example, Aptagen's GeneForge.® Codon optimization and custom gene synthesis platforms (Aptagen, 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or other public databases were used to determine optimized codons.

[0181] Many organisms favor using specific codons to encode specific amino acids for insertion into the peptide chain during growth. Codon preference, or codon bias (the difference in codon use between organisms), is provided by the degeneracy of the genetic code and is documented in many organisms. Codon bias is generally associated with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the characteristics of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of the chosen tRNA in the cell largely reflects the most frequently used codons in peptide synthesis. Therefore, it is possible to tailor genes based on codon optimization to optimize gene expression in a given organism.

[0182] Given the vast number of gene sequences available for use in a wide variety of animal, plant, and microbial species, it is possible to calculate the relative frequency of codon usage (Nakamura, Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucleic Acids Res., Vol. 28, p. 292 (2000)).

[0183] Inverted terminal repeat sequence ( ITR ) As described herein, ceDNA vectors are capsid-free linear double-stranded DNA molecules formed from a continuous strand of complementary DNA (linear, continuous, and capsid-free) with covalently closed ends, comprising distinct or asymmetric 5' inverted terminal repeat (ITR) sequences and 3' ITR sequences relative to each other. At least one ITR includes a functional terminal unwinding site and a replication protein binding site (RPS) (sometimes called a replication protein binding site), such as a Rep binding site. Typically, ceDNA vectors include at least one modified AAV inverted terminal repeat (ITR), i.e., a deletion, insertion, and / or substitution relative to another ITR, and an expressible transgene.

[0184] In any embodiment of any aspect or implementation thereof, at least one of the ITRs is an AAVITR, such as a wild-type AAV ITR. In any embodiment of any aspect or implementation thereof, at least one of the ITRs is a modified ITR relative to another ITR—that is, the ceDNA contains ITRs that are asymmetric relative to each other. In any embodiment of any aspect or implementation thereof, at least one ITR is a non-functional ITR.

[0185] In any embodiment of any aspect or embodiment 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 operatively linked to at least one transgene, and (3) two self-complementary sequences, such as ITRs, side-attached to the expression cassette, wherein the ceDNA vector does not associate with a capsid protein. In some embodiments of any aspect or embodiment herein, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, wherein at least one comprises an operational Rep-binding element (RBE) and a terminal unwinding site (TRS) or a functional variant of the RBE, and one or more cis-regulatory elements operatively linked to the transgene. In some embodiments of any aspect or embodiment herein, the ceDNA vector comprises additional components for regulating transgene expression, such as a regulatory switch for controlling and regulating transgene expression, and may include a regulatory switch that is a cytotoxic switch capable of causing controlled cell death in cells containing the ceDNA vector.

[0186] In any embodiment of any aspect or embodiment herein, the two self-complementary sequences may be ITR sequences from any known parvovirus, such as AAV-dependent viruses (e.g., AAV1-AAV12). Any AAV serotype may be used, including (but not limited to) modified AAV2 ITR sequences that, in addition to allowing for the formation of hairpin secondary structures, retain Rep binding sites (RBS), such as 5'-GCGCGCTCGCGCTCTC-3' and terminal unwinding sites (TRS). In some embodiments of any aspect or embodiment herein, the ITR may be synthetic. In one embodiment of any aspect or embodiment herein, the synthetic ITR is based on ITR sequences from more than one AAV serotype. In another embodiment, the synthetic ITR does not include AAV-based sequences. In yet another embodiment, the synthetic ITR retains the ITR structure described above, although it may have only some or no sequences derived from AAV. In some aspects, a synthetic ITR may preferentially interact with wild-type Rep or Rep of a specific serotype, or in some cases, may not be recognized by wild-type Rep and may only be recognized by mutated Rep. In some embodiments of any of the aspects and embodiments herein, the ITR is a synthetic ITR sequence that, in addition to a variable palindromic sequence that allows for the formation of a hairpin secondary structure, retains functional Rep binding sites (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' and terminal unwinding sites (TRS). In some examples, the modified ITR sequence retains the sequences of the RBS, TRS, and the structure and location of the Rep binding element from the corresponding sequence of the wild-type AAV2 ITR, forming a terminal loop portion of an ITR hairpin secondary structure within the ITR hairpin secondary structure. Exemplary ITR sequences for ceDNA vectors are disclosed in Tables 2 through 9, Tables 10A and 10B, SEQ ID NOs: 2, 52, 101-449, and 545-547, and partial ITR sequences are shown in Figures 26A and 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 include an ITR having modifications in the ITR corresponding to any of the modifications shown in any one or more of the ITR sequences or partial ITR sequences shown in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B of International Patent Application No. PCT / US2018 / 049996, filed September 7, 2018.

[0187] In any embodiment of any aspect or embodiment herein, the ceDNA vector may be generated from an expression construct that also includes a specific combination of cis-regulatory elements. Cis-regulatory elements include (but are not limited to): promoters, riboswitch, isolator, miR-regulatory elements, posttranscriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments of any aspect or embodiment herein, the ITR may act as a promoter for the transgene. In some embodiments of any aspect or embodiment herein, the ceDNA vector includes additional components for regulating transgene expression, such as a regulatory switch for regulating transgene expression as described in International Patent Application No. PCT / US2018 / 049996, filed September 7, 2018, or a killer switch capable of killing cells including the ceDNA vector.

[0188] In any embodiment of any aspect or embodiment of this document, the expression cassette may further include a post-transcriptional element to enhance transgene expression. In any embodiment of any aspect or embodiment of this document, a marmot hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to enhance transgene expression. Other post-transcriptional processing elements may be used, such as thymidine kinase genes from herpes simplex virus or post-transcriptional elements from hepatitis B virus (HBV). Secreted sequences may be linked to the transgene, for example, VH-02 and VK-A26 sequences. The expression cassette may include polyadenylated sequences or variants thereof known in the art, such as naturally occurring sequences isolated from bovine BGHpA or viral SV40pA, or synthetic sequences. Some expression cassettes may also include an upstream enhancer (USE) sequence for the SV40 late poly-A signal. The USE may be used in combination with SV40pA or a heterologous poly-A signal.

[0189] International application No. PCT / US2018 / 050042, filed on September 7, 2018, and incorporated herein by full reference. Figure 1 A-1C illustrates a schematic diagram of the corresponding sequence of a non-limiting exemplary ceDNA vector or ceDNA plasmid. The ceDNA vector is uncoated and can be obtained from a plasmid encoded in the following order: a first ITR, an expressible transgenic cassette, and a second ITR, wherein at least one of the first and / or second ITR sequences is mutated relative to the corresponding wild-type AAV2ITR sequence. The expressible transgenic cassette preferably includes one or more of the following in sequence: an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyadenylate).

[0190] promoter Suitable promoters, including those described above, can be derived from viruses and thus can be called viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression via any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter (such as the CMV immediate early promoter region (CMVTE), the Rous sarcoma virus (RSV) promoter, the human U6 small nucleus promoter (U6, e.g., Miyagishi et al., Nature Biotechnology, Vol. 20, pp. 497-500 (2002)), and enhanced U6 promoters (e.g., Xia et al., Nucleic Acids). (Res.) September 1, 2003; Volume 31, Issue 17), human H1 promoter (H1), CAG promoter, human α1-antitrypsin (HAAT) promoter (e.g., etc.). In any embodiment of any aspect or embodiment herein, these promoters are modified at their downstream intron-containing ends to include one or more nuclease cleavage sites. In any embodiment of any aspect or embodiment herein, the DNA containing the nuclease cleavage site is independent of the promoter DNA.

[0191] In any embodiment of any aspect or implementation thereof, the promoter may include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or transient expression. The promoter may also include terminal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. The promoter may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate the expression of a gene relative to the cell, tissue, or organ in which expression occurs, or relative 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 phage T7 promoter, phage T3 promoter, SP6 promoter, lac operon-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 promoters listed below. Such promoters and / or enhancers can be used to express any gene of interest (e.g., a therapeutic protein). For example, a vector may include a promoter operatively linked to a nucleic acid sequence encoding a therapeutic protein. In any embodiment of any aspect or embodiment herein, the promoter operatively linked to the therapeutic protein encoding the sequence may be a promoter derived from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter (such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter), a Molone virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter (such as the CMV immediate early promoter), an EBV promoter, or a Rae's sarcoma virus (RSV) promoter. In any embodiment of any aspect or embodiment herein, the promoter may also be a promoter derived from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human heme, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a liver-specific promoter, such as natural or synthetic human α1-antitrypsin (HAAT) or transthyretin (TTR). In any embodiment of any aspect or implementation thereof, the use of endogenous ApoE to specifically target hepatocytes via low-density lipoprotein (LDL) receptors present on the surface of hepatocytes enables delivery to the liver.

[0192] In any embodiment of any aspect or embodiment herein, the promoter used is a natural promoter of a gene encoding a therapeutic protein. Promoters and other regulatory sequences of the corresponding gene encoding the therapeutic protein are known and have been characterized. The promoter region used may also include one or more additional regulatory sequences (e.g., natural), such as enhancers known in the art (e.g., serine protease inhibitor enhancers).

[0193] Non-limiting examples of suitable promoters used according to the invention include, for example, the HAAT promoter, the human EF1-α promoter, or a CAG promoter consisting of fragments of the EF1-α promoter and the rat EF1-α promoter.

[0194] Polyadenylated sequence The ceDNA vector may include a sequence encoding a polyadenylated sequence to stabilize the mRNA expressed by the ceDNA vector and facilitate nuclear export and translation. In any embodiment of any aspect or embodiment herein, the ceDNA vector does not include a polyadenylated sequence. In other embodiments, the vector includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments of any aspect or embodiment herein, the polyadenylated sequence includes about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0195] In any embodiment of any aspect or implementation thereof, ceDNA may be obtained from a vector polynucleotide encoding a heteronucleotide operably located between two distinct inverted terminal repeat sequences (ITRs) (e.g., AAV ITRs), wherein at least one ITR includes a terminal unwinding site and a replication protein binding site (RPS), such as a Rep binding site (e.g., wt AAV ITR), and one of the ITRs includes deletions, insertions, and / or substitutions relative to the other ITR (e.g., a functional ITR).

[0196] In any embodiment of any aspect or embodiment herein, the host cell does not express viral capsid proteins and the polynucleotide vector template does not contain any viral capsid coding sequence. In any embodiment of any aspect or embodiment herein, the polynucleotide vector template does not contain the AAV capsid gene, nor does it contain capsid genes of other viruses. In any embodiment of any aspect or embodiment herein, the nucleic acid molecule also does not contain the AAVRep protein coding sequence. Therefore, in some embodiments of any aspect and embodiment herein, the nucleic acid molecule of the present invention does not contain either the functional AAV cap or AAV rep genes.

[0197] In any embodiment of any aspect of this document or of any of the embodiments, the ceDNA vector does not have a modified ITR.

[0198] In any embodiment of any aspect or implementation thereof, the ceDNA vector includes a regulatory switch as disclosed herein (or in International Patent Application No. PCT / US2018 / 049996, filed September 7, 2018).

[0199] V. Production of ceDNA vectors The method for generating ceDNA vectors comprising asymmetric or symmetric ITR pairs as defined herein is described in Part IV of PCT / US2018 / 049996, filed September 7, 2018, which is incorporated herein by reference in its entirety. As described herein, ceDNA vectors can be obtained, for example, by a method comprising the steps of: a) incubating a population of host cells (e.g., insect cells) having a polynucleotide expression construct template (e.g., ceDNA plasmid, ceDNA baculosome, and / or ceDNA baculovirus) without a viral capsid coding sequence in the presence of a Rep protein, under conditions sufficient to induce ceDNA vector generation within host cells and sustained for a time sufficient to induce ceDNA vector generation within host cells; and wherein the host cells do not contain a viral capsid coding sequence; and b) harvesting and isolating the ceDNA vector from the host cells. The presence of the Rep protein induces replication of the vector polynucleotide having a modified ITR, thereby generating the ceDNA vector in the host cells.

[0200] However, no viral particles (e.g., AAV viral particles) are expressed. Therefore, there are no size limitations, such as those naturally imposed in AAV or other virus-based vectors.

