Lipid nanoparticle compositions comprising closed-end DNA and cleavable lipids and methods of using the same

Abstract

The present invention provides a lipid formulation comprising lipid and capsid-free non-viral vector (for example, ceDNA).The lipid particle (for example, lipid nanoparticle) of the present invention comprises the lipid formulation that can be used to deliver capsid-free non-viral DNA vector to the target site (for example, cell, tissue, organ, etc.).The present invention provides, for example, a pharmaceutical composition comprising lipid nanoparticle (LNP), wherein the LNP comprises SS-cleavable lipid and closed-end DNA (ceDNA).

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 896,980, filed Sep. 6, 2019; U.S. Provisional Application No. 62 / 910,720, filed Oct. 4, 2019; and U.S. Provisional Application No. 62 / 940,104, filed Nov. 25, 2019, the contents of each of which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format. The ASCII copy, created on Sep. 3, 2020, is named 131698-07520_SL.txt and is 556 bytes in size.

Background Art

[0003] Gene therapy aims to improve the clinical outcomes for patients suffering from either a hereditary disorder or an acquired disease caused by an abnormal gene expression profile. Various types of gene therapies have been developed to deliver therapeutic nucleic acids to a patient's cells as a drug for treating the disease. Generally, gene therapy includes the treatment or prevention of a medical condition resulting from a defective gene or abnormal regulation or expression, e.g., under-expression or over-expression that can lead to a disorder, disease, or malignancy. For example, a disease or disorder caused by a defective gene can be treated by delivering repair genetic material to a subject to supplement the defective gene and provide a wild-type copy of the gene to enhance the wild-type copy of the gene. In some cases, treatment is achieved by delivery of a therapeutic nucleic acid molecule that regulates the expression of the defective gene at the translational level, either by providing an antisense nucleic acid that binds to the target DNA or mRNA and reduces the expression level of the defective gene, or by transcribing the wild-type mRNA to increase the correct copy of the gene.

[0004] In particular, human single-gene disorders are being treated by the delivery and expression of normal genes into target cells. The introduction and expression of repair genes into a patient's target cells can be carried out via a number of methods, including the use of engineered viral gene delivery vectors, and potentially plasmids, minigenes, oligonucleotides, minicircles, or various closed-ended DNAs. Among the many virus-derived vectors available (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated virus (rAAV) has emerged as a versatile and relatively safe vector in gene therapy. However, viral vectors such as adeno-associated vectors are highly immunogenic and can induce humoral and cellular immunity that may compromise efficacy, particularly upon readministration.

[0005] The molecular sequences and structural features encoded in the AAV viral genome / vector have evolved to promote episomal stability, viral gene expression, and interact with the host immune system. AAV vectors contain a hairpin DNA structure conserved throughout the AAV family and play an important role in the key function of AAV, the ability to utilize the host genome for replication while evading the host surveillance system.

[0006] However, among these gene therapies, some are greatly troubled by immune-related adverse events that are closely related to the host's own defense mechanisms against therapeutic nucleic acids. For example, the immune system has two general mechanisms for fighting infections that are associated with causing adverse events in the therapy recipient. The first is known as the "innate" immune response and is typically triggered within minutes of infection and serves to limit the spread of pathogens in vivo. The host recognizes conserved determinants expressed by a diverse range of infectious microorganisms but not present in the host, and these determinants stimulate elements of the host's innate immune system to produce immunomodulatory cytokines and polyreactive IgM antibodies. The second and subsequent mechanisms are known as the "adaptive" or antigen-specific immune response and are typically generated against determinants uniquely expressed by the pathogen. The innate and adaptive immune responses are mainly activated and regulated by a series of type I interferons (IFNs) via a series of signaling pathways activated by specific types of nucleic acids.

[0007] Non-viral gene delivery circumvents the specific disadvantages associated with viral transduction, particularly the humoral and cellular immune responses against viral structural proteins that form vector particles, and the disadvantages resulting from any novel viral gene expression. Non-viral gene delivery typically uses bacterial plasmids to introduce foreign DNA into recipient cells. In addition to the transgene of interest, such DNA routinely contains foreign sequence elements necessary for the selection and amplification of bacterial plasmid DNA (pDNA), such as antibiotic resistance genes and prokaryotic origins of replication. For example, plasmids produced in E. coli contain elements necessary for growth in prokaryotes, such as the origin of prokaryotic DNA replication and selectable markers, as well as unique prokaryotic modifications to DNA that are unnecessary and potentially harmful for the expression of the transgene in mammalian cells.

[0008] Conceptually clear, the prospect of using nucleic acid molecules for gene therapy to treat human diseases remains uncertain. The main cause of this uncertainty is the obvious adverse events related to the host's innate immune response to nucleic acid therapeutics, and thus, the way these materials regulate the expression of their intended targets in the context of the immune response. The current state of the art regarding the creation, function, behavior, and optimization of nucleic acid molecules that can be employed for clinical applications focuses particularly on the following aspects: (1) antisense oligonucleotides and double-stranded RNAs that directly regulate translation and gene expression, (2) transcriptional gene silencing RNAs that bring about long-term epigenetic modifications, (3) antisense oligonucleotides that interact with and modify gene splicing patterns, (4) the creation of synthetic or viral vectors that mimic the physiological functions of naturally occurring AAV or lentiviral genomes, and (5) the in vivo delivery of therapeutic oligonucleotides. However, despite the progress in the development of nucleic acid therapeutics evident in recent clinical outcomes, the field of gene therapy remains severely limited by the undesirable adverse events in the recipient induced by the therapeutic nucleic acids themselves.

[0009] Therefore, there is a strong need in this field for new technologies that can effectively reduce, improve, alleviate, prevent, or maintain the immune response systems induced by nucleic acid therapeutics. Summary of the Invention Means for Solving the Problems

[0010] Provided herein are pharmaceutical compositions comprising a cationic lipid, e.g., an ionizable cationic lipid, e.g., an SS-cleavable lipid, and a capsid-free non-viral vector (e.g., ceDNA) that can be used to deliver a capsid-free non-viral DNA vector to a target site of interest (e.g., a cell, tissue, organ, etc.), as well as methods of using and manufacturing the same. Surprisingly, as shown herein, lipid nanoparticles (LNPs) comprising a cleavable lipid provide more efficient delivery of a therapeutic nucleic acid, e.g., ceDNA, to target cells (including, e.g., hepatocytes). In particular, ceDNA particles comprising ceDNA and a cleavable lipid showed fewer copies of liver tissue samples showing equivalent protein expression compared to other lipids, such as MC3. The mechanism has not yet been determined and is not bound by theory, but ceDNA containing lipid particles (e.g., lipid nanoparticles) comprising an SS-cleavable lipid is thought to improve delivery to hepatocytes compared to non-parenchymal cells and transport more efficiently to the nucleus. Another advantage of the ceDNA lipid particles (e.g., lipid nanoparticles) comprising a cleavable lipid described herein is better tolerability as shown by a reduction in weight loss and a decrease in cytokine release compared to other lipids (e.g., other ionizable cationic lipids, such as MC3). The beneficial effect on tolerability can be further enhanced by adding an immunosuppressant conjugate (e.g., dexamethasone palmitate) or a tissue-specific ligand (e.g., N-acetylgalactosamine (GalNAc)) to the LNPs of the present disclosure. Surprisingly, ceDNA formulated with the SS-cleavable lipids described herein was found to successfully avoid phagocytosis by immune cells (see, e.g., FIGS. 13-15) and have the potential for higher expression per copy number in target cells or organs (e.g., the liver) compared to ceDNA formulated with other lipids, such as MC3.Indeed, there may be a synergistic effect between ceDNA formulated with an SS-cleavable lipid (e.g., ss-OP4) and GalNAc, and ceDNA-LNPs containing an SS-cleavable lipid and GalNAc may exhibit hepatocyte targeting up to approximately 4000-fold greater compared to that seen with ceDNA formulated with the SS-cleavable lipid alone (ss-OP4) (Figures 18A and 18B), while ceDNA formulated with a typical cationic lipid having GalNAc showed only approximately 10-fold greater hepatocyte targeting. Furthermore, it has been discovered that ceDNA formulated with an SS-cleavable lipid (ss-OP4) having GalNAc showed an improved safety profile with respect to complement and cytokine responses.

[0011] In one aspect, disclosed herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises an SS-cleavable lipid and a therapeutic nucleic acid (TNA). In another aspect, disclosed herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises an SS-cleavable lipid and an mRNA. In one aspect, disclosed herein is a pharmaceutical composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises an SS-cleavable lipid and a closed-ended DNA (ceDNA). According to some embodiments, the SS-cleavable lipid comprises a disulfide bond and a tertiary amine. According to some embodiments of any of the aspects or embodiments herein, the SS-cleavable lipid comprises an ss-OP lipid of Formula I.

Chemical formula

[0012] According to some embodiments of any of the aspects or embodiments of this specification, the LNP further comprises a sterol. According to some embodiments, the sterol is cholesterol. According to some embodiments of any of the aspects or embodiments of this specification, the LNP further comprises polyethylene glycol (PEG). According to some embodiments, the PEG is 1-(monomethoxy-polyethylene glycol)-2,It is 3-dimyristoyl-sn-glycero-3-phosphoethanolamine (PEG-DMG). According to some embodiments of any of the aspects or embodiments herein, the LNP further comprises a non-cationic lipid. According to some embodiments, the non-cationic lipid is distearoyl-sn-glycero-3-phosphoethanolamine, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dieleoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,It is selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dipalmitoyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, or a mixture thereof. According to some embodiments, the non-cationic lipid is selected from the group consisting of dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).

[0013] According to some embodiments, the PEG or PEG-lipid conjugate is present at about 1.5% to about 3%, such as about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%. According to some embodiments, the PEG or PEG-lipid conjugate is present at about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9% or about 3%. According to some embodiments, cholesterol is present at a molar percentage of about 20% to about 40%, such as about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 35%, about 25% to about 30% or about 30% to about 35%, and the SS-cleavable lipid is present at a molar percentage of about 80% to about 60%, such as about 80% to about 65%, about 80% to about 70%, about 80% to about 75%, about 75% to about 60%, about 75% to about 65%, about 75% to about 70%, about 70% to about 60% or about 70% to about 60%. According to some embodiments, cholesterol is present at a molar percentage of about 20% to about 40%, such as about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40%, and the SS-cleavable lipid is present at a molar percentage of about 80% to about 60%, such as about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61% or about 60%. According to some embodiments, cholesterol is present at a molar percentage of about 40%, and the SS-cleavable lipid is present at a molar percentage of about 50%. According to some embodiments of any of the aspects or embodiments herein, the composition further comprises cholesterol, a PEG or PEG-lipid conjugate, and a non-cationic lipid.According to some embodiments, the PEG or PEG-lipid conjugate is present at about 1.5% to about 3%, such as about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%, about 2.25% to about 3%, about 2.25% to about 2.75% or about 2.25% to about 2.5%. According to some embodiments, the PEG or PEG-lipid conjugate is present at about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9% or about 3%. According to some embodiments, cholesterol is present at a molar percentage of about 30% to about 50%, such as about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50% or about 45% to about 50%. According to some embodiments, cholesterol is present at a molar percentage of about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49% or about 50%. According to some embodiments, the SS-cleavable lipid is present at a molar percentage of about 42.5% to about 62.5%. According to some embodiments, the SS-cleavable lipid is present at a molar percentage of about 42.5%, about 43%, about 43.5%, about 44%, about 44.5%, about 45%, about 45.5%, about 46%, about 46.5%, 47.5%, about 48%, about 48.5%, about 49%, about 49.5%, about 50%, about 50.5%, about 51%, about 51.5%, about 47% about 52%, about 52.5%, about 53%, about 53.5%, about 54%, about 54.5%, about 55%, about 55.5%, about 56%, about 56.5%, about 57%, 57.5%, about 58%, about 58.5%, about 59%, about 59.5%, about 60%, about 60.5%, about 61%, about 61.5%, about 62% or about 62.5%.According to some embodiments of any of the aspects or embodiments of this specification, the non-cationic lipid is present in a molar percentage of about 2.5% to about 12.5%. According to some embodiments of any of the aspects or embodiments of this specification, cholesterol is present in a molar percentage of about 40%, the SS-cleavable lipid is present in a molar percentage of about 52.5%, the non-cationic lipid is present in a molar percentage of about 7.5%, and PEG is present at about 3%. According to some embodiments of any of the aspects or embodiments of this specification, the composition further comprises dexamethasone palmitate. According to some embodiments of any of the aspects or embodiments of this specification, the LNP has a diameter in the range of about 50 nm to about 110 nm, such as about 50 nm to about 100 nm, about 50 nm to about 95 nm, about 50 nm to about 90 nm, about 50 nm to about 85 nm, about 50 nm to about 80 nm, about 50 nm to about 75 nm, about 50 nm to about 70 nm, about 50 nm to about 65 nm, about 50 nm to about 60 nm, about 50 nm to about 55 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 95 nm, about 60 nm to about 90 nm, about 60 nm to about 85 nm, about 60 nm to about 80 nm, about 60 nm to about 75 nm, about 60 nm to about 70 nm, about 60 nm to about 65 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 nm to about 95 nm, about 70 nm to about 90 nm, about 70 nm to about 85 nm, about 70 nm to about 80 nm, about 70 nm to about 75 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, about 80 nm to about 95 nm, about 80 nm to about 90 nm, about 80 nm to about 85 nm, about 90 nm to about 110 nm, or about 90 nm to about 100 nm. According to some embodiments of any of the aspects or embodiments of this specification, the LNP has a size of less than about 100 nm, such as a size of less than about 105 nm, less than about 100 nm, less than about 95 nm, less than about 90 nm, less than about 85 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm, less than about 15 nm or less than about 10 nm.According to some embodiments, the LNP has a size of less than about 70 nm, such as less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm, less than about 15 nm or less than about 10 nm. According to some embodiments, the LNP has a size of less than about 60 nm, such as less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 20 nm, less than about 15 nm or less than about 10 nm. According to some embodiments of any of the aspects or embodiments herein, the composition has a total lipid to ceDNA ratio of about 15:1. According to some embodiments of any of the aspects or embodiments herein, the composition has a total lipid to ceDNA ratio of about 30:1. According to some embodiments of any of the aspects or embodiments herein, the composition has a total lipid to ceDNA ratio of about 40:1. According to some embodiments of any of the aspects or embodiments herein, the composition has a total lipid to ceDNA ratio of about 50:1. According to some embodiments of any of the aspects or embodiments herein, the composition further comprises N-acetylgalactosamine (GalNAc). According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.2% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.3% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.4% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.5% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.6% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.7% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.8% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.9% of the total lipid.According to some embodiments, GalNAc is present in the LNP at a molar percentage of 1.0% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of about 1.5% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of 2.0% of the total lipid. According to some embodiments of any of the aspects or embodiments herein, the composition further comprises from about 10 mM to about 30 mM malic acid, such as from about 10 mM to about 25 mM, from about 10 mM to about 20 mM, from about 10 mM to about 15 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, or from about 20 mM to about 25 mM. According to some embodiments of any of the aspects or embodiments herein, the composition further comprises about 10 mM malic acid, about 11 mM malic acid, about 12 mM malic acid, about 13 mM malic acid, about 14 mM malic acid, about 15 mM malic acid, about 16 mM malic acid, about 17 mM malic acid, about 18 mM malic acid, about 19 mM malic acid, about 20 mM malic acid, 21 mM malic acid, about 22 mM malic acid, about 23 mM malic acid, about 24 mM malic acid, about 25 mM malic acid, about 26 mM malic acid, about 27 mM malic acid, about 28 mM malic acid, about 29 mM malic acid, or about 30 mM malic acid. According to some embodiments, the composition comprises about 20 mM malic acid. According to some embodiments of any of the aspects or embodiments herein, the composition further comprises from about 30 mM to about 50 mM NaCl, such as from about 30 mM to about 45 mM NaCl, from about 30 mM to about 40 mM NaCl, from about 30 mM to about 35 mM NaCl, from about 35 mM to about 45 mM NaCl, from about 35 mM to about 40 mM NaCl, or from about 40 mM to about 45 mM NaCl. According to some embodiments of any of the aspects or embodiments herein, the composition further comprises about 30 mM NaCl, about 35 mM NaCl, about 40 mM NaCl, or about 45 mM NaCl. According to some embodiments, the composition comprises about 40 mM NaCl.According to some embodiments, the composition is about 20 mM to about 100 mM MgCl2, such as about 20 mM to about 90 mM MgCl2, about 20 mM to about 80 mM MgCl2, about 20 mM to about 70 mM MgCl2, about 20 mM to about 60 mM MgCl2, about 20 mM to about 50 mM MgCl2, about 20 mM to about 40 mM MgCl2, about 20 mM to about 30 mM MgCl2, about 320 mM to about 90 mM MgCl2, about 30 mM to about 80 mM MgCl2, about 30 mM to about 70 mM MgCl2, about 30 mM to about 60 mM MgCl2, about 30 mM to about 50 mM MgCl. 2. Further comprising from about 30 mM to about 40 mM MgCl2, from about 40 mM to about 90 mM MgCl2, from about 40 mM to about 80 mM MgCl2, from about 40 mM to about 70 mM MgCl2, from about 40 mM to about 60 mM MgCl2, from about 40 mM to about 50 mM MgCl2, from about 50 mM to about 90 mM MgCl2, from about 50 mM to about 80 mM MgCl2, from about 50 mM to about 70 mM MgCl2, from about 50 mM to about 60 mM MgCl2, from about 60 mM to about 90 mM MgCl2, from about 60 mM to about 80 mM MgCl2, from about 60 mM to about 70 mM MgCl2, from about 70 mM to about 90 mM MgCl2, from about 70 mM to about 80 mM MgCl2 or from about 80 mM to about 90 mM MgCl2. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a closed-ended linear double-stranded DNA. According to some embodiments of any of the aspects or embodiments herein, the ceDNA comprises an expression cassette comprising a promoter sequence and a transgene. According to some embodiments, the ceDNA comprises an expression cassette comprising a polyadenylation sequence. According to some embodiments of any of the aspects or embodiments herein, the ceDNA comprises at least one inverted terminal repeat (ITR) adjacent to either the 5' or 3' end of the expression cassette. According to some embodiments, the expression cassette is flanked by two ITRs, and the two ITRs comprise one 5' ITR and one 3' ITR. According to some embodiments, the expression cassette is ligated to an ITR (3' ITR) at the 3' end. According to some embodiments, the expression cassette is ligated to an ITR (5' ITR) at the 5' end. According to some embodiments, at least one of the 5' ITR and the 3' ITR is a wild-type AAV ITR. According to some embodiments, at least one of the 5' ITR and the 3' ITR is a modified ITR. According to some embodiments, the ceDNA further comprises a spacer sequence between the 5' ITR and the expression cassette. According to some embodiments, the ceDNA further comprises a spacer sequence between the 3' ITR and the expression cassette. According to some embodiments, the spacer sequence is at least 5 base pairs in length.According to some embodiments, the spacer array has a length of at least 5 to 100 base pairs. According to some embodiments, the spacer array has a length of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 base pairs. According to some embodiments, the spacer array has a length of 5 to 500 base pairs. According to some embodiments, the spacer array has a length of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490 or 495 base pairs. According to some embodiments of any of the aspects or embodiments herein, the ceDNA has a nick or a gap. According to some embodiments, the ITR is an ITR derived from an AAV serotype, derived from the ITR of the goose parvovirus, derived from the B19 virus ITR, and is a wild-type ITR from parvovirus. According to some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. According to some embodiments, the ITR is a mutant ITR, and the ceDNA optionally includes additional ITRs different from the first ITR. According to some embodiments, the ceDNA includes two mutant ITRs at both the 5' and 3' ends of the expression cassette, and optionally, the two mutant ITRs are symmetric mutants.According to some embodiments of any of the aspects or embodiments of this specification, the ceDNA is CELiD, a DNA-based minicircle, MIDGE, ministering DNA, a dumbbell-shaped linear double-stranded closed-ended DNA containing two hairpin structures of ITR at the 5' and 3' ends of an expression cassette, or doggybone (trademark) DNA. According to some embodiments of any of the aspects or embodiments of this specification, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0014] According to some aspects, the present disclosure provides a method of treating a genetic disorder in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any of the aspects or embodiments of this specification. According to some embodiments, the subject is a human. According to some embodiments, the genetic disorder is sickle cell anemia, melanoma, hemophilia A (factor VIII (FVIII) deficiency), 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), Scheie syndrome (MPS I S type), Hurler-Scheie syndrome (MPS I H-S type), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D types (MPS III A, B, C, and D types), Morquio A and B types (MPS IVA and MPS (IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick disease A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis I, II / III and IV, sialidosis I and II, glycogen storage disease I and II (Pompe disease), Gaucher disease I, II and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, and cathepsin A deficiency. According to some embodiments, the genetic disorder is Leber congenital amaurosis (LCA). According to some embodiments, the LCA is LCA10. According to some embodiments, the genetic disorder is Niemann-Pick disease. According to some embodiments, the genetic disorder is Stargardt macular dystrophy. According to some embodiments, the genetic disorder is glucose-6-phosphatase (G6Pase) deficiency (glycogen storage disease type I) or Pompe disease (glycogen storage disease type II). According to some embodiments, the genetic disorder is hemophilia A (factor VIII deficiency). According to some embodiments, the genetic disorder is hemophilia B (factor IX deficiency). According to some embodiments, the genetic disorder is Hunter syndrome (mucopolysaccharidosis type II).According to some embodiments, the genetic disorder is cystic fibrosis. According to some embodiments, the genetic disorder is dystrophic epidermolysis bullosa (DEB). According to some embodiments, the genetic disorder is phenylketonuria (PKU). According to some embodiments, the genetic disorder is hyaluronidase deficiency. According to some embodiments of any of the aspects or embodiments herein, the method further comprises administering an immunosuppressant. According to some embodiments, the immunosuppressant is dexamethasone. According to some embodiments of any of the aspects or embodiments herein, the subject exhibits a reduced immune response level to the pharmaceutical composition as compared to the immune response level observed with an LNP comprising MC3 as the primary cationic lipid, and the immune response level to the pharmaceutical composition is at least 50% lower than the level observed with the LNP comprising MC3. According to some embodiments, the immune response is measured by detecting the level of an inflammatory cytokine or chemokine. According to some embodiments, the inflammatory cytokine or chemokine is selected from the group consisting of IL-6, IFNα, IFNγ, IL-18, TNFα, IP-10, MCP-1, MIP1α, MIP1β, and RANTES. According to some embodiments, at least one of the inflammatory cytokines is below a detectable level in the subject's serum 6 hours after administration of the pharmaceutical composition. According to some embodiments of any of the aspects or embodiments herein, the LNP comprising an SS-cleavable lipid and a closed-ended DNA (ceDNA) exhibits a phagocytosis level that is at least 50% lower than the phagocytosis level of an LNP comprising MC3 as the primary cationic lipid administered under similar conditions or is not phagocytosed. According to some embodiments, the SS-cleavable lipid is ss-OP of formula I. According to some embodiments, the LNP further comprises cholesterol and a PEG-lipid conjugate. According to some embodiments, the LNP further comprises a non-cationic lipid.According to some embodiments, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE). According to some embodiments, the LNP further comprises N-acetylgalactosamine (GalNAc). According to some embodiments, GalNAc is present in the LNP at a molar percentage of 0.5% of the total lipid.