[0201] The presence of the ceDNA vector isolated from the host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme having a single recognition site on the ceDNA vector, and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of characteristic bands of linear and continuous DNA compared with linear and discontinuous DNA.

[0202] In any embodiment of any aspect or implementation thereof, the present invention provides the use of a host cell line in the production of a nonviral DNA vector in which a DNA vector polynucleotide expression template (ceDNA template) has been stably integrated into its own genome, as described, for example, in Lee, L. et al. (2013) Plos One 8(8): e69879. Preferably, Rep is added to the host cell at an MOI of about 3. When the host cell line is a mammalian cell line, such as 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 the Rep protein into the cell, such that the ceDNA is excised and amplified in the presence of Rep and a helper virus.

[0203] In one embodiment of any aspect or implementation thereof, the host cell used to prepare the ceDNA vector described herein is an insect cell, and a baculovirus is used to deliver a polynucleotide encoding the Rep protein and a template for a nonviral DNA vector polynucleotide expression construct for ceDNA. In some embodiments of any aspect or implementation thereof, the host cell is engineered to express the Rep protein.

[0204] The ceDNA vector is then harvested and isolated from the host cells. The timing of cell collection and harvesting of the ceDNA vector described herein can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvesting time can be selected based on cell viability, cell morphology, cell growth, etc. In any embodiment of any aspect or embodiment herein, cells are grown under conditions sufficient for ceDNA vector production and harvested after baculovirus infection for a time sufficient for ceDNA vector production, but before most cells begin to die due to baculovirus toxicity. The DNA vector can be isolated using plasmid purification kits, such as the Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation are also applicable to DNA vectors. Generally, any nucleic acid purification method can be used.

[0205] DNA vectors can be purified by any means known to those skilled in the art for purifying DNA. In any embodiment of any aspect or embodiment herein, the ceDNA vector is purified into DNA molecules. In any embodiment of any aspect or embodiment herein, the ceDNA vector is purified into exosomes or microparticles. The presence of the ceDNA vector can be confirmed by digesting the vector DNA isolated from cells using a restriction enzyme having a single recognition site for the DNA vector, and by analyzing the digested and undigested DNA material using gel electrophoresis, thereby confirming the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[0206] VI. Preparation of lipid particles Lipid particles (e.g., lipid nanoparticles) can spontaneously form when TNA (e.g., ceDNA) and lipids are mixed. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane (e.g., 100 nm cutoff) using, for example, a hot barrel 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.

[0207] Typically, lipid particles (e.g., lipid nanoparticles) can be formed by any method known in the art. For example, lipid particles (e.g., lipid nanoparticles) can be prepared by methods described, for example, in U.S. Patent Application Publications US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129, and US2010 / 0130588, the entire contents of each of which are incorporated herein by reference. In some embodiments of any of the aspects and embodiments herein, lipid particles (e.g., lipid nanoparticles) can be prepared using a continuous mixing method, a direct dilution method, or an online dilution method. Methods and apparatus for preparing lipid nanoparticles using direct dilution and online dilution methods are described in US2007 / 0042031, the contents of which are incorporated herein by reference in their entirety. U.S. Patent Application Publication No. US2004 / 0142025 describes a method and apparatus for preparing lipid nanoparticles using a stepwise dilution method, the entire contents of which are incorporated herein by reference.

[0208] In any embodiment of any aspect or embodiment herein, lipid particles (e.g., lipid nanoparticles) may be prepared by an impact jetting method. Typically, the particles are formed by mixing lipids dissolved in an alcohol (e.g., ethanol) with ceDNA dissolved in a buffer, such as citrate buffer, sodium acetate buffer, sodium acetate and magnesium chloride buffer, malate buffer, malate and sodium chloride buffer, or sodium citrate and sodium chloride buffer. The lipid-to-ceDNA mixing ratio may be approximately 45%–55% lipids and approximately 65%–45% ceDNA.

[0209] The lipid solution may contain disclosed cationic lipids, non-cationic lipids (e.g., phospholipids, such as DSPC, DOPE, and DOPC), one or more polyethylene glycol-modified lipids, and sterols (e.g., cholesterol), with a total lipid concentration in an alcohol (e.g., ethanol) of 5 mg / mL to 30 mg / mL, more likely 5 mg / mL to 15 mg / mL, and most likely 9 mg / mL to 12 mg / mL. In the lipid solution, the molar ratio of lipids may be approximately 25% to 98% for cationic lipids, such as approximately 35% to 65%; approximately 0% to 15% for nonionic lipids, such as approximately 0% to 12%; approximately 0% to 15% for polyethylene glycol-modified lipids, such as approximately 1% to 6%; and approximately 0% to 75% for sterols, such as approximately 30% to 50%.

[0210] The ceDNA solution may include a ceDNA buffer solution with a concentration range of 0.3 mg / mL to 1.0 mg / mL, preferably 0.3 mg / mL to 0.9 mg / mL, and a pH range of 3.5 to 5.

[0211] To form LNPs, in one exemplary but non-limiting embodiment, two liquids are heated to a temperature of about 15°C-40°C, preferably about 30°C-40°C, and then mixed, for example, in an impingement jet mixer, to immediately form LNPs. The mixing flow rate can be in the range of 10 mL / min-600 mL / min. The tube ID can have a range of 0.25 mm to 1.0 mm and a total flow rate of 10 mL / min-600 mL / min. The combination of flow rate and tube ID can have the effect of controlling the particle size of LNPs between 30 nm and 200 nm. The solution can then be mixed with a buffer solution of higher pH in a mixing ratio in the range of 1:1 to 1:3 vol:vol, preferably about 1:2 vol:vol. If desired, the temperature of this buffer solution can be in the range of 15°C-40°C or 30°C-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 min to 2 hours. The temperature during incubation can be in the range of 15℃-40℃ or 30-40℃. After incubation, the solution is filtered through a filter, such as a 0.8µm filter, which includes an anion exchange separation step. This process can be performed using tube inner diameters of 1mmID to 5mmID and flow rates of 10 to 2000 mL / min.

[0212] After formation, LNP can be concentrated and percolated through an ultrafiltration process, in which the alcohol is removed and the buffer is replaced with a final buffer solution, such as phosphate-buffered saline (PBS) at approximately pH 7 (e.g., approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4).

[0213] Ultrafiltration can be performed using tangential flow filtration (TFF) with membranes having a nominal molecular weight cutoff of 30kD-500kD. The membrane form can be hollow fiber or a plate-and-sheet container. A TFF process with an appropriate molecular weight cutoff retains LNPs in the osmotic buffer, and the filtrate or permeate contains alcohol, citrate buffer waste, and final buffer waste. The TFF process is a multi-step process with an initial ceDNA concentration of 1 mg / mL-3 mg / mL. After concentration, the LNP solution is perfused with the final buffer at 10-20 volumes to remove alcohol and perform buffer exchange. The material can then be concentrated 1-3 times. The concentrated LNP solution can be aseptically filtered.

[0214] VII. Pharmaceutical compositions and formulations This document also provides pharmaceutical compositions comprising TNA lipid particles and pharmaceutically acceptable carriers or excipients. In any embodiment of any aspect or embodiment of this document, the invention also relates to pharmaceutical compositions comprising cationic lipids as described in any embodiment of any aspect or embodiment of this document, or lipid nanoparticles as described in any embodiment of any aspect or embodiment of this document, and pharmaceutically acceptable excipients.

[0215] Typically, the average diameter of the lipid particles (e.g., lipid nanoparticles) of the present invention is selected to achieve the desired therapeutic effect.

[0216] Depending on the intended use of the lipid particles (e.g., lipid nanoparticles), the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using, for example, endosome release parameter (ERP) assays.

[0217] In any embodiment of any aspect or embodiment herein, ceDNA may be complexed with or encapsulated at lipid sites of the lipid particles (e.g., lipid nanoparticles). In any embodiment of any aspect or embodiment herein, ceDNA may be completely encapsulated at lipid sites of the lipid particles (e.g., lipid nanoparticles), thereby protecting it from degradation by nucleases, for example, in aqueous solutions. In any embodiment of any aspect or embodiment herein, the ceDNA in the lipid particles (e.g., lipid nanoparticles) is substantially undegraded after the lipid particles (e.g., lipid nanoparticles) are exposed to nucleases at 37°C for at least about 20, 30, 45, or 60 minutes. In some embodiments of any of the aspects and embodiments herein, the ceDNA in the lipid particles (e.g., lipid nanoparticles) is substantially not degraded after the particles are incubated in serum at 37°C for at least about 30 minutes, 45 minutes, or 60 minutes, or at least about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.

[0218] In any embodiment of any aspect or implementation thereof, the lipid particles (e.g., lipid nanoparticles) are substantially nontoxic to the subject, such as to mammals like humans.

[0219] In any embodiment of any aspect or embodiment herein, a pharmaceutical composition comprising the therapeutic nucleic acid of this disclosure may be formulated in lipid particles (e.g., lipid nanoparticles). In some embodiments of any aspect and embodiment herein, the lipid particles comprising the therapeutic nucleic acid may be formed from the disclosed cationic lipids. In some other embodiments, the lipid particles comprising the therapeutic nucleic acid may be formed from non-cationic lipids. In a preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles formed from the disclosed cationic lipids comprising a therapeutic nucleic acid selected from the group consisting of: mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), cleats-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), small circular DNA, small genes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, terminally closed linear double-helix DNA (ceDNA / CELiD), plasmids, rod particles, doggybone ™ DNA vectors, simplified immunologically defined gene expression (MIDGE) vectors, nonviral mini-string DNA vectors (linearly covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors (“dumbbell DNA”).

[0220] In another preferred embodiment, the lipid particles of the present invention are lipid particles containing nucleic acids, which are formed from non-cationic lipids and optionally polyethylene glycol-modified lipids or other forms of conjugated lipids that prevent particle aggregation.

[0221] In any embodiment of any aspect or implementation thereof, the lipid particle formulation is an aqueous solution. In any embodiment of any aspect or implementation thereof, the lipid particle (e.g., lipid nanoparticle) formulation is a lyophilized powder.

[0222] According to some aspects, this disclosure provides a lipid particle formulation that further includes one or more pharmaceutical excipients. In any embodiment of any aspect or embodiment herein, the lipid particle (e.g., lipid nanoparticle) formulation further includes sucrose, tris, trehalose, and / or glycine.

[0223] In any embodiment of any aspect or embodiment herein, the lipid particles (e.g., lipid nanoparticles) disclosed herein may 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 any embodiment of any aspect or embodiment herein, the TNA lipid particles (e.g., lipid nanoparticles) disclosed herein may be incorporated into a pharmaceutical composition suitable for a desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via hyperbaric intravenous or intra-arterial infusion and intracellular injection such as intranuclear microinjection or intracytoplasmic injection is also covered. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA carrier concentrations. Sterile injectable solutions can be prepared by incorporating the desired amount of the ceDNA carrier compound, as needed, together with one or a combination of the ingredients listed above, into an appropriate buffer solution, followed by filtration sterilization.

[0224] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intraamniotic, intrasheath, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intrabrain), intrasheath, intrabladder, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, anterior chamber, and vitreous), cochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via hyperbaric intravenous or intraarterial infusion and intracellular injection such as intranuclear microinjection or intracytoplasmic injection is also covered.

[0225] Pharmaceutically active compositions comprising TNA lipid particles (e.g., lipid nanoparticles) can be formulated to deliver transgenes in nucleic acids into recipient cells, thereby inducing therapeutic expression of the transgenes therein. The compositions may also include pharmaceutically acceptable carriers.

[0226] Pharmaceutical compositions intended for therapeutic purposes are generally sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA carrier concentrations. Sterile injectable solutions can be prepared by incorporating the desired amount of the ceDNA carrier compound, as needed, together with one or a combination of the ingredients listed above, into an appropriate buffer solution, followed by filtration sterilization.