[0015] According to another aspect, the present disclosure provides a method of reducing a complement response in a subject in need of treatment with a therapeutic nucleic acid, the method comprising administering to the subject an effective amount of lipid nanoparticles (LNP) comprising the therapeutic nucleic acid, an ss-cleavable lipid, a sterol, and polyethylene glycol (PEG) and N-acetylgalactosamine (GalNAc). According to some embodiments, the subject suffers from a genetic disorder. According to some embodiments, the genetic disorder is sickle cell anemia, melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency) and hemophilia B (coagulation factor IX (FIX) deficiency), cystic fibrosis (CFTR), 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 I type), Scheie syndrome (MPS I S type), Hurler-Scheie syndrome (MPS Iselected from the group consisting of Hurler-Scheie syndrome (MPS I-H), Hunter syndrome (MPS II), Sanfilippo A, B, C, and D syndromes (MPS III A, B, C, and D), Morquio A and B syndromes (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick diseases A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis I, II / III, and IV, sialidosis I and II, glycogen storage diseases I and II (Pompe disease), Gaucher diseases I, II, and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, and cathepsin A deficiency.According to some embodiments, the therapeutic nucleic acid is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), ribozymes, ceDNAs, ministrings, doggybone™, telomere-capped DNA, or dumbbell linear DNA, dicer substrates dsRNA, small hairpin RNAs (shRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), mRNAs, tRNAs, rRNAs, DNA virus vectors, viral RNA vectors, non-viral vectors, and any combination thereof. According to some embodiments, the ceDNA is selected from the group consisting of CELiD, MIDGE, ministring DNA, dumbbell-shaped linear double-stranded capped DNA containing two hairpin structures of ITR at the 5' and 3' ends of the expression cassette, or doggybone™ DNA, and the ceDNA is capsid-free and linear double-stranded DNA. According to some embodiments, the PEG is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG). According to some embodiments, the PEG is present in the LNP at a molecular percentage of about 2% to 4%, such as about 2% to about 3.5%, about 2% to about 3%, about 2% to about 2.5%, about 2.5% to about 4%, about 2.5% to about 3.5%, about 2.5% to about 3%, about 3% to about 4%, about 3.5% to about 4%, or about 2%, about 2.25%, about 2.5%, about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75% or about 4%. According to some embodiments, the PEG is present in the LNP at a molecular percentage of about 3%. According to some embodiments, the LNP further comprises a non-cationic lipid. According to some embodiments, the non-cationic lipid is selected from the group consisting of dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).According to some embodiments, GalNAc is present in the LNP at a molar percentage of about 0.3% to 1% of the total lipid, such as about 0.3% to about 0.9%, about 0.3% to about 0.8%, about 0.3% to about 0.7%, about 0.3% to about 0.6%, about 0.3% to about 0.5%, about 0.3% to about 0.4%, about 0.4% to about 0.9%, about 0.4% to about 0.8%, about 0.4% to about 0.7%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, about 0.5% to about 0.9%, about 0.5% to about 0.8%, about 0.5% to about 0.7%, about 0.5% to about 0.6%, about 0.6% to about 0.9%, about 0.6% to about 0.8%, about 0.6% to about 0.7%, about 0.7% to about 0.9%, about 0.7% to about 0.8%, about 0.8% to about 0.9%, or at a molar percentage of about 0.3%, about 0.4, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1% of the total lipid. According to some embodiments, GalNAc is present in the LNP at a molar percentage of about 0.5% of the total lipid.

[0016] The embodiments of the present disclosure briefly summarized above and discussed in more detail below can be understood by reference to the exemplary embodiments of the present disclosure depicted in the accompanying drawings. However, the accompanying drawings show only typical embodiments of the present disclosure and, therefore, should not be regarded as limiting the scope, as the present disclosure can recognize other equally effective embodiments.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] The present disclosure provides a lipid-based platform for delivering nucleic acids, such as therapeutic nucleic acids (TNAs), such as circular episomal DNA (ceDNA), that can move from the cytoplasm to the nucleus of a cell without a viral capsid component. Immunogenicity associated with virus vector-based gene therapy significantly limits the number of patients due to existing background immunity and precludes readministration to patients. Due to the absence of existing immunity, the therapeutic nucleic acids described herein that contain lipid particles (e.g., lipid nanoparticles) enable additional doses of the therapeutic nucleic acid as needed and further expand patient access, including pediatric populations that may require subsequent administrations as they grow. Further, it is a discovery of the present disclosure that lipid particles (e.g., lipid nanoparticles) containing cleavable lipids having one or more tertiary amino groups and therapeutic nucleic acids containing disulfide bonds provide efficient delivery of therapeutic nucleic acids with improved tolerability and safety profiles. The therapeutic nucleic acids containing lipid particles (e.g., lipid nanoparticles) described herein are not subject to the packaging constraints imposed by the space within a viral capsid and thus, theoretically, the only size limitation for a therapeutic nucleic acid containing a lipid particle (e.g., lipid nanoparticle) lies in the DNA replication efficiency of the host cell.

[0019] As described and exemplified herein, the therapeutic nucleic acid can be circular episomal DNA (ceDNA). According to some embodiments, the therapeutic nucleic acid can be mRNA.

[0020] I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It is to be understood that the invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein and can vary as such. 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. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), Robert S. Porter et al. (eds.), Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8), Immunology by Werner Luttmann, published by Elsevier, 2006, Janeway’s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305), Lewin’s Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055), Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414), Davis et al. Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X), Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542), Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005, and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), and all of the contents thereof are hereby incorporated by reference in their entirety into this specification.

[0021] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

[0022] The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with "for example".

[0023] The use of alternatives (e.g., "or") is to be understood to mean any one, both, or any combination of the alternatives.

[0024] As used herein, the term "about", when referring to a measurable value such as an amount, a time duration, etc., means an inclusion of variations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% so as to be suitable for carrying out the disclosed method.

[0025] As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include any integer value within the recited range and, where appropriate, its fractions (such as tenths and hundredths of an integer) unless otherwise specified.

[0026] As used herein, the terms "comprise", "comprising", "comprises", and "comprised of" are meant to be synonymous with the terms "include", "including", "includes", or "contain", "containing", "contains", and are inclusive or open-ended terms specifying the presence of what follows the component, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps that are known in or disclosed in the art.

[0027] The term "consisting of" refers to the compositions, methods, processes, and their respective components described herein, excluding any elements not recited in the description of the embodiments.

[0028] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiments of the present invention.

[0029] As used herein, terms such as "such as" and "for example" are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are described herein.

[0031] As used herein, the terms "administer", "administering" and variations thereof refer to introducing a composition or agent (e.g., a nucleic acid, particularly ceDNA) to a subject and include simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo therapies. Introduction of a composition or agent to a subject can be by any suitable route including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another person. Administration can be carried out by any suitable route. A suitable route of administration enables the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0032] As used herein, phrases such as "anti-therapeutic nucleic acid immune response", "anti-metastasis vector immune response", "immune response to a therapeutic nucleic acid", "immune response to a metastasis vector" mean any undesirable immune response to a therapeutic nucleic acid, whether of viral or non-viral origin. In some embodiments, the undesirable immune response is an antigen-specific immune response to the viral metastasis vector itself. In some embodiments, the immune response is specific to a metastasis vector that can be double-stranded DNA, single-stranded RNA, or double-stranded RNA. In other embodiments, the immune response is specific to the sequence of the metastasis vector. In other embodiments, the immune response is specific to the CpG content of the metastasis vector.

[0033] As used herein, the term "aqueous solution" means a composition that contains water, either in whole or in part.

[0034] As used herein, "base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include modifications placing new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0035] As used herein, the term "carrier" means including any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce a toxic reaction, an allergic reaction, or a similar adverse reaction when administered to a host.

[0036] As used herein, the term "ceDNA" means a capsid-free closed-ended linear double-stranded (ds) duplex DNA for synthetic or other non-viral gene delivery. According to some embodiments, the ceDNA is a closed-ended linear double-stranded (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimally immunologically defined gene expression (MIDGE) vector. According to some embodiments, the ceDNA is a ministring DNA. According to some embodiments, the ceDNA is a dumbbell-shaped linear double-stranded closed-ended DNA containing two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette. According to some embodiments, the ceDNA is a doggybone (trademark) DNA. A detailed description of ceDNA is provided in the international application PCT / US2017 / 020828, filed Mar. 3, 2017, the entire content of which is hereby expressly incorporated by reference. Certain methods for the production of ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Application No. 18 / 49996, filed Sep. 7, 2018, and International Application No. 2018 / 064242, filed Dec. 6, 2018, each of which is hereby incorporated by reference in its entirety. Certain methods for the production of synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application No. 2019 / 14122, filed Jan. 18, 2019, the entire content of which is hereby incorporated by reference.

[0037] As used herein, the term "closed-ended DNA vector" refers to a capsid-free DNA vector having at least one covalently closed end and at least a portion of the vector having an intramolecular duplex structure.

[0038] As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed - end DNA vector containing at least one terminal palindrome. In some embodiments, the ceDNA contains two covalently closed ends.

[0039] As used herein, the term "ceDNA - bacmid" means a molecule that contains a ceDNA genome as an intermolecular double - strand that can propagate as a plasmid in E. coli and thereby can act as a shuttle vector for baculovirus, referring to an infectious baculovirus genome.

[0040] As used herein, the term "ceDNA - baculovirus" means a baculovirus that contains a ceDNA genome as an intermolecular double - strand within the baculovirus genome.

[0041] As used herein, the terms "ceDNA - baculovirus - infected insect cell" and "ceDNA - BIIC" are used interchangeably and mean an invertebrate host cell (including, but not limited to, insect cells such as Sf9 cells) infected with a ceDNA - baculovirus.

[0042] As used herein, the term "ceDNA genome" means an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further contain one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or virus genome as an intermolecular double - strand polynucleotide of DNA.

[0043] As used herein, the terms "DNA regulatory sequence", "control element", and "regulatory element" are used interchangeably herein and refer to transcriptional and translational control sequences such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., which provide and / or regulate the transcription of non-coding sequences (e.g., DNA-targeted RNA) or coding sequences (e.g., site-specific modified polypeptides or Cas9 / Csn1 polypeptides), and / or regulate the translation of the encoded polypeptide.

[0044] As used herein, the phrase "effective amount" or "therapeutically effective amount" of a therapeutic agent, such as an activator or therapeutic nucleic acid, is an amount sufficient to effect a desired effect, e.g., inhibition of the expression of a target sequence, as compared to the expression level detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include, for example, protein or RNA level assays using techniques known to those of skill in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0045] As used herein, the term "exogenous" means a substance present in a cell other than its natural source. The term "exogenous" as used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide introduced into a biological system such as a cell or an organism by a process involving human intervention that is not normally found and for which it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide introduced into a biological system such as a cell or an organism by a process involving human intervention for which it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to result in ectopic expression or levels that are relatively low. In contrast, as used herein, the term "endogenous" refers to a substance that is native to a biological system or cell.

[0046] As used herein, the term "expression" means the cellular processes involved in the production of RNA and proteins, and, optionally, secreted proteins including, but not limited to, transcription, transcriptional processing, translation, and protein folding, modification, and processing. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene), and polypeptides obtained by translation of mRNA transcribed from a gene.

[0047] As used herein, the term "expression vector" means a vector that directs the expression of RNA or polypeptides from sequences ligated to transcriptional regulatory sequences on the vector. The sequences to be expressed are often, but not necessarily, heterologous to the host cell. Expression vectors can contain additional elements, e.g., expression vectors can have two replication systems so that they can be maintained in two organisms, e.g., human cells in the case of expression, and a prokaryotic host in the case of cloning and amplification. Expression vectors can be recombinant vectors.

[0048] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and mean a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequences sufficient to direct the transcription of a transgene of a DNA vector, e.g., a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0049] As used herein, the term "terminal repeat" or "TR" includes any viral or non-viral terminal or synthetic sequence that includes at least one minimal origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence (also referred to as "RBS" or Rep binding element (RBE)) and the terminal resolution site ("TRS") together constitute the "minimal origin of replication" of AAV, and thus, a TR includes at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically each referred to as an "inverted terminal repeat" or "ITR". In the context of a virus, the ITRs play important roles in mediating replication, viral particle and DNA packaging, DNA integration, and rescue of genomes and proviruses. A TR that is not a reverse complement (palindrome) over its full length can still perform the conventional functions of an ITR, and thus, the term ITR is used to refer to a TR in a viral or non-viral AAV vector that can mediate replication in a host cell. It will be understood by those of skill in the art that in a complex AAV vector construct, there can be more than three ITRs or asymmetric ITR pairs.

[0050] "ITR" can be artificially synthesized using a set of oligonucleotides that contain one or more desirable functional arrays (e.g., palindromic arrays, RBS). The ITR sequence can be an AAV ITR, an artificial non-AAV ITR, or an ITR that is physically derived from a viral AAV ITR (e.g., an ITR fragment removed from a viral genome). For example, the ITR can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, murine parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that serves as the origin of SV40 replication can be further modified by cleavage, substitution, deletion, insertion, and / or addition and used as an ITR. Parvoviridae viruses are composed of two subfamilies, the Parvovirinae subfamily that infects vertebrates and the Densovirinae subfamily that infects invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) viral family, which is capable of replicating in vertebrate hosts including, but not limited to, humans, primates, cattle, dogs, horses, and sheep species. Typically, the ITR sequence can be derived from not only AAV but also parvovirus, lentivirus, fowlpox virus, and B19 in a configuration of wild-type, "doggy bone" and "dumbbell type", symmetric or asymmetric ITR orientation. The ITR is typically present at both the 5' and 3' ends of the AAV vector, but the ITR can be present at only one end of the linear vector. For example, the ITR can be present only at the 5' end. In some other cases, the ITR can be present only at the 3' end of the synthetic AAV vector. For convenience herein, the ITR located 5' (upstream thereof) with respect to the expression cassette in the synthetic AAV vector is referred to as the "5' ITR" or "left ITR", and the ITR located 3' (downstream thereof) with respect to the expression cassette in the vector or synthetic AAV is referred to as the "3' ITR" or "right ITR".

[0051] "Wild-type ITR" or "WT-ITR" refers to the sequence of the naturally occurring ITR sequence in an AAV genome or other dependent virus that, for example, maintains Rep binding activity and Rep nicking ability. The nucleotide sequence of WT-ITR from any AAV serotype may vary slightly from the canonical sequence that occurs naturally due to degeneracy of the genetic code or drift, and thus the WT-ITR sequences included for use herein include WT-ITR sequences as a result of naturally occurring variations (e.g., replication errors).

[0052] As used herein, the terms "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refer to a pair of WT-ITRs within a synthetic AAV vector that are both wild-type ITRs having inverted complementary sequences over their entire length. For example, an ITR can be considered to be a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, provided that the changes do not affect the physical and functional properties of the sequence and the overall three-dimensional structure (two-dimensional and three-dimensional structures). In some embodiments, the deviating nucleotides represent conservative sequence changes. As a non-limiting example, the sequences have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical sequence (e.g., measured using BLAST with default settings), and have a three-dimensional spatial configuration that is symmetric with respect to other WT-ITRs such that their three-dimensional structures have the same shape in geometric space. Substantially symmetric WT-ITRs have the same A, C-C', and B-B' loops in three-dimensional space. Substantially symmetric WT-ITRs can be functionally confirmed as WT by determining that they have an operable Rep binding site (RBE or RBE') and a terminal resolution site (trs) that pair with the appropriate Rep protein. Optionally, other functions, including transgene expression under permissive conditions, can be tested.

[0053] As used herein, the terms "modified ITR", "mod-ITR", or "variant ITR" are used interchangeably and refer to an ITR having mutations in at least one or more nucleotides as compared to a WT-ITR from the same serotype. The mutations can result in a change in one or more of the A, C, C', B, B' regions of the ITR as compared to the three-dimensional spatial conformation of the WT-ITR of the same serotype, and can result in a change in the three-dimensional spatial conformation (i.e., its three-dimensional structure in geometric space).

[0054] As used herein, the term "asymmetric ITR", also referred to as "asymmetric ITR pair", refers to a pair of ITRs within a single-stranded synthetic AAV genome that are not reverse complementary over their entire length. As a non-limiting example, an asymmetric ITR pair does not have a symmetric three-dimensional spatial conformation with respect to its cognate ITRs such that their three-dimensional structures are different shapes in geometric space. In other words, the asymmetric ITR pair has a different overall geometric structure, i.e., the configuration of their A, C-C', and B-B' loops in three-dimensional space is different (e.g., compared to the cognate ITR, one ITR can have a shorter C-C' arm and / or a shorter B-B' arm). The sequence difference between the two ITRs can be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair can be a wild-type AAV ITR sequence and the other ITR can be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence and the two ITRs are modified ITRs having different shapes (i.e., different overall geometric structures) in geometric space. In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm and the other ITR can have a different modification (e.g., a single arm, or a short B-B' arm, etc.) such that they have a different three-dimensional spatial conformation as compared to their cognate asymmetric mod-ITR.