[0227] In any embodiment of any aspect or embodiment of this document, the lipid particle (e.g., lipid nanoparticle) is a solid core particle having at least one lipid bilayer. In any embodiment of any aspect or embodiment of this document, the lipid particle (e.g., lipid nanoparticle) has a non-bilayer structure, i.e., a non-layered (i.e., non-bilayer) morphology. Without limitation, this non-bilayer morphology may include, for example, three-dimensional tubes, rods, cubic symmetry, etc. The non-layered morphology (i.e., non-bilayer structure) of the 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 particle (layered versus non-layered) can be readily evaluated and characterized using, for example, Cryo-TEM analysis as described in content US2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0228] In any embodiment of any aspect or implementation thereof, lipid particles having a non-sheet morphology (e.g., lipid nanoparticles) are electronically dense.

[0229] In any embodiment of any aspect or embodiment herein, this disclosure provides lipid particles (e.g., lipid nanoparticles) that are structurally monolayered or multilayered. In some aspects, this disclosure provides formulations of lipid particles (e.g., lipid nanoparticles) comprising multivesicular particles and / or foam-based particles. By controlling the composition and concentration of the lipid components, the rate at which conjugated lipids are exchanged from the lipid particles can be controlled, thereby controlling the rate of fusion of the lipid particles (e.g., lipid nanoparticles). Furthermore, other variables, including, for example, pH, temperature, or ionic strength, can be used to alter and / or control the rate of fusion of the lipid particles (e.g., lipid nanoparticles). Based on this disclosure, those skilled in the art will appreciate other methods that can be used to control the rate of fusion of lipid particles (e.g., lipid nanoparticles). It is also apparent that the size of the lipid particles can be controlled by controlling the composition and concentration of the conjugated lipids.

[0230] In any embodiment of any aspect or embodiment herein, the pKa of the formulated cationic lipid may be correlated with the efficacy of LNP delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), Vol. 51, No. 34, pp. 8529-8533; Semil et al., Nature Biotechnology, Vol. 28, pp. 172-176 (2010), both of which are incorporated herein by reference in their entirety). In any embodiment of any aspect or embodiment herein, the preferred range of pKa is from about 5 to about 8. In any embodiment of any aspect or embodiment herein, the preferred range of pKa is from about 6 to about 7. In any embodiment of any aspect or embodiment herein, the preferred pKa is about 6.5. In any embodiment of any aspect or embodiment herein, the pKa of the cationic lipid in lipid particles (e.g., lipid nanoparticles) can be determined using a fluorescence assay based on 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS).

[0231] In any embodiment of any aspect of this document or of any of the embodiments, the encapsulation of ceDNA in lipid particles (e.g., lipid nanoparticles) can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as Oligreen. ® Determination method or PicoGreen ® The assay utilizes a dye that enhances fluorescence upon association with nucleic acids. Encapsulation is typically determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to fluorescence observed after adding a small amount of nonionic detergent. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ceDNA, allowing it to interact with the dye on the impermeable membrane. The encapsulation of ceDNA can be calculated as E = (Io - I) / Io, where I and Io represent the fluorescence intensity before and after the addition of the detergent.

[0232] unit dose In any embodiment of any of the aspects or embodiments described herein, the pharmaceutical composition may be present in a unit dosage form. The unit dose will generally be suitable for one or more specific routes of administration of the pharmaceutical composition. In some embodiments of any of the aspects and embodiments described herein, the unit dosage form is suitable for administration by inhalation. In some embodiments of any of the aspects and embodiments described herein, the unit dosage form is suitable for administration by a vaporizer. In some embodiments of any of the aspects and embodiments described herein, the unit dosage form is suitable for administration by a nebulizer. In some embodiments of any of the aspects and embodiments described herein, the unit dosage form is suitable for administration by aerosol. In some embodiments of any of the aspects and embodiments described herein, the unit dose is suitable for oral administration, buccal administration, or sublingual administration. In some embodiments of any of the aspects and embodiments described herein, the unit dosage form is suitable for intravenous, intramuscular, or subcutaneous administration. In some embodiments of any of the aspects and embodiments described herein, the unit dosage form is suitable for intrathecal or intraventricular administration. In some embodiments of any of the aspects and embodiments described herein, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient that can be combined with a carrier material to produce a single dose will generally be the amount by which the compound produces a therapeutic effect.

[0233] VIII. Treatment Methods The lipid nanoparticles and methods described herein (e.g., TNA lipid particles (e.g., lipid nanoparticles) as described herein) can be used to introduce nucleic acid sequences (e.g., therapeutic nucleic acid sequences) into host cells. In any embodiment of any aspect or implementation thereof, the introduction of nucleic acid sequences into host cells using TNALNP (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles) as described herein) can be monitored for gene expression assessment using appropriate biomarkers from the treated patient.

[0234] The LNP compositions provided herein can be used to deliver transgenic (nucleic acid sequences) for a variety of purposes. In any embodiment of any aspect or embodiment herein, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein (e.g., lipid nanoparticles)) can be used in a variety of ways, including, for example, non-in situ, in vitro and in vivo applications, methods, diagnostic procedures and / or gene therapy regimens.

[0235] This document provides a method for treating a disease or condition in a subject, comprising introducing a therapeutically effective amount of a TNAL NP (e.g., a ceDNA carrier lipid particle (e.g., a lipid nanoparticle) described herein), optionally with a pharmaceutically acceptable carrier, into target cells (e.g., hepatocytes, muscle cells, kidney cells, neurons, or other affected cell types) desired by the subject. The implemented TNA LNP (e.g., a ceDNA carrier lipid particle (e.g., a lipid nanoparticle) described herein) contains a nucleotide sequence of interest for treating the disease. Specifically, the TNA may include a desired exogenous DNA sequence operatively linked to a control element capable of directing the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into the subject. The TNA LNP (e.g., a ceDNA carrier lipid particle (e.g., a lipid nanoparticle) described herein) may be administered via any suitable route described herein and known in the art. In any embodiment of any aspect or embodiment described herein, the target cells are in a human subject.

[0236] This document provides a method for providing a diagnostically or therapeutically effective amount of TNA LNPs (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles) as described herein) to a subject in need. The method comprises providing a specified amount of TNA LNPs (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles) as described herein) to the intracellular, tissue, or organ of the subject in need; and sustaining this for a period of time to enable transgene expression from the TNA LNPs, thereby providing the subject with a diagnostically or therapeutically effective amount of proteins, peptides, or nucleotides expressed by the TNA LNPs (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles) as described herein). In any embodiment of any aspect or implementation thereof, the subject is a human.

[0237] This document provides methods for diagnosing, preventing, treating, or improving at least one or more symptoms of a disease, condition, dysfunction, injury, abnormality, or trauma in a subject. Typically, the method includes at least the step of administering a TNA LNP (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles)) to a subject in need, in an amount and for a duration sufficient to diagnose, prevent, treat, or improve at least one or more symptoms of the subject's disease, condition, dysfunction, injury, abnormality, or trauma. In any embodiment of any aspect or implementation thereof, the subject is a human.

[0238] This document provides methods that use TNA LNPs as a tool to treat symptoms of one or more diseases or disease states. Defective genes in many genetic diseases are known and are generally classified into two categories: defective states, typically enzymes, which are generally inherited in a recessive manner; and imbalanced states, which may involve regulatory or structural proteins and are generally, but not always, inherited in a dominant manner. For defective state diseases, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be used to deliver transgenes to introduce normal genes into affected tissues for alternative therapy, and in some embodiments of any of the aspects and embodiments described herein, to establish animal disease models using antisense mutations. For imbalanced disease states, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to establish the disease state in a model system, which can then be used to counteract the disease state. Therefore, the TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) and methods disclosed herein are capable of treating genetic diseases. As used herein, disease states can be treated by partially or completely rescuing defects or imbalances that cause or exacerbate the disease.

[0239] Generally, TNA LNPs (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles)) can be used to deliver any transgene as described above to treat, prevent, or improve symptoms associated with any condition involving gene expression. Descriptive 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 dystrophy (e.g., Duchenne muscular dystrophy, Becker's disease), Heller's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondrial diseases (e.g., Lebow's hereditary optic neuropathy (LHON), Reye's syndrome, and subacute sclerotic encephalopathy), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathy), diseases of solid organs (e.g., brain, liver, kidney, heart), etc. In some embodiments of any of the aspects and implementations herein, such as the ceDNA vector disclosed herein, the ceDNA vector may be advantageously used to treat subjects suffering from metabolic disorders (e.g., ornithine carbamoyltransferase deficiency).

[0240] In any embodiment of any aspect or implementation thereof, the TNA LNP described herein may be used to treat, improve, and / or prevent diseases or conditions caused by mutations in genes or gene products. Examples of diseases or conditions that can be treated with TNA LNPs (e.g., ceDNA carrier lipid particles as described herein (e.g., lipid nanoparticles)) include, but are not limited to, metabolic diseases or conditions (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage diseases); urea cycle diseases or conditions (e.g., ornithine carbamoyltransferase (OTC) deficiency); lysosomal storage diseases or conditions (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Hunter syndrome)); liver diseases or conditions (e.g., progressive familial intrahepatic cholestasis (PFIC)); blood diseases or conditions (e.g., hemophilia A and B, thalassemia and anemia); cancers and tumors; and genetic diseases or conditions (e.g., cystic fibrosis).

[0241] In any embodiment of any aspect or implementation thereof, a TNA LNP (e.g., a ceDNA carrier lipid particle) may be used to deliver a heterologous nucleotide sequence when it is necessary to regulate the expression level of a transgene (e.g., a transgene encoding a hormone or growth factor).

[0242] In any embodiment of any aspect or implementation thereof, a TNA LNP (e.g., a ceDNA carrier lipid particle (e.g., a lipid nanoparticle)) may be used to correct abnormal levels and / or functions (e.g., protein deletion or defect) of gene products that cause a disease or condition. A TNA LNP (e.g., a ceDNA carrier lipid particle (e.g., a lipid nanoparticle)) may produce functional proteins and / or alter protein levels to alleviate or reduce symptoms of a specific disease or condition caused by protein deficiency or defect, or to provide benefits thereof. For example, treatment for OTC deficiency can be achieved by producing functional OTC enzymes; treatment for hemophilia A and B can be achieved by altering the levels of factors VIII, IX, and X; treatment for PKU can be achieved by altering the level of phenylalanine hydroxylase; treatment for Fabry disease or Gaucher disease can be achieved by producing functional α-galactosidase or β-glucocerebrosidase, respectively; treatment for MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronic acid-2-sulfatase, respectively; treatment for cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conduction regulators; treatment for glycogen storage diseases can be achieved by restoring the function of functional G6Pase enzymes; and treatment for PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

[0243] In any embodiment of any aspect or implementation thereof, a TNA LNP (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles)) can be used to deliver RNA-based therapeutic agents to cells in vitro or in vivo. Examples of RNA-based therapies include, but are not limited to, mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), cleats-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). For example, a TNA LNP (e.g., ceDNA carrier lipid particles (e.g., lipid nanoparticles)) can be used to deliver antisense nucleic acids to cells in vitro or in vivo. For example, in the case where the transgene is an RNAi molecule, expression of the antisense nucleic acid or RNAi in the target cells can attenuate the expression of a specific protein. Therefore, to reduce the expression of a specific protein in a subject in need, a transgene, either an RNAi molecule or an antisense nucleic acid, can be administered. Antisense nucleic acids can also be administered in vitro to modulate cell physiology, for example, to optimize cell or tissue culture systems.

[0244] In any embodiment of any aspect or implementation thereof, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver DNA-based therapeutic agents to cells in vitro or in vivo. Examples of DNA-based therapeutic agents include, but are not limited to, small circular DNA, small genes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, terminally closed linear double-stranded DNA (ceDNA / CELiD), plasmids, rod particles, and dog bone. ™ DNA vectors, simplified immunologically defined gene expression (MIDGE) vectors, nonviral mini-string DNA vectors (linearly-covalently blocked DNA vectors), or dumbbell-shaped DNA minimal vectors (“dumbbell DNA”). For example, in any embodiment of any aspect or embodiment herein, a ceDNA vector (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver small loops to cells in vitro or in vivo. For example, in the case where the transgene is a small loop DNA, expression of the small loop DNA in target cells attenuates the expression of a specific protein. Therefore, to reduce the expression of a specific protein in a subject in need, the transgene, as a small loop DNA, can be administered. Small loop DNA can also be administered to cells in vitro to modulate cell physiology, for example, to optimize cell or tissue culture systems.