[0055] As used herein, the term "symmetric ITR" refers to a pair of ITRs within a single-stranded AAV genome that are wild-type or mutant (e.g., modified relative to wild-type) adeno-associated virus ITR sequences and are reverse complements over their entire lengths. In a non-limiting example, both ITRs are wild-type ITR sequences from AAV2. In another example, neither ITR is a wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as variant ITRs) and may differ in sequence from the wild-type ITR due to nucleotide addition, deletion, substitution, cleavage, or point mutation. For convenience herein, the ITR located 5' (upstream) of the expression cassette in a synthetic AAV vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' (downstream) of the expression cassette in a synthetic AAV vector is referred to as the "3' ITR" or "right ITR."

[0056] As used herein, the terms "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refer to a pair of modified ITRs within a synthetic AAV, both of which have inverted complementary sequences over their entire lengths. For example, a modified ITR can be considered substantially symmetric even if there are some nucleotide sequences that deviate from the inverted complementary sequence, as long as the variations do not affect the properties and overall shape. As a non-limiting example, the sequences have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) to the reference sequence and have a three-dimensional spatial configuration that is symmetric with respect to their cognate modified ITRs such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops configured in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair can have different inverted complementary nucleotide sequences but can still have the same symmetric three-dimensional spatial configuration. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR of a mod-ITR pair (e.g., the 5' ITR) can be derived from one serotype, and the other ITR (e.g., the 3' ITR) can be derived from a different serotype, but both can have the same corresponding mutations (e.g., if the 5' ITR has a deletion in the C region, the cognate modified 3' ITR of a different serotype has a deletion at the corresponding position in the C' region), thereby resulting in the modified ITR pair having the same symmetric three-dimensional spatial configuration. In such embodiments, each ITR of the modified ITR pair can be derived from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and the modification of one ITR is reflected at the corresponding position of the cognate ITR of a different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) where the differences in the nucleotide sequences between the ITRs do not affect the properties or overall shape and they have substantially the same shape in three-dimensional space.As a non-limiting example, the mod-ITR has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical mod-ITR determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and has a symmetric three-dimensional spatial configuration such that their three-dimensional structures have the same shape in geometric space. Substantially symmetric mod-ITR pairs have the same A, C-C’, and B-B’ loops in three-dimensional space. For example, if a modified ITR of a substantially symmetric mod-ITR pair has a deletion in the C-C’ arm, the cognate mod-ITR has a corresponding deletion in the C-C’ loop and has a similar three-dimensional structure of the remaining A and B-B’ loops of the same shape in the geometric space of the cognate mod-ITR.

[0057] As used herein, the term "adjacent" means referring to the relative position of a nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, A and C are adjacent to B on both sides. The same applies to the arrangement A×B×C. Thus, an adjacent sequence follows before or after the sequence to which it is adjacent, but does not need to be contiguous or immediately adjacent to the sequence to which it is adjacent. In one embodiment, the term "adjacent" refers to the terminal repeats at each end of a linear single-stranded synthetic AAV vector.

[0058] As used herein, the term "gap" means referring to the interrupted portion of the synthetic DNA vector of the present invention, and a stretch of single-stranded DNA is created in other double-stranded ceDNA. The gap can have a length of single-stranded double-stranded DNA from 1 base pair to 100 base pairs. Typical gaps designed and created by the methods described herein, and synthetic vectors generated by such methods can, for example, have lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 bp. Exemplary gaps in the present disclosure can have lengths of 1 bp to 10 bp, 1 to 20 bp, 1 to 30 bp.

[0059] As used herein, the term "nick" typically refers to a discontinuity in a double-stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of a single strand due to damage or enzymatic action. It is understood that one or more nicks allow for the release of torsional strain during DNA replication and that nicks also play a role in facilitating the binding of the transcriptional machinery.

[0060] As used herein, the terms "neDNA", "ceDNA with a nick" refer to closed-ended DNA having a nick or gap in the stem region or spacer region upstream of an open reading frame of 1 to 100 base pairs (e.g., a promoter and transgene to be expressed).

[0061] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid related to the expression of a given RNA or protein, in vitro or in vivo. Thus, genes include regions encoding expressed RNA (typically including polypeptide coding sequences), and often the regulatory sequences required for their expression. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and may include sequences designed to have particular desired parameters.

[0062] As used herein, the term "gene delivery" means the process by which foreign DNA is introduced into a host cell for use in gene therapy.

[0063] As used herein, the phrase "hereditary disease" or "hereditary disorder" means a disease caused, in whole or in part, directly or indirectly, by one or more abnormalities in the genome, particularly a condition that is present from birth. The abnormality can be a mutation, insertion, or deletion in a gene. The abnormality can affect the coding sequence of a gene or its regulatory sequences.

[0064] As used herein, the terms "heterologous nucleotide sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that can be incorporated into a vector, such as a ceDNA vector disclosed herein, and thereby delivered and expressed. A heterologous nucleic acid sequence can be linked (e.g., by genetic manipulation) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterologous nucleic acid sequence can be linked (e.g., by genetic manipulation) to a variant polypeptide to generate a nucleotide sequence encoding a fusion variant polypeptide.

[0065] As used herein, the term "homology" or "homologous" means, after aligning the sequences as necessary and introducing gaps to achieve the maximum percent sequence identity, the percentage of nucleotide residues of a homologous arm that are identical to the nucleotide residues of the corresponding sequence on the target chromosome. Alignment for the purpose of determining the percent nucleotide sequence homology can be achieved in a variety of ways within the skill in the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. In some embodiments, for example, the nucleic acid sequence (e.g., DNA sequence) of the homology arm of the repair template is considered "homologous" if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding unmodified or unedited nucleic acid sequence (e.g., genomic sequence) of the host cell.

[0066] As used herein, the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc. by the nucleic acid therapeutics of the present disclosure. By way of non-limiting example, the host cell can be any of an isolated primary cell, a pluripotent stem cell, a CD34 + cell, an induced pluripotent stem cell, or some immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell can be an in situ or in vivo cell in a tissue, organ, or organism. Further, the host cell can be, for example, a target cell of a mammalian subject (e.g., a human patient in need of gene therapy).

[0067] As used herein, the term "inducible promoter" refers to that which is characterized by initiating or enhancing transcriptional activity in the presence of, affected by, or contacted by an inducer or inducing agent. As used herein, an "inducer" or "inducing agent" can be endogenous or a normally exogenous compound or protein administered in such a manner as to be active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducing agent, i.e., a chemical, compound, or protein, can itself be the result of the transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of an inducible promoter. In some embodiments, the inducible promoter is induced in the absence of a particular agent such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionein, ecdysone, mammalian viruses (e.g., adenovirus late promoter, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), and other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0068] As used herein, the term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes such as cell extracts, and can refer to introducing a programmable synthetic biological circuit into a cell-free system, e.g., a medium that does not contain cells or cell lines such as cell extracts.

[0069] As used herein, the term "in vivo" means referring to an assay or process occurring in or within an organism such as a multicellular animal. In some of the embodiments described herein, a method or use can be said to occur "in vivo" when a unicellular organism such as bacteria is used. The term "ex vivo" refers to methods and uses performed using viable cells having an intact membrane outside the body of a multicellular animal or plant, such as, among others, explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells).

[0070] As used herein, the term "lipid" means referring to a group of organic compounds including but not limited to esters of fatty acids, and is characterized by being insoluble in water but soluble in many organic solvents. Lipids are typically classified into at least three classes: (1) "simple lipids" including fats, oils, and waxes; (2) "complex lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0071] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, sphingoglycolipid families, diacylglycerol, and β-acyl oxy acids, are also included in the group called amphiphilic lipids. Furthermore, the above amphiphilic lipids can be mixed with other lipids including triglycerides and sterols.

[0072] In one embodiment, the lipid composition contains one or more tertiary amino groups, one or more phenyl ester bonds, and disulfide bonds.

[0073] As used herein, the term "lipid conjugate" means a conjugated lipid that inhibits aggregation of lipid particles (e.g., lipid nanoparticles). Such lipid conjugates include, for example, PEG conjugated to dialkyloxypropyl (e.g., PEG-DAA conjugate), PEG conjugated to diacylglycerol (e.g., PEG-DAG conjugate), PEG conjugated to cholesterol, PEG conjugated to phosphatidylethanolamine, and PEG conjugated to ceramide (see, e.g., U.S. Patent No. 5,885,613), such as PEG-lipid conjugates, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugate, see, e.g., U.S. Provisional Application No. 61 / 294,828, filed January 13, 2010, and U.S. Provisional Application No. 61 / 295,140, filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugate), and mixtures thereof, but are not limited thereto. Additional examples of POZ-lipid conjugates are described in PCT Publication No. 2010 / 006282. PEG or POZ can be conjugated directly to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for conjugating PEG or POZ to a lipid can be used, including non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties such as amide or carbamate are used. The disclosures of each of the above patent documents are hereby incorporated by reference in their entirety for all purposes.

[0074] As used herein, the term "encapsulated in lipid" means a lipid particle that provides an active or therapeutic agent, such as a nucleic acid (e.g., ceDNA), by 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).

[0075] As used herein, the terms "lipid particle" or "lipid nanoparticle" mean lipid formulations that can be used to deliver therapeutic agents, such as nucleic acid therapeutics, to a target site of interest (e.g., cells, tissues, organs, etc.). In one embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles, which are typically formed from cationic lipids, non-cationic lipids, and optionally conjugated lipids that prevent aggregation of the particles. In other preferred embodiments, therapeutic agents such as therapeutic nucleic acids can be encapsulated in the lipid portion of the particles, thereby protecting them from enzymatic degradation. In one embodiment, the lipid particles comprise a nucleic acid (e.g., ceDNA) and a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds, and disulfide bonds.