[0245] In any embodiment of any aspect or implementation thereof, exemplary transgenes encoded by a TNA vector including an expression cassette include, but are not limited to: X, lysosomal enzymes (e.g., hexosaminease A associated with Tay-Sachs disease or Hunter syndrome / MPS). II-related iduronate sulfatase, erythropoietin, angiostatin, endostatin, superoxide dismutase, globulins, leptin, catalase, tyrosine hydroxylase, and cytokines (e.g., interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors and hormones (e.g., growth hormone, insulin, insulin-like growth factor 1 and 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic 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 to one or more desired targets. In some exemplary embodiments, the ceDNA vector encodes more than one transgene. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects and embodiments herein, the transgene encodes an antibody as defined herein, including a full-length antibody or an antibody fragment. In some embodiments of any of the aspects and embodiments herein, the antibody is an antigen-binding domain or immunoglobulin variable domain sequence as defined herein. Other illustrative 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 therapies, and tumor suppressor gene products.

[0246] In any embodiment of any aspect or embodiment herein, this disclosure relates to a method of treating a subject (e.g., a human) with a genetic condition, the method comprising administering to the subject an effective amount of lipid nanoparticles or a pharmaceutical composition thereof as described in any aspect or embodiment herein. In any embodiment of any aspect of this document or of any embodiment thereof, the genetic disease is selected from the group consisting of: sickle cell anemia, melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Schie syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type IHS), Hunter syndrome (MPS type II), Sanfilippo A, B, C and D (MPS type IHS) Types IIIA, 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, mucinous storage disease types I, II / III, and IV, sialic acid storage disease types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, cystinosis, Barton disease, aspartic glucosamineuria, Sala disease, Danon disease (LAMP-2 deficiency), lysosomal acidic lipidosis. LAL deficiency, neuronal ceroid lipofuscin deposition (CLN1-8, INCL, and LINCL), sphingolipid syndrome, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), epidermolysis bullosa (DEB), exonucleotide pyrophosphatase 1 deficiency, systemic arterial calcification in infants (GACI), and Leber congenital amaurosis.Stargardt's macular dystrophy (ABCA4), ornithine transcarbamate (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis of disease (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency. In any embodiment of any aspect or embodiment herein, the genetic condition is hemophilia A. In any embodiment of any aspect or embodiment herein, the genetic condition is hemophilia B. In any embodiment of any aspect or embodiment herein, the genetic condition is phenylketonuria (PKU). In any embodiment of any aspect or embodiment herein, the genetic condition is Wilson's disease. In any embodiment of any aspect or embodiment herein, the genetic condition is Gaucher disease type I, II, or III. In any embodiment of any aspect or embodiment herein, the genetic condition is Stargardt's macular dystrophy. In any embodiment of any aspect of this document or any of the embodiments, the genetic condition is LCA10. In any embodiment of any aspect of this document or any of the embodiments, the genetic condition is Usher syndrome. In any embodiment of any aspect of this document or any of the embodiments, the genetic condition is wet AMD.

[0247] In any of the aspects or embodiments described herein, this disclosure relates to the use of lipid nanoparticles or pharmaceutical compositions thereof as described in any of the aspects or embodiments described herein for the manufacture of a medicament for treating a subject (e.g., a human) with a genetic condition. Exemplary genetic conditions are as described above. In any of the aspects or embodiments described herein, the genetic condition treated by medication is Stargardt's macular dystrophy. In any of the aspects or embodiments described herein, the genetic condition treated by medication is LCA10. In any of the aspects or embodiments described herein, the genetic condition treated by medication is Usher syndrome. In any of the aspects or embodiments described herein, the genetic condition treated by medication is wet AMD.

[0248] In any embodiment of any of the aspects or embodiments herein, this disclosure relates to lipid nanoparticles or pharmaceutical compositions thereof as described in any of the aspects or embodiments herein for treating genetic conditions in a subject (e.g., a human). Exemplary genetic conditions are as described above. In any embodiment of any of the aspects or embodiments herein, the genetic condition treated by the above-described use is Stargardt's macular dystrophy. In any embodiment of any of the aspects or embodiments herein, the genetic condition treated by the above-described use is LCA10. In any embodiment of any of the aspects or embodiments herein, the genetic condition treated by the above-described use is Usher syndrome. In any embodiment of any of the aspects or embodiments herein, the genetic condition treated by the above-described use is wet AMD.

[0249] application In any embodiment of any aspect or embodiment herein, a TNA LNP (e.g., ceDNA carrier lipid particles as described herein) may be administered to an organism for in vivo transduction of cells. In any embodiment of any aspect or embodiment herein, a TNA LNP (e.g., ceDNA carrier lipid particles) may be administered to an organism for in vitro transduction of cells.

[0250] Generally, administration is carried out via any route typically used to bring the molecule into final contact with blood or tissue cells. Suitable methods of administration of such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route generally provides a more direct and efficient response than another. Exemplary administration modes of TNALNP (e.g., ceDNA carrier lipid particles) include oral, rectal, mucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, percutaneous, intraendothelial, intrauterine (or, oocyte), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to skeletal muscle, diaphragmatic muscle, and / or myocardium), intrapleural, intracerebral, and intraarticular), surface (e.g., both skin and mucosal surfaces, including airway surfaces and percutaneous administration), intralymphatic, etc., and direct tissue or organ injection (e.g., to the liver, eyes, skeletal muscle, myocardium, diaphragmatic muscle, or brain).

[0251] TNA LNPs, such as ceDNA vectors (e.g., ceDNA LNPs), can be administered to any site of the subject, including but not limited to sites selected from the group consisting of: brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eyes. In any embodiment of any aspect or implementation thereof, ceDNA LNPs may also be administered to tumors (e.g., within or near tumors or lymph nodes). The most suitable route in any given case will depend on the nature and severity of the symptom being treated, improved, and / or prevented, and the nature of the specific ceDNA LNP used. Additionally, ceDNA allows for the administration of more than one transgene via a single vector or multiple ceDNA vectors (e.g., a mixture of ceDNA vectors).

[0252] In any embodiment of any aspect or implementation thereof, administration of ceDNA LNP to skeletal muscle includes, but is not limited to, administration to skeletal muscle in the limbs (e.g., upper arm, lower arm, thigh, and / or calf), back, neck, head (e.g., tongue), chest, abdomen, pelvis / perineum, and / or fingers. ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be delivered to skeletal muscle via intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion (optionally, isolated limb perfusion of the leg and / or arm; see, for example, Arruda et al., (2005) Blood, Vol. 105, pp. 3458-3464), and / or direct intramuscular injection. In a particular embodiment, ceDNA LNP is administered to the limbs (arms and / or legs) of a subject (e.g., a subject with muscular dystrophy such as DMD) via limb perfusion, optionally isolated limb perfusion (e.g., intravenous or intra-articular administration). In any embodiment of any aspect or implementation thereof, ceDNA LNP may be administered without employing a “hydrodynamic” technique.

[0253] Application of TNA LNPs (e.g., ceDNA LNPs) to the myocardium includes application to the left atrium, right atrium, left ventricle, right ventricle, and / or diaphragm. TNA LNPs (e.g., ceDNA LNPs) can be delivered to the myocardium via 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. Application to the diaphragm muscle can be performed by any suitable method, including intravenous, intra-arterial, and / or intraperitoneal administration. Application to smooth muscle can be performed by any suitable method, including intravenous, intra-arterial, and / or intraperitoneal administration. In any embodiment of any aspect or embodiment herein, application can be made to endothelial cells present within, near, and / or on the smooth muscle.

[0254] In any embodiment of any aspect or implementation thereof, TNA LNP (e.g., ceDNALNP) is applied to skeletal muscle, diaphragm and / or myocardium (e.g., to treat, improve and / or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure).

[0255] TNA LNPs (e.g., ceDNA LNPs) can be administered to the CNS (e.g., to the brain or eye). TNA LNPs (e.g., ceDNA LNPs) can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, including the occipital, temporal, parietal, and frontal lobes of the cerebrum, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and hypothalamus. 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 into the cerebrospinal fluid (e.g., via lumbar puncture). In cases where the blood-brain barrier has been disturbed (e.g., brain tumor or cerebral infarction), TNA LNPs (e.g., ceDNA carrier lipid particles) can be further administered intravascularly to the CNS.

[0256] In any embodiment of any aspect or implementation thereof, TNA LNP (e.g., ceDNALNP) may be administered to the desired region of the CNS via any route known in the art, including but not limited to: intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of sugars such as mannitol), intranasal, intraauricular, intraocular (e.g., vitreous, subretinal, anterior chamber), and periocular (e.g., subcapsular region of the ocular fascia) delivery, as well as intramuscular delivery via retrograde delivery to motor neurons.

[0257] According to some embodiments of any aspect or implementation thereof, a TNA LNP (e.g., ceDNA LNP) is administered as a liquid formulation to a desired area or compartment in the CNS via direct injection (e.g., stereotactic injection). According to other embodiments, a TNA LNP (e.g., ceDNA LNP) can be delivered by local application to the desired area or by intranasal administration of an aerosol formulation. It can also be administered to the eye by local application of droplets. As another alternative, the ceDNA vector can be administered as a solid, sustained-release formulation (see, for example, U.S. Patent No. 7,201,898, the entire contents of which are incorporated herein by reference). In one embodiment of any aspect or implementation thereof, a TNA LNP (e.g., ceDNA LNP) can be used for retrograde transport to treat, improve, and / or prevent diseases and conditions involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.). For example, a TNA LNP (e.g., ceDNA LNP) can be delivered to muscle tissue, from which it can migrate to neurons.

[0258] In any embodiment of any aspect or implementation thereof, the therapeutic product may be repeatedly administered until an appropriate expression level is achieved. Therefore, in any embodiment of any aspect or implementation thereof, the therapeutic nucleic acid may be administered and repeatedly administered. For example, the therapeutic nucleic acid may be administered on day 0. After initial treatment on day 0, the therapeutic nucleic acid may be administered for approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, or approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 7 months, approximately 6 months, approximately 7 months, approximately 8 years, approximately 9 years, approximately 10 years, approximately 11 years, approximately 12 years, approximately 13 years, approximately 14 years, approximately 15 years, approximately 16 years, approximately 17 years, approximately 18 years, approximately 1 ... A second administration (repeated dose) may be administered within approximately 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 years.

[0259] In any embodiment of any aspect or embodiment described herein, one or more additional compounds may also be included. These compounds may be applied alone, or additional compounds may be included in the lipid particles (e.g., lipid nanoparticles) of the invention. In other words, the lipid particles (e.g., lipid nanoparticles) may include compounds other than TNA or a second TNA that is at least different from the first TNA. Without limitation, other additional compounds may be selected from the group consisting of: small or large molecules, organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptide mimics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0260] In any embodiment of any aspect or embodiment herein, one or more additional compounds may be therapeutic agents. Therapeutic agents may be selected from any class suitable for a therapeutic purpose. Therefore, therapeutic agents can be selected from any class suitable for a therapeutic purpose. Therapeutic agents can be selected based on the therapeutic purpose and the desired biological effect. For example, in any of the embodiments or implementations herein, if the TNA within the LNP is available for treating cancer, the additional compound may be an anticancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates)). In any of the embodiments or implementations herein, if the LNP containing TNA is available for treating infection, the additional compound may be an antibacterial agent (e.g., an antibiotic compound or an antiviral compound). In any of the embodiments or implementations herein, if the LNP containing TNA is available for treating an immune disease or condition, the additional compound may be a compound that modulates an immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In any of the embodiments or implementations herein, different mixtures of different lipid particles containing different compounds, such as TNAs encoding different proteins, or different compounds, such as therapeutic agents, may be used in the compositions and methods of the present invention. In any of the embodiments or implementations herein, the additional compound is an immunomodulator. For example, another compound is an immunosuppressive formulation. In some embodiments of any of the embodiments or implementations herein, the additional compound is immunostimulatory.

[0261] Example The following embodiments are provided by way of illustration and not limitation. Those skilled in the art will understand that the range of lipids contemplated in this disclosure can be designed and synthesized using the general synthetic methods described below.

[0262] Example 1: General Synthesis The lipids of synthetic formula I were designed and synthesized using a similar synthetic method as described in Scheme 1 below. All variables in the compounds are shown in Scheme 1, i.e., R. 1 R 2 R 3 R 4 R 5 R 6a R 6b X and n are as defined in Equation I. x Is it as defined by R? 4 However, it is missing one carbon atom in the aliphatic chain.

[0263] Option 1 The monoester lipid disclosed herein, namely Formula I, was designed and synthesized using a similar synthetic method as described in Scheme 2 below, where X is -C(=O)-. All variables in the compound are shown in Scheme 1, i.e., R 1 R 2 R 3 R 4 R 5 R 6a R 6b X and n are as defined in Equation I. x Is it as defined by R? 4 However, it is missing one carbon atom in the aliphatic chain.

[0264] Option 2 plan 1 and plan 2 Referring to Schemes 1 and 2, in step 1, 4-dimethylaminopyridine (DMAP) was added to a stirred solution of acid 2 in dichloromethane (DCM), followed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). The resulting mixture was stirred at room temperature for 15 min under a nitrogen (N2) atmosphere. Then, compound 1 was added dropwise and the mixture was stirred overnight. On the second day, the reaction was diluted with DCM and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and evaporated to dryness. The crude product was purified by silica gel column chromatography using a 0%–10% methanol DCM solution as eluent. Fractions containing the desired compound were combined and evaporated to give compound 3 (0.78 g, 54%).

[0265] In step 2, lithium aluminum hydride (LiAlH4) was added to the tetrahydrofuran (THF) solution in step 3. The reaction mixture was heated at 50°C overnight. On the second day, the reaction was cooled to 0°C and quenched by adding water dropwise. Subsequently, the reaction was filtered through diatomaceous earth to give crude product 4. The product was used in the next step without further purification.

[0266] In step 3, compound 5 or 5' (synthesized according to the procedure described in International Patent Application Publication No. WO2017 / 049245, the entire contents of which are incorporated herein by reference) was dissolved in a dimethylformamide / methanol mixture DMF:MeOH (1:1) and 4 was added. The reaction was stirred overnight at room temperature. The product was extracted with ethyl acetate (EtOAc), and the organic layer was washed with a saturated aqueous solution of sodium bicarbonate (NaHCO3 (aqueous solution)) and brine, and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using a 0%–10% MeOH DCM solution as eluent to give the cationic lipid of formula I.

[0267] Compound 5 or 5' can alternatively be synthesized according to the steps described in Scheme 3 below. y Is it as defined by R? 5 However, it is missing one carbon atom in the aliphatic chain.

[0268] Option 3 According to Scheme 3, pure phosphoric anhydride solution 7 was added dropwise to an ice-cold solution of 9-heptadecene 6 in anhydrous tetrahydrofuran. The reaction mixture was stirred for 30 min, and then NaH was added in portions. The reaction mixture was refluxed, cooled to 0°C, quenched with water, and extracted with ether. The organic layer was washed several times with water and brine, dried over Na2SO4, and concentrated. The crude product was purified by column chromatography to provide 7.1 g (93% yield) of pure product 8.

[0269] Compound 8 was dissolved in an EtOAc / MeOH mixture and reduced with H2 using a wet 10% Pd / C- catalyst. Clean conversion yielded compound 9.

[0270] Compound 9 (THF, cooled, and LiAlH4 added dropwise. The reaction mixture was stirred overnight, heated to room temperature, and then quenched with a THF / H2O mixture (1:1 by volume). The reaction mixture was extracted with EtOAc and filtered through diatomaceous earth. The organic phase was washed twice with water and brine, dried over Na2SO4, and concentrated. Purification by column chromatography (CH2Cl2-EtOAc) yielded compound 10.

[0271] Compound 10 and alkanonic acid 11 were dissolved in DCM, and then DMAP and EDCI were added to this solution at room temperature. After stirring overnight, the reaction was quenched with water, diluted with DCM, and washed with NaHCO3 (saturated aqueous solution) and brine. The organic phase was dried over Na2SO4 and concentrated. Column chromatography purification (hexane-EtOAc) yielded 3.8 g of compound 5 or 5'.

[0272] Example 2: Synthesis of Lipid 6 The procedure for synthesizing lipid 6 is also described below with reference to Scheme 4, which is also provided below.

[0273] Option 4 step 1 : N-(2-( dimethylamino ) Ethyl ) nonanamide ( 3a Synthesis of ) DMAP (0.91 g, 7.5 mmol) was added to a stirred solution of nonanoic acid (2a) (1.0 g, 6.3 mmol) in 60 mL of DCM, followed by EDCI (1.44 g, 7.5 mmol). The resulting mixture was stirred at room temperature for 15 min under a nitrogen atmosphere. Then, N2 was added dropwise. 1 N 1 -Dimethylethane-1,2-diamine (1a) (0.66 g, 7.5 mmol) was reacted and the mixture was stirred overnight. On the second day, the reaction mixture was diluted with DCM and washed with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using a 0%–10% methanol-DCM solution as eluent. Fractions containing the desired compound were combined and evaporated to give compound 3a (0.78 g, 54%).

[0274] step 2 : N 1 ,N 1 - dimethyl -N2- nonyl ethane -1,2- diamine ( 4a Synthesis of ) LiAlH4 was added to a THF solution of 3a (0.78 g, 3.4 mmol). The reaction mixture was heated at 50 °C overnight. On the second day, the reaction was cooled to 0 °C and quenched dropwise with water. The reaction was then filtered through diatomaceous earth to give crude product 4a (0.6 g, 82%). The product was used in the next step without further purification.

[0275] step 3 : 8-((2-( Dimethylamino ) Ethyl )( Renji ) amino ) Heptadecanoate -9- esters or lipids 6 Synthesis Compound 5a (synthesized according to the procedure described in International Patent Application Publication No. WO2017 / 049245, the entire contents of which are incorporated herein by reference) (0.6 g, 1.3 mmol) was dissolved in 20 mL of DMF:MeOH (1:1) and 4a (0.35 g, 1.5 mmol) was added. The reaction was stirred overnight at room temperature. The product was extracted with EtOAc (200 mL), and the organic layer was washed with saturated NaHCO3 (aqueous solution) and brine, and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and purified by column chromatography using a 0%–10% methanol-DCM solution as eluent to give lipid 6 (0.062 g, 10%). 1 ¹H NMR (300 MHz, chloroform-d) δ 4.85 (quintet) J = 6.2Hz, 1H), 2.57 – 2.48 (m, 2H), 2.43 –2.32 (m, 6H), 2.31 – 2.25 (m, J = 7.5Hz, 2H), 2.23 (s, 6H), 1.66 – 1.34 (m,8H), 1.24 (s, 47H), 0.86 (t, J = 6.6Hz, 9H).

[0276] Example 3: Synthesis of Lipid 1 The procedure for synthesizing lipid 1 is described below with reference to Scheme 5, which is also provided below.

[0277] Option 5 Steps 1 and 2 of scheme 5 are as described in embodiment 2.

[0278] 8- Bromooctanoic acid dodecane -11- esters ( 5b Synthesis of ) EDCI (6.1 g, 32.0 mmol) was added to a stirred solution of 10.0 g (10.0 g, 32.0 mmol) and 8-bromooctanoic acid (7.1 g, 44.8 mmol) (both commercially available) in 250 mL of dichloromethane (DCM), followed by DMAP (392 mg, 3.21 mmol). The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. On the second day, the reaction mixture was diluted with DCM and washed with an aqueous solution of NaHCO3 (250 mL) and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using a 0%–10% hexane solution of EtOAc as the eluent. Fractions containing the desired compounds were combined and evaporated to give 5b (6.3 g, 38%). ¹H NMR (300MHz, chloroform-d) δ 4.84–4.88 (m, ¹H), 3.39 (t, J = 6.0Hz, 2H), 2.28 (t, J = 6.0Hz, 2H), 1.80–1.89 (m, 2H), 1.25–1.62 (m, 43H), 0.86 (t, J = 6.0Hz, 6H).

[0279] step 3 : 8-((2-( dimethylamino ) Ethyl )( Renji ) amino ) Octanodecane -11- esters or lipids 1 of synthesis Compound 5b (4.34 g, 8.41 mmol) was dissolved in 5.0 mL of DMF:MeOH (1:1) and 4a (2.0 g, 9.35 mmol) was added. The reaction was stirred overnight at room temperature. The solvent was evaporated under vacuum and purified by column chromatography using a 0%–10% methanol-DCM solution as the eluent to give lipid 1 (330 mg, 11%). 1 ¹H NMR (300MHz, chloroform-d) δ 4.84–4.93 m (¹H), 3.51–3.55 m (4H), 2.98–3.03 m (4H), 2.83 s (6H), 2.26 t (t). J = 6.0Hz, 2H), 1.48 – 1.77 (m, 8H), 1.23-1.44 (m, 57H), 0.86 (t, J =6.0Hz, 9H).

[0280] Example 4: Synthesis of Lipid 3 The procedure for synthesizing lipid 3 is described below with reference to Scheme 6, which is also provided below.

[0281] Option 6 Steps 1 and 2 of Scheme 6 are as described in Example 3.

[0282] 8- Bromooctanoic acid pentadecane -13- esters ( 5c Synthesis of ) Compound 5c was synthesized using a similar procedure to that described above for the synthesis of 8-bromooctanoic acid dodecane-11-yl ester (5b), by replacing the starting material dodecane-11-ol with a commercially available pentadecane-13-ol.

[0283] step 3 : 8-((2-( Dimethylamino ) Ethyl )( Renji ) amino ) Octanodecane -13- esters or lipids 3 The combination become Lipid 3 was prepared by replacing starting material 5b with compound 5c using a similar procedure to that described above for the synthesis of lipid 1.

[0284] Example 5: Synthesis of Lipid 7 The procedure for synthesizing lipid 7 is described below with reference to Scheme 7, which is also provided below.

[0285] Option 7 step 1 : N-(2-( dimethylamino ) Ethyl ) heptamethamide ( 3b Synthesis of ) EDCI (20 g, 104 mmol) was added to a stirred solution of heptanoic acid (2b) (7.0 g, 80 mmol) in 20 mL of DCM. The resulting mixture was stirred at room temperature under a N2 atmosphere for 15 min. Then, 1a (7.1 g, 80 mmol) dissolved in 10 mL of DCM was added, followed by DMAP (0.3 g, 2.5 mmol), and stirring was continued overnight. On the second day, the reaction mixture was diluted with DCM and washed with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using a 0%–10% methanol DCM solution as eluent. Fractions containing the desired compounds were combined and evaporated to give compound 3b (9.5 g, 59%). 1 ¹H NMR (300MHz, chloroform-d) δ 6.0 (wide s, 1H), 3.3 (dd, 2H), 2.4 (dd, J= 6, 2H), 2.2 (s, 6H), 2.16 (dd, J=6, 2H), 1.9–1.5 (m, 4H), 1.3–1.2 (m, 7H), 0.87 (t, 3H).

[0286] step 2 : N 1 - Gengji -N 2 ,N 2 - dimethyl ethane -1,2- diamine ( 4b Synthesis of ) A solution of LiAlH42M in THF (15 mL, 30 mmol) at 0 °C was added to a solution of 3b (3 g, 15 mmol) in THF (80 mL). The reaction mixture was heated to reflux overnight. On the second day, the reaction was cooled to 0 °C and quenched dropwise with water (3 mL). The reaction was then filtered through diatomaceous earth to give crude product 4b. The crude product was purified by silica gel column chromatography using a 0%–10% methanol / NH3 (0.1%) DCM solution as eluent. Fractions containing the desired compounds were combined and evaporated to give 4b (1.3 g, 46%). 1 ¹H NMR (300MHz, chloroform-d) δ 2.67 (dd, J=6, 2H), 2.59 (dd, J=7, 2H), 2.40 (dd, J=6, 2H), 2.20 (s, 6H), 1.50–1.40 (m, 3H), 1.30–1.15 (m, 9H), 0.87 (t, 3H). [C 11 H 26 The measured MS value of N2 was 187.2 [M+H]. + The calculated value is 186.3.