[0076] The lipid particles of the present invention typically have an average diameter of about 20 nm to about 120 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm or about 150 nm.

[0077] As used herein, the term "cationic lipid" refers to any lipid that is positively charged at physiological pH. The cationic lipid in the lipid particle can include one or more cationic lipids such as, for example, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), "SS-cleavable lipid", or mixtures thereof. In some embodiments, the cationic lipid is also an ionizable lipid, i.e., an ionizable cationic lipid.

[0078] As used herein, the term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups conjugated to neutral lipids.

[0079] As used herein, the term "hydrophobic lipid" refers to a compound having a nonpolar group that includes, but is not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0080] As used herein, the term "ionizable lipid" means a lipid having at least one protonatable or deprotonatable group, such as a cationic lipid, such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably at or above physiological pH. The addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the dominant species, and it will be understood by those skilled in the art that not all lipids need to be present in a charged or neutral form. Generally, ionizable lipids have a pKa of protonatable groups in the range of about 4 to about 7. In some embodiments, the ionizable lipid may include a "cleavable lipid" or an "SS-cleavable lipid".

[0081] As used herein, the term "neutral lipid" means any of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

[0082] As used herein, the term "non-cationic lipid" means any amphiphilic lipid, and any other neutral or anionic lipid.

[0083] As used herein, the terms "cleavable lipid" or "SS-cleavable lipid" refer to a lipid that contains a unit capable of cleaving a disulfide bond. The cleavable lipid may include a cleavable disulfide bond ("ss") that contains a lipid-like substance that includes a pH-sensitive tertiary amine and a self-degrading phenyl ester. For example, the SS-cleavable lipid may be an ss-OP lipid (COATSOME® SS-OP), an ss-M lipid (COATSOME® SS-M), an ss-E lipid (COATSOME® SS-E), an ss-EC lipid (COATSOME® SS-EC), an ss-LC lipid (COATSOME® SS-LC), an ss-OC lipid (COATSOME® SS-OC), and an ss-PalmE lipid (see, for example, Formulas I-IV), or a lipid described by Togashi et al., (2018) Journal of Controlled Release "A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug" 279:262-270. Additional examples of cleavable lipids are described in U.S. Patent No. 9,708,628 and U.S. Patent No. 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, the cleavable lipid includes a tertiary amine that responds to a disulfide bond that can be cleaved in an acidic compartment, such as an endosome or lysosome for membrane destabilization, and a reducing environment such as the cytoplasm. In one embodiment, the cleavable lipid is a cationic lipid. In one embodiment, the cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

[0084] As used herein, the term "organic lipid solution" is meant to refer to a composition that includes an organic solvent that has a lipid, either in whole or in part.

[0085] As used herein, the term "liposome" means a spherical structure assembled from lipid molecules that encloses an internal aqueous volume separated from the aqueous exterior. A liposome is a vesicle having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of pharmaceutical development. They act by fusing with cell membranes and repositioning their lipid structure to deliver a drug or active pharmaceutical ingredient. Liposome compositions for such delivery typically consist of compounds having phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0086] As used herein, the term "local delivery" means the direct delivery of an active agent, such as interfering RNA (e.g., siRNA), to a target site within an organism. For example, a drug can be delivered locally by direct injection to a disease site such as a tumor, or to other target sites such as an inflammatory site, or to a target organ such as the liver, heart, pancreas, or kidney.

[0087] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically modified or biochemically modified, non-natural, or derivatized nucleotide bases. "Oligonucleotide" generally refers to polynucleotides of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of oligonucleotides. Oligonucleotides are also known as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" are to be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides as applicable to the described embodiments. DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-ended linear double-stranded DNA (CELiD or ceDNA), doggybone (trademark) DNA, dumbbell-shaped DNA, minimally immunologically defined gene expression (MIDGE)-vectors, viral vectors or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have binding properties similar to those of the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioate, phosphorodiamidate morpholino oligomers (morpholino), phosphoramidate, methylphosphonate, chiral methylphosphonate, 2'-O-methyl ribonucleotide, locked nucleic acid (LNA™), and peptide nucleic acid (PNA). Unless otherwise limited, the term includes nucleic acids containing known analogs of natural nucleotides having binding properties similar to those of the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly shown sequences.

[0088] As used herein, the terms "nucleic acid therapy," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any modality of therapy using a nucleic acid as the active ingredient of a therapeutic agent for treating a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrates dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutic agents include minicircle DNA, minigenes, viral DNA (e.g., lentiviral or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimally immunologically defined gene expression (MIDGE)-vectors, non-viral miniring DNA vectors (linearly covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA").

[0089] As used herein, "inhibitory polynucleotide" refers to a DNA or RNA molecule that reduces or prevents the expression (transcription or translation) of a second (target) polynucleotide. Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences. The term "inhibitory polynucleotide" further includes RNAi that encodes an actual inhibitory species such as DNA and RNA molecules, for example, a DNA molecule encoding a ribozyme.

[0090] As used herein, "gene silencing" or "gene silenced" with respect to the activity of an RNAi molecule, such as siRNA or miRNA, refers to at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% reduction in the mRNA level of a target gene in a cell compared to the mRNA level seen in a cell in which the miRNA or RNA interference molecule is absent. In a preferred embodiment, the mRNA level is reduced by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.

[0091] As used herein, the terms "interfering RNA", "RNAi", or "interfering RNA sequence" include single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide), double-stranded RNA (i.e., double-stranded RNA such as siRNA, dicer substrate dsRNA, shRNA, aiRNA, or pre-miRNA), DNA-RNA hybrid (see, e.g., PCT Publication No. 2004 / 078941), or, when the interfering RNA is present in the same cell as the target gene or sequence, a DNA-DNA hybrid (see, e.g., PCT Publication No. 2004 / 104199) that can reduce or inhibit the expression of the target gene or sequence (e.g., by mediating degradation or inhibiting translation of mRNA complementary to the interfering RNA sequence). Thus, interfering RNA refers to single-stranded RNA complementary to the target mRNA sequence or double-stranded RNA formed by two complementary strands or a single self-complementary strand. The interfering RNA can have substantial or complete identity to the target gene or sequence or can contain regions of mismatch (i.e., mismatch motifs). The sequence of the interfering RNA can correspond to the full-length target gene or a subsequence thereof. Preferably, the interfering RNA molecule is chemically synthesized. The disclosures of each of the above patent documents are hereby incorporated by reference in their entirety for all purposes. The term "RNAi" can include both gene-silencing RNAi molecules and RNAi effector molecules that activate gene expression. In some embodiments, RNAi agents that inhibit or function in gene silencing are useful, for example, in the methods, kits, and compositions disclosed herein to inhibit the innate immune response.

[0092] Interfering RNAs include "small interfering RNAs" or "siRNAs", e.g., interfering RNAs that are about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of a double-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length). The siRNA duplex may include 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini. Examples of siRNAs include double-stranded polynucleotide molecules assembled from two separate strand molecules (one strand is the sense strand and the other is the complementary antisense strand), double-stranded polynucleotide molecules assembled from a single-strand molecule (the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker), double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions, and circular single-stranded polynucleotide molecules having a stem with two or more loop structures and self-complementary sense and antisense regions (the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule), but are not limited thereto. As used herein, the term "siRNA" includes RNA-RNA duplexes as well as DNA-RNA hybrids (see, e.g., PCT Publication No. 2004 / 078941).

[0093] As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain segments of nucleic acids in a manner that would not otherwise occur in nature, or is synthetic. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences necessary for the expression of the coding sequences of the present disclosure. An "expression cassette" includes a DNA coding sequence operably linked to a promoter.

[0094] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) non-covalently binds, i.e., forms Watson-Crick base pairs and / or G / U base pairs, and contains a nucleotide sequence that allows it to "anneal" or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength (i.e., the nucleic acid specifically binds to a complementary nucleic acid). As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and cytosine (C) pairing with guanine (G). In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), a guanine (G) base pairs with uracil (U). For example, G / U base pairing is partly responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodon base pairing with codons in mRNA. In the context of this disclosure, a guanine (G) in the protein-binding segment (dsRNA duplex) of a DNA-targeting RNA molecule of interest is considered complementary to uracil (U), and vice versa. Thus, if a G / U base pair can be made at a given nucleotide position in the protein-binding segment (dsRNA duplex) of a DNA-targeting RNA molecule of interest, that position is not considered non-complementary, but rather is considered complementary.

[0095] As used herein, "nucleotide" includes the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through phosphate groups.

[0096] "Operably linked" means that the components so described are in a juxtaposition that permits them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. A promoter can be said to drive the expression of, or drive the transcription of, the nucleic acid sequence it regulates. The phrases "operably linked," "operatively positioned," "operably connected," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional position and / or orientation with respect to the nucleic acid sequence and is regulated to control the initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which the nucleic acid sequence is in an inverted orientation such that what was the coding strand is now the non-coding strand and vice versa. Inverted promoter sequences are used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in combination with an enhancer.

[0097] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length that can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0098] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil / water or water / oil, and various types of wetting agents. This term also includes agents approved by the regulatory authorities of the United States Federal Government or listed in the United States Pharmacopeia for use in animals including humans, and carriers or diluents that do not cause significant irritation to the subject and do not impair the biological activity and properties of the administered compound.

[0099] As used herein, the term "promoter" means any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of a nucleic acid sequence, which can be a heterologous target gene encoding a protein or RNA. A promoter can be constitutive, inducible, repressive, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remaining nucleic acid sequence are controlled. A promoter can also contain gene elements to which regulatory proteins and molecules such as RNA polymerase and other transcription factors can bind. Within a promoter sequence, a transcription start site, as well as protein-binding domains involved in the binding of RNA polymerase, will be found. Eukaryotic promoters often, but not always, contain 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. A promoter sequence is joined at its 3' end by a transcription start site and extends upstream (5' orientation) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background.