[0287] step 3 : 8-((2-( dimethylamino ) Ethyl )( Gengji ) amino ) Heptadecanoate -9- esters or lipids 7 The combination become Compound 5a (6 g, 13 mmol) was dissolved in 20 mL of DMF:MeOH (1:1), and 4b (2.65 g, 14 mmol) was added. The reaction was stirred overnight at room temperature. The solvent was evaporated under vacuum and purified by column chromatography using a 0%–10% methanol-DCM solution as the eluent to give lipid 7 (0.5 g, 6%). 1 ¹H NMR (300MHz, chloroform-d) δ 4.85 (quintet) J = 6.2Hz, 1H), 3.10-2.90 (m, 2H), 2.88-2.80 (m, 6H), 2.43 (s, 6H), 2.27 (dd, 2H), 1.67-1.34 (m, 8H), 1.30-1.2 (m, 45 H), 0.86 (t, 9H). [C 36 H 74 The measured MS value of NO2 was 567.5 [M+H]. + The calculated value is 566.6.

[0288] Example 6: Synthesis of Lipid 10 The procedure for synthesizing lipid 10 is described below with reference to Scheme 8, which is also provided below.

[0289] Option 8 step 1 : N-(2-( dimethylamino ) Ethyl ) Undecylamide ( 3c Synthesis of ) DMAP (4.49 g, 36.8 mmol) was added to undecanoic acid (2c) (5.27 g, 28.3 mmol) in 250 mL of stirred solution of DCM, followed by EDCI (6.3 g, 36.0 mmol). The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 15 min. Then, 1a (3.03 g, 34.4 mmol) was added dropwise and stirring continued overnight. On the second day, the reaction mixture was diluted with DCM and washed with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography using a 0%–10% methanol solution of DCM as eluent. Fractions containing the desired compound were combined and evaporated to give 3 (6.94 g, 95% yield). 1 ¹H NMR (300MHz, chloroform-d) δ ppm: 3.25-3.35 (m, 2H), 2.38-2.44 (m, 2H), 2.22 (s, 6H), 2.12-2.22 (m, 2H), 1.55-1.62 (m, 2H), 1.18-1.32 (brs, 14H), 0.80-0.90 (m, 3H).

[0290] step 2 : N 1 ,N 1 - dimethyl -N 2 - Undecyl ethane -1,2- diamine ( 4c Synthesis of ) Add 23.3 mL of a solution of 2N LiAlH4 in THF (46.6 mmol) to an ice-cold solution of 3c (5.97 g, 23.3 mmol) in 90 mL of THF. Stir the reaction mixture overnight at 80 °C. Cool the reaction to 0 °C and quench it by adding water dropwise. Subsequently, filter the reaction through diatomaceous earth, concentrate the filtrate, and purify it by chromatography (DMC-MeOH-NH3) to provide 4.2 g of compound 4c (4.2 g, 75% yield). 1 ¹H NMR (300MHz, chloroform-d) δ: 2.66 (t, J = 6.3Hz, 2H), 2.58(t, J = 7.14Hz, 2H), 2.40 (t, J = 6.3Hz, 2H), 2.21 (s, 6H), 1.42 – 1.54 (m, 2H), 1.41 – 1.54 (m, 16H), 1.24 (0.86 (t, J = 6.3Hz, 3H).

[0291] step 3 Lipids 10 of 8-((2-( dimethylamino ) Ethyl )( Renji ) amino ) Octanodecane -11- ester Synthesis Compound 5a (0.91 g, 2.0 mmol) was dissolved in 50 mL of EtOH, and 4c (1.6 g, 7.0 mmol) was added. The reaction mixture was stirred overnight at 65–75 °C. The reaction mixture was concentrated and purified by column chromatography using DCM-MeOH-NH3 as the eluent to give lipid 10 (142 mg, 12%). 1 H NMR (300MHz, dmso-d6) δ: 4.70 – 4.82 (m,1H), 2.90 – 3.0 (m, 2H), 2.78 – 2.88 (m, 2H), 2.52 – 2.62 (m, 10H), 2.20 –2.30 (m, 2H), 1.55 – 1.35 (m, 10H), 1.15 – 1.35 (m, 46H), 0.75 – 0.90 (9H). [C 40 H 82 The MS value of N₂O₂ was 623.6 [M+H]. + The calculated value is 622.6 (precise mass).

[0292] Example 7: Synthesis of Lipid 11 The procedure for synthesizing lipid 11 is described below with reference to Scheme 9, which is also provided below.

[0293] Option 9 Steps 1 and 2 of scheme 9 are as described in embodiment 3.

[0294] step 3 : 6-((2-( dimethylamino ) Ethyl )( Renji ) amino ) hexanoic acid 3- Octyl undecyl ester or lipid 11 Synthesis Compound 5d (1.36 g, 2.95 mmol - synthesis as described below) was dissolved in 13 mL of EtOH and 4a (1.21 g, 5.89 mmol) was added. The reaction mixture was stirred overnight at 65–75 °C. The reaction mixture was concentrated and purified twice by column chromatography using DCM-MeOH-NH3 as eluent to give pure lipid 11 (142 mg, 12%). 1 H NMR (300MHz,dmso-d6) δ: 4.02 (t, J = 6.6Hz, 2H), 2.90 – 3.00 (m, 2H), 2.75 – 2.85 (m, 2H), 2.61 (s, 6H), 2.50-2.60 (m, 4H), 2.27 (t,J = 7.4Hz, 2H), 1.15-1.60 (m, 53H), 0.80-0.90 (m, 9H). [C 38 H 78 The MS value of [N2O2] was 595.2 [M+H]. + The calculated value is 594.61 (precise mass).

[0295] 3- Octyl undecane -2- Ethyl acrylate ( 8a Synthesis of ) To an ice-cold solution of 9-heptadecane (6a, 5.98 g, 23.5 mmol) in 200 mL of anhydrous THF, pure ethyl 2-(diethoxyphosphoryl)acetate (7a) (40.0 g, 178 mmol) was added dropwise. The reaction mixture was stirred for 30 min, and then NaH (6.25 g, 157 mmol, 60%, as an oil) was added in portions. 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 and brine, dried over Na₂SO₄, and concentrated. The crude product was purified by column chromatography to provide 7.1 g (93% yield) of pure product 8a. 1 ¹H NMR (300MHz, d-chloroform) δ ppm: 5.60 (s, 1H), 4.14 (q, J = 7.1Hz, 2H), 2.60 - 2.54 (m, 2H), 2.12 - 2.08 (m, 2H), 1.50 - 1.20 (m, 27H), 0.95 - 0.82 (m, 6H).

[0296] 3- Ethyl octyl undecanoate 9a Synthesis Compound 9a (7.05 g, 21.7 mmol) was dissolved in 220 mL of EtOAc and 100 mL of MeOH and reduced with H2 (1 atm) using 1.2 g of wet 10% Pd / C catalyst. Clean conversion yielded 7.0 g (99% yield) of compound 7. 1 ¹H NMR (300MHz, d-chloroform) δ (ppm), J (Hz): 4.12 (q, J = 7.1Hz, 2H), 2.20 (d, J= 6.9,2H), 1.90 – 1.80 (m, 1H), 1.35 – 1.20 (m, 33H), 1.90 – 1.81 (m, 6H).

[0297] 3- Octyl undecane -1- alcohol( 10a Synthesis of ) Compound 9a (7.0 g, 21.4 mmol) was dissolved in 16 mL of THF, cooled to 0 °C, and LiAlH4 (16 mL, 2 M THF solution, 32.2 mmol) was added dropwise. The reaction mixture was stirred overnight, heated to room temperature, and then quenched at 0 °C by adding 30 mL of a THF / H2O mixture (1:1 by volume). The reaction mixture was extracted with EtOAc and filtered through diatomaceous earth. The organic phase was washed twice with water and brine, dried over Na2SO4, and concentrated. Purification by column chromatography (CH2Cl2-EtOAc) yielded 6.0 g of compound 10a in 97% yield. 1 ¹H NMR (300MHz, d-chloroform) δ ppm: 3.66 (t, J = 6.9Hz, 2H), 1.51 (m, 2H), 1.41(br s, 1H), 1.10-1.29 (m, 29H), 1.81-1.90 (m, 6H).

[0298] 6- Bromohexanoic acid 3- Octyl undecyl ester ( 5d Synthesis of ) Compounds 10a (3.5 g, 12.3 mmol) and 11a (2.9 g, 14.9 mmol – commercially available) were dissolved in 25 mL of dichloromethane, and then DMAP (190 mg, 1.55 mmol) and EDCI (2.95 g, 15.4 mmol) were added to this solution at room temperature. After stirring overnight, the reaction was quenched with water, diluted with dichloromethane, and washed with NaHCO3 (saturated aqueous solution) and brine. The organic phase was dried over Na2SO4 and concentrated. Column chromatography (hexane-EtOAc) purified 3.8 g of compound 5d in 67% yield. 1 ¹H NMR (300MHz, d-chloroform) δ ppm: 4.08 (t, J = 7.14Hz, 2H), 3.40(t, J = 6.6Hz, 2H), 2.30 (t, J = 7.14Hz, 2H), 1.92 – 1.80 (m, 2H), 1.70- 1.20 (m, 36H), 1.92 – 1.80 (m, 6H) Example 8: Synthesis of cationic lipids containing quaternary ammonium or quaternary ammonium cations Each of the lipids 1 to 11 described above and the lipid of Formula I can be converted into their corresponding lipids containing quaternary ammonium or quaternary ammonium cations by treatment with chloromethane (CH3CN) in acetonitrile (CH3CN) and chloroform (CHCl3).

[0299] Example 9: Preparation of lipid nanoparticles Lipid nanoparticles (LNPs) are prepared at a total lipid to ceDNA weight ratio of approximately 10:1 to 30:1. Briefly, cationic lipids, non-cationic lipids (e.g., distearate phosphatidylcholine (DSPC)), components providing membrane integrity (e.g., sterols, such as cholesterol), and conjugated lipid molecules (e.g., polyethylene glycol-modified lipid conjugates), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol with an average PEG molecular weight of 2000 (“PEG-DMG”), are dissolved in an alcohol (e.g., ethanol) at molar ratios, for example, 47.5:10.0:40.7:1.8, 47.5:10.0:39.5:3.0, or 47.5:10.0:40.2:2.3. The ceDNA is diluted to the desired concentration in a buffer solution. For example, ceDNA is diluted to a concentration of 0.1 mg / mL to 0.25 mg / mL in a buffer solution comprising sodium acetate, sodium acetate and magnesium chloride, citric acid, malic acid, or malic acid and sodium chloride. In one example, ceDNA is diluted to 0.2 mg / mL in 10 mM to 50 mM citrate buffer (pH 4). Using, for example, a syringe pump or impingement mixer, the alcohol-containing lipid solution is mixed with the aqueous ceDNA solution at a ratio of approximately 1:5 to 1:3 (volume / volume) at a total flow rate greater than 10 ml / min. In one example, the alcohol-containing lipid solution is mixed with the aqueous ceDNA solution at a ratio of approximately 1:3 (volume / volume) at a flow rate of 12 ml / min. The alcohol is removed, and the buffer is replaced with PBS by dialysis. Alternatively, the buffer is replaced with PBS using centrifuge tubes. Alcohol removal and simultaneous buffer exchange are achieved by, for example, dialysis or tangential flow filtration. The resulting lipid nanoparticles are filtered through a sterile filter with a 0.2 µm pore size.

[0300] In one study, lipid nanoparticles comprising exemplary ceDNA were prepared using a lipid solution comprising reference lipid A, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 47.5:10.0:40.7:1.8 as a control. In some studies, tissue-specific target ligands such as N-acetylgalactosamine (GalNAc) are included in formulations comprising reference lipid A, reference lipid B, MC3, or cationic lipids disclosed herein. MC3 is (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate, also known as DLin-MC3-DMA, and has the following structure: GalNAc ligands such as triantennary GalNAc (GalNAc3) or tetraantennary GalNAc (GalNAc4) can be synthesized as is known in the art (see, for example, WO2017 / 084987 and WO2013 / 166121) and chemically conjugated to lipids or PEG as is well known in the art (see Resen et al., *Journal of Biochemistry* (2001), “Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo,” Vol. 276, pp. 375577-37584). Aqueous solutions of ceDNA in buffered buffers were prepared. The lipid and ceDNA solutions were mixed on a NanoAssembler using an internal program at a total flow rate of 12 mL / min and a lipid-to-ceDNA ratio of 1:3 (v / v).

[0301] Table 1A: Test Material Application - Study 1 Comparative Formula (I) Cationic Lipids vs. Reference Lipids A

[0302] Table 1B: Test Material Application - Comparison of various (I) cationic lipids with each other and with reference lipids A and B A study comparing it with MC3

[0303] No. = Number; IV = Intravenous; ROA = Route of administration; LNP = Lipid nanoparticles; IVIS = In vivo imaging phase; BW = Body weight Table 2A: Description of LNP Compositions - Study 1 Comparative Formula (I) Cationic Lipids with Reference Lipid A

[0304] DSPC = distearylphosphatidylcholine; Chol = cholesterol; DMG-PEG2000 = l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG) 2000 -DMG); GalNAc = N-acetylgalactosamine; GalNAc4 = tetraanthaline GalNAc Table 2B: Description of LNP compositions - Study 2 compares various formula (I) cationic lipids with each other and with reference. Comparison of lipids A, B and MC3

[0305] DSPC = distearylphosphatidylcholine; Chol = cholesterol; DMG-PEG2000 = l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG)2000 -DMG); GalNAc = N-acetylgalactosamine; GalNAc4 = tetraanthaline GalNAc LNPs containing reference lipid A, reference lipid B, and MC3 were used as positive controls.

[0306] Example 10: Preclinical in vivo study of lipid nanoparticles Preclinical studies were conducted to evaluate the in vivo expression and tolerability of ceDNA-luciferase formulated with LNPs in mice. These LNPs comprised reference lipid A, reference lipid B, or MC3 as a positive control, or cationic lipids of this disclosure. The study design and procedures involved in these preclinical studies are described below.

[0307] Materials and methods Species (number, sex, age): CD-1 male mice, approximately 4 weeks old in Study 1 and approximately 6–8 weeks old in Study 2.

[0308] Cage side observation: Conduct cage side observations daily.

[0309] Clinical observation: In both Study 1 and Study 2, clinical observation was conducted on days 0, 1, 2, 3, 4, and 7 (before euthanasia). Additional observation was conducted for each exception. If applicable, the weight of all animals was recorded on the same days as described above. Additional weight was recorded as needed.

[0310] Dosage administration: The test product (LNP:ceDNA-Luc) in all groups was administered intravenously via the caudal vein on day 0 at a volume of 5 mL / kg. The dose level was 0.25 mg / kg in Study 1 and 0.5 mg / kg in Study 2.

[0311] Vital imaging: On day 4, all animals were administered 150 mg / kg (60 mg / mL) of fluorescein via intraperitoneal (IP) injection at 2.5 mL / kg. ≤15 minutes after each fluorescein administration, all animals underwent the IVIS imaging phase according to the in vivo imaging protocol described below.

[0312] In vivo IVIS imaging protocol ● The fluorescein stock powder should be stored at a nominal temperature of -20°C.

[0313] ● Store the prepared fluorescein in 1 mL aliquots at 2℃-8℃ in the dark.

[0314] ● The prepared fluorescein is stable for up to 3 weeks in the dark at 2℃-8℃ and for about 12 hours at room temperature (RT).

[0315] ● Dissolve a sufficient volume of fluorescein in PBS to achieve the target concentration of 60 mg / mL, and adjust the pH to 7.4 as needed using 5-M NaOH (approximately 0.5 µl / mg fluorescein) and HCl (approximately 0.5 µl / mg fluorescein).

[0316] ● Prepare an appropriate amount according to the protocol, including at least about 50% excess.

[0317] Injection and Imaging ● Shave off the animal's fur (as needed).

[0318] ● According to the protocol, 150 mg / kg fluorescein was injected into PBS at a rate of 60 mg / mL via IP.

[0319] ● Imaging can be performed immediately after application or up to 15 minutes later.

[0320] ● Set the isoflurane vaporizer to 1%-3% (typically 2.5%) to anesthetize the animal during the imaging phase.

[0321] ● Isoflurane anesthesia during the imaging phase: o Place the animal in the isoflurane chamber and wait for the isoflurane to take effect, approximately 2-3 minutes.

[0322] o Ensure that the anesthesia level on the side of the IVIS machine is in the "on" position.

[0323] o Place the animal into the IVIS machine Perform the required acquisition scheme using the highest sensitivity settings.

[0324] Results and discussion Research 1 The purpose of Study 1 was to evaluate the ability of the exemplary lipid of this disclosure (i.e., lipid 6) to be formulated as LNP, and its in vivo expression and tolerability when the LNP-ceDNA-luciferase composition was administered to mice at a dose of 0.25 mg / kg.

[0325] As a general rule, a polydispersity index (PDI) of 0.15 or lower indicates good uniformity in the size of the formed LNPs, and an encapsulation efficiency (EE) of 90% indicates a satisfactory encapsulation rate. LNPs 2, 3, and 4, all formulated with lipid 6 but with different amounts of DMG-PEG2000 and correspondingly adjusted cholesterol levels, exhibited excellent PDI values ​​below 0.1 and EE values ​​greater than 95%.

[0326] like Figure 1As shown, LNP 2, LNP 3 and LNP 4 (i.e., LNPs containing lipid 6 as a cationic lipid and ceDNA-luciferase as a nucleotide cargo) exhibited good in vivo luciferase expression levels on day 4 equivalent to those of LNP1 formulated with reference lipid A and ceDNA-luciferase.

[0327] Research 2 The purpose of Study 2 was to evaluate the ability of several exemplary lipids of this disclosure, namely lipids 1, 7, and 11, to be formulated as LNPs (i.e., LNP10, LNP8, and LNP9, respectively), to express and tolerate LNP-ceDNA-luciferase compositions in mice at a dose of 0.5 mg / kg. The expression and tolerance of these LNP compositions of the present invention were also compared with LNP compositions formulated with reference lipid A, reference lipid B, and MC3 (all having a head group different from that of lipids of formula (I)). All formulated LNP compositions exhibited satisfactory encapsulation efficiency and polydispersity index.

[0328] like Figure 2A As shown, LNP 8, LNP 9, and LNP 10 (i.e., LNPs containing lipids 7, 11, and 1, respectively) exhibited good in vivo luciferase expression levels on day 4. Notably, the luciferase expression levels of LNP 8 and LNP 9, formulated with lipids 7 and 11, respectively, were higher than those of LNP 6, formulated with MC3. Furthermore, Figure 2B The results showed that even at 0.5 mg / kg (twice the dose level applied in Study 1), LNP 8, LNP 9 and LNP 10, each formulated with the cationic lipids of this disclosure, achieved complete weight recovery by day 4 post-treatment, indicating that these LNP compositions were well tolerated in mice.

[0329] References and equivalent literature All patents and other publications (including references, granted patents, published patent applications, and co-pending patent applications) cited throughout this application are expressly incorporated herein by reference to describe and disclose methods that can be used in conjunction with the techniques described herein, such as those described in such publications. These publications are provided solely for their disclosure prior to the filing date of this application. In this regard, nothing should be construed as an admission that the inventor had no right to such disclosure prior to it by virtue of a prior invention or for any other reason. All statements regarding the dates of these documents or statements regarding their content are based on information available to the applicant and do not constitute an admission of the accuracy of the dates or content of these documents.

[0330] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the precise forms disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of this disclosure, as will be recognized by those skilled in the art. For example, although method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially simultaneously. The teachings of this disclosure provided herein can be suitably applied to other procedures or methods. The various embodiments described herein can be combined to provide other embodiments. If desired, aspects of this disclosure may be modified to employ the compositions, functions, and concepts of the foregoing references and applications to provide yet another embodiment of this disclosure. Moreover, due to considerations of biological functional equivalence, some changes to the protein structure may be made without affecting the type or amount of biological or chemical action. These and other changes may be made to this disclosure based on the detailed description. It is intended that all such modifications be included within the scope of the appended claims.

[0331] Specific elements of any of the foregoing embodiments may be combined or substituted for elements in other embodiments. Furthermore, although advantages associated with certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need to exhibit such advantages in order to fall within the scope of this disclosure.

[0332] The techniques described herein are further illustrated by the following examples, which should not be construed as further limitations. It should be understood that the invention is not limited in any way to the specific methods, schemes, and reagents described herein, and therefore variations are possible. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims.

Claims

1. A cationic lipid represented by Formula I: I Or its pharmaceutically acceptable salt, wherein: R' is absent, and is either hydrogen or a C1-C3 alkyl group; the prerequisite is that when R' is hydrogen or a C1-C3 alkyl group, R' and R... 1 and R 2 All the attached nitrogen atoms were protonated; R 1 and R 2 Each is independently hydrogen or C1-C3 alkyl; R 3 For C3-C 10 Alkylene or C3-C 10 alkenyl; R 4 For C1-C 16 Non-branched alkyl, C2-C 16 non-branched alkenyl or ;in: R 4a and R 4b Each independently is C1-C 16 Non-branched alkyl or C2-C 16 Non-branched alkenyl groups; R 5 It does not exist; it is either a C1-C8 alkylene group or a C2-C8 alkenylene group. R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 alkenyl; X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -S-S-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -O-N=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2)C(=O)O- or OC(=O)(CR a )2)C(=O)-; wherein: R a Each time it appears, it is independently either hydrogen or C. 1-6 Alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6.

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

3. The cationic lipid according to claim 1 or claim 2, wherein the lipid is represented by formula II: II Or a pharmaceutically acceptable salt thereof, where n is an integer selected from 1, 2, 3, and 4.

4. The cationic lipid according to any one of claims 1 to 3, wherein the lipid is represented by formula III: III Or its pharmaceutically acceptable salt.

5. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein R 1 and R 2 Each can be independently hydrogen, C1-C2 alkyl, or C2-C3 alkenyl.

6. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R', R 1 and R 2 Each is independently hydrogen or C1-C2 alkyl.

7. The cationic lipid according to any one of claims 1 to 6, wherein the lipid is represented by formula IV: IV Or its pharmaceutically acceptable salt.

8. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein R 5 It is either absent or is a C1-C8 alkylene group.

9. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein R 5 It is either absent or is a C2 alkylene group.

10. The cationic lipid according to any one of claims 1 to 9, wherein the lipid is represented by formula V: V Or its pharmaceutically acceptable salt.

11. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein R 4 For C1-C 14 Non-branched alkyl, C2-C 14 non-branched alkenyl or ;where R 4a and R 4b Each independently is C1-C 12 Non-branched alkyl or C2-C 12 Unbranched alkenyl groups.

12. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein R 4 For C2-C 12 Non-branched alkyl or C2-C 12 Unbranched alkenyl groups.

13. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein R 4 C5-C 12 Non-branched alkyl groups.

14. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R 4 It is a C6 non-branched alkyl, C7 non-branched alkyl, C8 non-branched alkyl, C9 non-branched alkyl, C 10 Non-branched alkyl, C 11 Non-branched alkyl or C 12 Non-branched alkyl groups.

15. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, wherein R 4 It is a C9 non-branched alkyl group.

16. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, wherein R 3 It is a C3-C8 alkylene or C3-C8 alkenylene.

17. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, wherein R 3 It is a C3-C7 alkylene group.

18. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein R 3 It is a C7 alkylene group.

19. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, wherein R 3 It is a C5 alkylene group.

20. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 19, wherein R 6a and R 6b Each independently is C7-C 12 Alkyl or C7-C 12 Alkenyl group.

21. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein R 6a and R 6b Each is independently a C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C8 alkenyl, C 10 alkenyl, C 11 alkenyl or C 12 Alkenyl group.

22. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 21, wherein R 6a and R 6b Each is independently a C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl or C 12 alkyl.

23. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 22, wherein R 6a and R 6b It contains an equal number of carbon atoms.

24. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23, wherein R 6a and R 6b They are the same.

25. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, wherein R 6a and R 6b Both are C7 alkyl, or C8 alkyl, or C9 alkyl, or C 10 Alkyl, or C 11 Alkyl, or C 12 alkyl.

26. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, wherein R 6a and R 6b Both are C8 alkyl groups.

27. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, wherein R 6a and R 6b Both are C9 alkyl groups.

28. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, wherein R 6a and R 6b Both are C 10 alkyl.

29. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, wherein R 6a and R 6b Both are C 11 alkyl.

30. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, wherein R 6a and R 6b Both are C 12 alkyl.

31. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 22, wherein R 6a and R 6b Each contains a different number of carbon atoms.

32. The cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 31, wherein R' is absent.

33. The cationic lipid according to claim 1, wherein the lipid is: 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid, 11-yl dodecanoic acid ester (Lipid 1); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid tridecyl-12-yl ester (Lipid 2); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid pentadecane-13-yl ester (Lipid 3); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid nonadecanoyl ester (Lipid 4); 6-((2-(dimethylamino)ethyl)(nonyl)amino)hexanoic acid 3-decyltridecyl ester (Lipid 5); 8-((2-(dimethylamino)ethyl)(nonyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 6); 8-((2-(dimethylamino)ethyl)(heptyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 7); 8-((2-(dimethylamino)ethyl)(octyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 8); 8-(decyl(2-(dimethylamino)ethyl)amino)octanoic acid heptadecano-9-yl ester (Lipid 9); 8-((2-(dimethylamino)ethyl)(undecyl)amino)octanoic acid heptadecanyl-9-yl ester (Lipid 10); 6-((2-(dimethylamino)ethyl)(nonyl)amino)hexanoic acid 3-octyl undecyl ester (Lipid 11); Or its pharmaceutically acceptable salt.

34. A lipid nanoparticle (LNP) comprising a cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 33; and a therapeutic nucleic acid.

35. The lipid nanoparticles of claim 34, wherein the therapeutic nucleic acid is encapsulated in the lipid.

36. The lipid nanoparticles according to claim 34 or claim 35, wherein the therapeutic nucleic acid is selected from the group consisting of: small genes, plasmids, small loops, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, ceDNA, mini-strings, and doggybone. ™ DNA or dumbbell-shaped linear DNA with ends blocked by anterior granules, cleavage enzyme-substrate dsRNA, hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, DNA viral vectors, viral RNA vectors, non-viral vectors, and any combination thereof.

37. The lipid nanoparticles according to any one of claims 34 to 36, wherein the therapeutic nucleic acid is terminally blocked DNA (ceDNA).

38. The lipid nanoparticles according to any one of claims 34 to 36, wherein the lipid nanoparticles further comprise sterols.

39. The lipid nanoparticles of claim 38, wherein the sterol is cholesterol or β-sitosterol.

40. The lipid nanoparticles according to any one of claims 34 to 39, wherein the lipid nanoparticles further comprise non-cationic lipids.

41. The lipid nanoparticles of claim 40, wherein the non-cationic lipid is selected from the group consisting of: distearyl-sn-glycerol-ethanolamine phosphate (DSPE), distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoyl oleoyl phosphatidyl ethanolamine (POPE), dioleoyl phosphatidyl ethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphatidyl ethanolamine (DMPE), distearate phosphatidyl ethanolamine (DSPE), monomethyl phosphatidyl ethanolamine (such as 16-O-monomethyl PE), dimethyl phosphatidyl ethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), lecithin choline (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyroxyphosphatidylcholine (DMPC), dimyroxyphosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), disqualylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), ditrans oleoylphosphatidyl... Acylethanolamine (DEPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE); 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphatidylphosphate, lysophosphatidylcholine, dilinoleylphosphatidylcholine, and mixtures thereof.

42. The lipid nanoparticles according to claim 40 or claim 41, wherein the non-cationic lipid is selected from the group consisting of: dioleoylphosphatidylcholine (DOPC), distearate phosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE).

43. The lipid nanoparticles according to any one of claims 34 to 42, wherein the lipid nanoparticles further comprise at least one polyethylene glycol-modified lipid.

44. The lipid nanoparticles of claim 43, wherein the at least one polyethylene glycol-modified lipid is selected from the group consisting of: PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dispalmityloxypropyl; PEG-distearyloxypropyl; 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol-PEG (DMG-PEG); distearyl-racemic-glycerol-PEG (DSG-PEG); PEG-dilaurylglycerol; PEG-dispalmitoylglycerol; PEG-distearylglycerol; PEG-dilauryloxyglucamide; PEG-dimyristoylglucamide; PEG - Dipalmitoyl saccharamide; PEG-distearate saccharamide; (l-[8'-(cholesterol-5-en-3[β]-oxy)formamido-3',6'-dioxooctanoyl]carbamoyl-[ω]-methyl-poly(ethylene glycol) (PEG-cholesterol); 3,4-bistetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether (PEG-DMB), l,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N[methoxy(poly(ethylene glycol)) (DSPE-PEG), and l,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-poly(ethylene glycol)-hydroxy (DSPE-PEG-OH).

45. The lipid nanoparticles according to claim 43 or claim 44, wherein the at least one polyethylene glycol-modified lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, DSG-PEG, or a combination thereof.

46. ​​The lipid nanoparticles according to any one of claims 43 to 45, wherein the at least one polyethylene glycol-modified lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSG-PEG2000, or a combination thereof.

47. The lipid nanoparticles according to any one of claims 34 to 46, wherein the lipid nanoparticles further comprise a tissue-specific targeting ligand.

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

49. The lipid nanoparticles of claim 47 or claim 48, wherein the tissue-specific targeting ligand is covalently linked to the at least one polyethylene glycol-modified lipid to form a polyethylene glycol-modified lipid conjugate.

50. The lipid nanoparticles of claim 49, wherein the polyethylene glycol-modified lipid conjugate comprises a tetrapod GalNAc covalently linked to DSPE-PEG2000.

51. The lipid nanoparticles according to any one of claims 34 to 50, wherein the cationic lipid is present in a molar percentage of about 30% to about 80%.

52. The lipid nanoparticles according to any one of claims 38 to 51, wherein the sterol is present in a molar percentage of about 20% to about 50%.

53. The lipid nanoparticles according to any one of claims 40 to 52, wherein the non-cationic lipid is present in a molar percentage of about 2% to about 20%.

54. The lipid nanoparticles according to any one of claims 43 to 53, wherein the at least one polyethylene glycol-modified lipid is present in a molar percentage of about 2.1% to about 10%.

55. The lipid nanoparticles according to any one of claims 49 to 54, wherein the polyethylene glycol-modified lipid conjugate is present in a molar percentage of about 0.1% to about 10%.

56. The lipid nanoparticles according to claim 34, wherein the lipid nanoparticles further comprise sterols, non-cationic lipids, polyethylene glycol-modified lipids, and polyethylene glycol-modified lipid conjugates.

57. The lipid nanoparticles according to any one of claims 34 to 56, wherein the lipid nanoparticles further comprise dexamethasone palmitate.

58. The lipid nanoparticles according to any one of claims 34 to 57, wherein the ratio of total lipids to ceDNA in the particles is about 10:1 to about 40:

1.

59. The lipid nanoparticles according to any one of claims 34 to 58, wherein the diameter of the nanoparticles ranges from about 40 nm to about 120 nm.

60. The lipid nanoparticles according to any one of claims 34 to 59, wherein the diameter of the nanoparticles is less than about 100 nm.

61. The lipid nanoparticles according to any one of claims 34 to 60, wherein the diameter of the nanoparticles is about 60 nm to about 80 nm.

62. The lipid nanoparticles according to any one of claims 34 to 61, wherein the ceDNA is a terminally closed linear double-stranded DNA.

63. The lipid nanoparticle of claim 62, wherein the ceDNA comprises an expression cassette, and wherein the expression cassette comprises a promoter sequence and a transgene.

64. The lipid nanoparticles of claim 63, wherein the expression cassette comprises a polyadenylated sequence.

65. The lipid nanoparticles according to any one of claims 62 to 64, wherein the ceDNA comprises at least one inverted terminal repeat (ITR) sequence, the ITR being attached to the 5' or 3' end of the expression cassette.

66. The lipid nanoparticles of claim 65, wherein the expression cassette is side-connected to two ITRs, wherein the two ITRs comprise a 5' ITR and a 3' ITR.

67. The lipid nanoparticles of claim 65, wherein the expression cassette is connected to the 3' end ITR (3'ITR).

68. The lipid nanoparticles of claim 65, wherein the expression cassette is connected to the 5' end of the ITR (5'ITR).

69. The lipid nanoparticles of claim 65, wherein the at least one ITR is an ITR derived from an AAV serotype, an ITR derived from a goose virus, an ITR derived from a B19 virus, or a wild-type ITR derived from a parvovirus.

70. The lipid nanoparticles of claim 69, wherein the AAV serotype is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

71. The lipid nanoparticles according to any one of claims 66 to 70, wherein at least one of the 5' ITR and the 3' ITR is a wild-type AAV ITR.

72. The lipid nanoparticles according to any one of claims 66 to 71, wherein at least one of the 5' ITR and the 3' ITR is a modified or mutated ITR.

73. The lipid nanoparticles according to any one of claims 66 to 72, wherein the 5' ITR and the 3' ITR are symmetrical.

74. The lipid nanoparticles according to any one of claims 66 to 73, wherein the 5' ITR and the 3' ITR are asymmetric.

75. The lipid nanoparticles according to any one of claims 66 to 74, wherein the ceDNA further comprises a spacer sequence between the 5' ITR and the expression cassette.

76. The lipid nanoparticles according to any one of claims 66 to 75, wherein the ceDNA further comprises a spacer sequence between the 3' ITR and the expression cassette.

77. The lipid nanoparticles according to claim 75 or claim 76, wherein the length of the spacer sequence is at least 5 base pairs.

78. The lipid nanoparticles according to any one of claims 37 to 77, wherein the ceDNA has nicks or vacancies.

79. The lipid nanoparticles according to any one of claims 37 to 78, wherein the ceDNA is CELiD, a DNA-based small loop, a midge, a mini-strand DNA, a dumbbell-shaped linear double-stranded DNA with two ITR hairpin structures at the 5' and 3' ends of the expression cassette, or a doggybone. ™ DNA.

80. A pharmaceutical composition comprising a cationic lipid or a lipid nanoparticle according to any one of claims 1 to 34 or any one of claims 34 to 79, and a pharmaceutically acceptable excipient.

81. A method of treating a subject with a genetic disorder, the method comprising administering to the subject an effective amount of lipid nanoparticles according to any one of claims 34 to 79, or an effective amount of a pharmaceutical composition according to claim 80.

82. The method of claim 81, wherein the subject is a human.

83. The method of claim 81 or claim 82, wherein the genetic disease is selected from the group consisting of: sickle cell anemia, melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Schie syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C and D (MPS type III)). Types 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 (MPSIX), 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, mucinous storage disease types I, II / III, and IV, sialic acid storage disease types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, cystinosis, Barton disease, aspartic glucosamineuria, Salla disease, Danon disease (LAMP) -2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscin deposition disease (CLN1-8, INCL, and LINCL), sphingolipid syndrome, galactosylsialic acidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich ataxia, Duchenne muscular dystrophy (DMD), Becker's muscular dystrophy (BMD), epidermolysis bullosa (DEB), exonucleotide pyrophosphatase 1 deficiency, systemic arterial calcification in infants (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4), ornithine transcarbamate (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), α-1 antitrypsin deficiency.Progressive familial intrahepatic cholestasis of blood (PFIC) includes type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2) and cathepsin A deficiency.

84. The method of claim 83, wherein the genetic disorder is hemophilia A.

85. The method of claim 83, wherein the genetic disorder is hemophilia B.

86. The method of claim 83, wherein the genetic condition is phenylketonuria (PKU).

87. The method of claim 83, wherein the genetic disease is Wilson's disease.

88. The method of claim 83, wherein the genetic disease is Gaucher disease type I, II or III.

89. The method of claim 83, wherein the genetic condition is Stargardt macular dystrophy.

90. The method of claim 83, wherein the genetic condition is LCA10.

91. The method of claim 83, wherein the genetic disorder is Usher syndrome.

92. The method of claim 83, wherein the genetic condition is wet AMD.

93. The method of claim 83, wherein the genetic disorder is dystrophic epidermolysis bullosa (DEB).

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