[0100] A promoter can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon of a given gene or sequence, and can be naturally associated with the gene or sequence. Such a promoter can be referred to as "endogenous". Similarly, in some embodiments, an enhancer can be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments, the coding nucleic acid segment is positioned under the control of a "recombinant promoter" or "heterologous promoter", both of which refer to a promoter that is not normally associated with the encoded nucleic acid sequence operably linked 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 a promoter or enhancer can include a promoter or enhancer from another gene, a promoter or enhancer isolated from any other prokaryotic, viral, or eukaryotic cell, and a synthetic promoter or enhancer that does not "naturally occur", i.e., it can include different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic manipulation known in the art. In addition to synthetically producing the nucleic acid sequences of promoters and enhancers, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques including PCR with respect to the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906, each of which is incorporated herein by reference in its entirety). Further, it is contemplated that control sequences that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc. can be used as well.

[0101] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase the transcriptional activation of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream or downstream of the gene start site they regulate. Enhancers can be located within the intron region or exon region of unrelated genes.

[0102] As used herein, "Rep binding site" ("RBS") and "Rep binding element" ("RBE") are used interchangeably and mean a binding site for a Rep protein (e.g., AAV Rep78 or AAV Rep68), such that upon binding by the Rep protein, the Rep protein is enabled to carry out its site-specific endonuclease activity on the sequence incorporating the RBS. The RBS sequence and its reverse complement together form a single RBS. RBS sequences are well known in the art and include, for example, the RBS sequence identified in AAV2, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO:1). Any known RBS sequence can be used in embodiments of the present invention, including other known AAV RBS sequences and other naturally known or synthetic RBS sequences. Without being bound by theory, the nuclease domain of the Rep protein binds to the double-stranded nucleotide sequence GCTC, and thus two known AAV Rep proteins are thought to bind directly and stably assemble to the double-stranded oligonucleotide, 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO:1). In addition, soluble aggregated conformational isomers (i.e., an indefinite number of interrelated Rep proteins) dissociate and bind to oligonucleotides containing the Rep binding site. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone on each strand. The interaction with the nitrogenous bases provides sequence specificity, while the interaction with the phosphodiester backbone is non-sequence specific or low-sequence specific and stabilizes the protein-DNA complex.

[0103] As used herein, the phrase "recombinant vector" means a vector that contains a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest in the subject as extrachromosomal DNA at high copy number, thereby eliminating the potential impact of chromosomal integration.

[0104] As used herein, the term "reporter" means a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism can be readily observed. For example, a fluorescent protein fluoresces a cell when excited with light of a specific wavelength, luciferase catalyzes a reaction that produces light in a cell, and an enzyme such as β-galactosidase converts a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP), and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0105] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, for example, as a reporter polypeptide or, more preferably, as a polypeptide that kills cells, such as a toxin, or a drug that renders cells more susceptible to killing by a selected agent or deletion thereof. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. For example, effector proteins include, but are not limited to, restriction endonucleases that target host cell DNA sequences (whether genomic or episomal), proteases that target polypeptides necessary for cell survival, DNA gyrase inhibitors, and ribonuclease type toxins. In some embodiments, the expression of an effector protein controlled by a synthetic biological circuit described herein can participate as a factor in another synthetic biological circuit, thereby expanding the range and complexity of the responsiveness of the biological circuitry.

[0106] Transcription regulators refer to transcription activators and repressors that activate or repress the transcription of a gene of interest. A promoter is a region of a nucleic acid that initiates the transcription of a particular gene. Transcription activators typically bind near a transcription promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to the transcription promoter and sterically hinder the initiation of transcription by RNA polymerase. Other transcription regulators can serve as either activators or repressors depending on where they bind and on cellular and environmental conditions. Non-limiting examples of the transcription regulator class include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine-zipper proteins.

[0107] As used herein, "repressor protein" or "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates transcription of a sequence operably linked to the regulatory sequence element, respectively. Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins described herein are, for example, modular in form, including separable DNA binding and input agent binding, or responsive elements or domains.

[0108] As used herein, an "input agent-responsive domain" is a domain of a transcription factor that binds to a condition or input agent or otherwise renders a linked DNA-binding fusion domain responsive to the presence of that condition or input. In one embodiment, the presence of the condition or input results in a conformational change in the input agent-responsive domain or the protein to which it is fused, which modifies the transcriptional regulatory activity of the transcription factor.

[0109] As used herein, the terms "sense" and "antisense" mean to refer to the orientation of structural elements on a polynucleotide. The sense and antisense versions of an element are reverse complements of each other.

[0110] As used herein, the term "sequence identity" means the relatedness between two nucleotide sequences. For the purposes of the present disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Any parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (number of identical deoxyribonucleotides × 100) / (length of the alignment - total number of gaps in the alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0111] As used herein, the term "spacer region" means an intervening sequence that separates functional elements within a vector or genome. In some embodiments, the AAV spacer region holds two functional elements for desired processing for optimal functionality. In some embodiments, the spacer region provides or increases the genetic stability of the vector or genome. In some embodiments, the spacer region facilitates easy genetic manipulation of the genome by providing a convenient position for cloning sites and gaps in the designed number of base pairs. For example, in certain embodiments, an oligonucleotide "polylinker" or "polycloning site" containing several restriction endonuclease sites, or a non-open reading frame sequence designed to have no known protein (e.g., transcription factor) binding sites, is positioned within the vector or genome to separate cis-acting factors, and for example, 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc. can be inserted.

[0112] As used herein, the term "subject" means a human or animal to whom treatment, including prophylactic treatment with a therapeutic nucleic acid according to the invention, is provided. Usually, the animal is a vertebrate such as, but not limited to, a primate, rodent, farm animal, or game animal. Examples of primates include, but are not limited to, chimpanzees, cynomolgus monkeys, rhesus monkeys, and macaques, such as the rhesus macaque. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of farm animals and game animals include cows, horses, pigs, deer, bison, buffalo, felines, such as house cats, canines, such as dogs, foxes, wolves, avians, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon, but are not limited thereto. In certain embodiments of 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, the subject can be a neonatal or fetal subject, for example, the subject is present in utero. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Non-human mammals can be advantageously used as subjects representing animal models of diseases and disorders. Additionally, the methods and compositions described herein can be used in farm animals and / or pets. Human subjects can be of any age, sex, race, or ethnic group, such as Caucasian, Asian, African, Black, African American, Afro-European, Latin American, Middle Eastern, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject has already received treatment. In some embodiments, the subject is an embryo, fetus, neonate, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonate, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.

[0113] As used herein, the phrase "subject in need" refers to a subject who is (i) scheduled to receive a ceDNA lipid particle (or a pharmaceutical composition comprising a ceDNA lipid particle) according to the described invention, (ii) receiving a ceDNA lipid particle (or a pharmaceutical composition comprising a ceDNA lipid particle) according to the described invention, or (iii) has received a ceDNA lipid particle (or a pharmaceutical composition comprising a ceDNA lipid particle) according to the described invention, provided that the context and usage of the phrase do not otherwise indicate.

[0114] As used herein, the terms "suppress", "reduce", "impede", "inhibit", and / or "decrease" (and similar terms) generally refer to the act of directly or indirectly reducing a concentration, level, function, activity, or behavior relative to a natural, expected, or average level, or relative to a control condition.

[0115] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vector" mean the AAV vector and its method of synthetic production in a completely cell-free environment.

[0116] As used herein, the term "systemic delivery" means the delivery of lipid particles that results in widespread in vivo distribution of an active agent, such as interfering RNA (e.g., siRNA), within an organism. Depending on the administration technique, systemic delivery of a particular agent may or may not be achieved. Systemic delivery means that a useful amount, preferably a therapeutically effective amount, of the agent is exposed to most parts of the body. To obtain widespread in vivo distribution, generally, the agent requires a blood lifespan such that it is not rapidly degraded or excreted (by first-pass organs (liver, lung, etc.) or by rapid non-specific cell binding) before reaching the disease site distal to the administration site. Systemic delivery of lipid particles (e.g., lipid nanoparticles) can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles (e.g., lipid nanoparticles) is by intravenous delivery.

[0117] As used herein, the terms "terminal resolution site" and "trs" are used interchangeably herein and mean the region where Rep forms a tyrosine-phosphodiester bond with 5'-thymidine to generate a 3'-OH that serves as a substrate for DNA elongation via a cellular DNA polymerase, such as DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex may be involved in a ligation reaction. In some embodiments, the TRS includes minimally unpaired thymidine. In some embodiments, the nicking efficiency of the TRS can be at least partially controlled by its distance within the same molecule from the RBS. When the acceptor substrate is a complementary ITR, the resulting product is an intermolecular duplex. TRS sequences are known in the art and include, for example, the hexanucleotide sequence 5'-GGTTGA-3' identified in AAV2. Any known TRS sequence can be used in embodiments of the present invention, including other known AAV TRS sequences and other synthetic TRS sequences that are naturally known or AGTT, GGTTGG, AGTTGG, AGTTGA, and other motifs such as RRTTRR.

[0118] As used herein, the terms "therapeutic amount," "therapeutically effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent (e.g., the ceDNA lipid particles described herein) are used interchangeably and refer to an amount sufficient to provide the intended benefit of treatment. However, dosage levels are based on a variety of factors including the type of injury, age, weight, gender, medical condition of the patient, severity of the medical condition, route of administration, and the particular active agent used. Thus, the dosing regimen can vary widely but can be routinely determined by a physician using standard methods. Additionally, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include a prophylactic or preventative amount of the described compositions of the invention. In the prophylactic or preventative use of the described invention, a pharmaceutical composition or agent is administered in an amount sufficient to eliminate or reduce the risk, or decrease the severity, or delay the onset of a disease, disorder or condition in a patient who is susceptible to, or otherwise at risk of, the disease, disorder or condition, including the biochemical, histological and / or behavioral symptoms of the disease, disorder or condition, its complications, and the intermediate pathological phenotypes that appear during the development of the disease, disorder or condition. In some medical judgment, it is generally preferred to use the maximum dose, i.e., the highest safe dose. The terms "dose" and "dosage" are used interchangeably herein.

[0119] As used herein, the term "therapeutic effect" refers to the result of treatment, which result is judged to be desirable and beneficial. Therapeutic effects can include, directly or indirectly, the arrest, reduction, or elimination of disease symptoms. Therapeutic effects can also include, directly or indirectly, the arrest, reduction, or elimination of the progression of disease symptoms.

[0120] For any therapeutic agent described herein, a therapeutically effective amount can first be determined from preliminary in vitro studies and / or animal models. A therapeutically effective dose can also be determined from human data. The doses applied can be adjusted based on the relative bioavailability and efficacy of the compound administered. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of those skilled in the art. The general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.

[0121] Pharmacokinetic principles provide a basis for altering the dosing regimen to obtain the desired degree of therapeutic effect while minimizing unacceptable side effects. The plasma concentration of a drug can be measured, and in situations where it is related to the therapeutic concentration range, additional guidance regarding changes in the dose can be obtained.

[0122] As used herein, the terms “treat,” “treating,” and / or “treatment” include obtaining a beneficial or desirable clinical result, which includes suppressing, substantially inhibiting, retarding, or reversing the progression of a condition, substantially ameliorating the clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms. Treating further refers to achieving one or more of the following: (a) reducing the severity of a disorder, (b) limiting the development of symptoms characteristic of the disorder being treated, (c) limiting the worsening of symptoms characteristic of the disorder being treated, (d) limiting the recurrence of a disorder in a patient who has previously had the disorder, and (e) limiting the recurrence of symptoms in a patient who was previously asymptomatic with respect to the disorder.

[0123] Beneficial or desired clinical outcomes, such as pharmacological and / or physiological effects, may include preventing the occurrence of a disease, disorder or condition (preventive treatment), alleviating the symptoms of a disease, disorder or condition, reducing the degree of a disease, disorder or condition, stabilizing (i.e., not worsening) a disease, disorder or condition, preventing the spread of a disease, disorder or condition, delaying or slowing the progression of a disease, disorder or condition, improving or alleviating a disease, disorder or condition, and combinations thereof, in a subject who may be predisposed to the disease, disorder or condition but has not yet experienced or manifested the symptoms of the disease, as well as extending survival compared to survival expected in the absence of treatment, but are not limited thereto.

[0124] The terms "vector" or "expression vector" mean a replicon, such as a plasmid, bacteriomid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an "insert", "transgene", or "expression cassette", can be attached to effect the expression or replication of the attached segment (the "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 ultimately be of viral origin or non-viral origin. However, for the purposes of the present disclosure, "vector" generally refers to a synthetic AAV vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that can replicate when associated with appropriate control elements and can transfer a gene sequence into a cell. In some embodiments, the vector can be a recombinant vector or an expression vector.

[0125] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Members of each group can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in or deleted from the group for reasons of convenience and / or patentability. If any such inclusion or deletion occurs, the specification is considered to include the modified group herein and thus meets the description of all Markush groups used in the appended claims.

[0126] In some embodiments of the aspects, the disclosure described herein is not related to the process of human cloning, the process of modifying the genetic identity of the human germ line, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without any substantial medical benefit to humans or animals, and processes for modifying the genetic identity of animals resulting from such processes.

[0127] Other terms are defined within the description of the various aspects of the invention herein.

[0128] All patents and other publications cited throughout this application, including literature references, issued patents, published patent applications, and pending patent applications, are hereby expressly incorporated by reference herein for the purpose of explaining and disclosing, for example, the methodologies described in such publications that may be used in connection with the technologies described herein. These publications are provided only for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0129] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but as will be recognized by those of ordinary skill in the art, various equivalent modifications are possible within the scope of the present disclosure. For example, method steps or functions are presented in a given order, but alternative embodiments may perform the functions in a different order or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to provide further embodiments of the present disclosure using the compositions, functions, and concepts of the above-referenced documents and applications. Additionally, in view of considerations of biological function equivalence, some changes can be made to the protein structure without affecting the type or amount of biological or chemical action. In light of the detailed description, these and other changes can be made to the present disclosure. All such modifications are intended to be included within the scope of the appended claims.

[0130] Any particular element of any of the foregoing embodiments can be combined with or replaced by an element of other embodiments. Additionally, while the advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments are necessarily required to exhibit such advantages since all embodiments are within the scope of the present disclosure.

[0131] The technology described in this specification is further illustrated by the following examples and should in no way be construed as further limiting them. It should be understood that the present invention is not limited in any way to the specific methodologies, protocols, reagents, etc. described herein and may vary as such. 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.

[0132] II. Cleavable Lipids Provided herein is a pharmaceutical composition comprising a cleavable lipid and a capsid-free non-viral vector (e.g., ceDNA) that can be used to deliver a capsid-free non-viral DNA vector to a target site of interest (e.g., cells, tissues, organs, etc.). As used herein, the term "cleavable lipid" refers to a cationic lipid that contains a disulfide bond ("SS") cleavable unit. In one embodiment, the SS-cleavable lipid comprises a tertiary amine that responds to disulfide bonds that can be cleaved in an acidic compartment, for membrane destabilization (e.g., endosome or lysosome), and in a reducing environment (e.g., cytoplasm). Examples of SS-cleavable lipids can include SS-cleavable and pH-activated lipid-like substances such as ss-OP lipid, ssPalm lipid, ss-M lipid, ss-E lipid, ss-EC lipid, ss-LC lipid, ss-OC lipid, etc. As shown herein, ceDNA lipid particles (e.g., lipid nanoparticles) comprising a cleavable lipid provide, for example, more efficient delivery of ceDNA to target cells, including hepatocytes. As reported by the present disclosure, ceDNA particles comprising ceDNA and a cleavable lipid showed fewer ceDNA copies in the liver with equivalent luciferase expression compared to other lipids such as MC3. Indeed, a synergistic effect between ceDNA and the cleavable lipid was observed, minimizing the phagocytosis effect (e.g., see Figures 14 - 17) while increasing ceDNA expression up to 4,000-fold compared to other lipids, such as MC3. As also reported by the present disclosure, lipid formulations comprising mRNA and a cleavable lipid resulted in increased transgene expression for up to 3 days after subretinal injection in rats compared to vehicle controls (Figures 24 and 25). Thus, the lipid particles described herein (e.g., ceDNA lipid particles or mRNA lipid particles) can be advantageously used to increase the delivery of nucleic acids (e.g., ceDNA or mRNA) to target cells / tissues compared to other conventional lipids with minimal or no phagocytosis effect. Thus, the lipid particles described herein (e.g., ceDNA lipid particles or mRNA lipid particles) provided enhanced nucleic acid delivery compared to conventional lipid nanoparticles known in the art.The mechanism has not yet been determined and is not bound by theory, but lipid particles containing cleavable lipids (e.g., ceDNA lipid particles or mRNA lipid particles) are thought to improve delivery to hepatocytes and avoid phagocytosis. Another advantage of ceDNA containing lipid particles with cleavable lipids described herein is that they exhibit excellent tolerance in vivo compared to other lipid nanoparticles, such as MC3.

[0133] In one embodiment, the cleavable lipid may comprise three components, namely, an amine terminal group, a linker group, and a hydrophobic tail. In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one or more tertiary amino groups, and disulfide bonds. The tertiary amino group provides pH responsiveness, induces endosomal escape, the phenyl ester bond enhances structural degradability (self-degradability), and the disulfide bond is cleaved in a reducing environment.

[0134] In one embodiment, the cleavable lipid is an ss-OP lipid. In one embodiment, the ss-OP lipid comprises a structure represented by the following Formula I.

Chemical formula

[0135] In one embodiment, the SS-cleavable lipid is an SS-cleavable and pH-activatable lipid-like substance (ssPalm). ssPalm lipids are well known in the art. See, for example, Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, ssPalm is an ssPalmM lipid having a structure of Formula II.

Chemical formula

[0136] In one embodiment, the ssPalmE lipid is an ssPalmE-P4-C2 lipid having a structure of Formula III. [Chemical formula]

[0137] In one embodiment, the ssPalmE lipid is an ssPalmE-Paz4-C2 lipid having the structure of formula IV. [Chemical formula]

[0138] In one embodiment, the cleavable lipid is an ss-M lipid. In one embodiment, the ss-M lipid has the structure shown in the following formula V. [Chemical formula]

[0139] In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, the ss-E lipid has the structure shown in the following formula VI. [Chemical formula]

[0140] In one embodiment, the cleavable lipid is an ss-EC lipid. In one embodiment, the ss-EC lipid has the structure shown in the following formula VII. [Chemical formula]

[0141] In one embodiment, the cleavable lipid is an ss-LC lipid. In one embodiment, the ss-LC lipid has the structure shown in the following formula VIII. [Chemical formula]

[0142] In one embodiment, the cleavable lipid is an ss-OC lipid. In one embodiment, the ss-OC lipid has the structure shown in the following formula IX. [Chem.]

[0143] In one embodiment, the lipid particle (lipid nanoparticle) formulation is made and loaded with ceDNA obtained by the process disclosed in International Application No. 2018 / 050042, filed on September 7, 2018, which is incorporated herein by reference in its entirety. This can be achieved by high-energy mixing of ethanol lipids at low pH with aqueous ceDNA, which protonates the lipids and provides a favorable energetics for particle ceDNA / lipid association and nucleation. The particles can be further stabilized by aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level. In one embodiment, the present disclosure provides ceDNA lipid particles comprising a lipid of Formula I prepared by the process described in Example 6.

[0144] Generally, lipid particles (e.g., lipid nanoparticles) are prepared at a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 60:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) can be in the range of about 1:1 to about 60:1, about 1:1 to about 55:1, about 1:1 to about 50:1, about 1:1 to about 45:1, about 1:1 to about 40:1, about 1:1 to about 35:1, about 1:1 to about 30:1, about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, about 6:1 to about 9:1, about 30:1 to about 60:1. According to some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with ceDNA (mass or weight) relative to a total lipid ratio of about 60:1. According to some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with ceDNA (mass or weight) relative to a total lipid ratio of about 30:1. The amounts of lipid and ceDNA can be adjusted to provide a desired N / P ratio, e.g., an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid particle formulation can be in the range of about 5 mg / mL to about 30 mg / mL.

[0145] In some embodiments, the lipid nanoparticles contain an agent for condensing and / or encapsulating nucleic acid cargo such as ceDNA. Such agents are also referred to herein as condensing agents or encapsulating agents. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acids can be used as long as it is non-fusogenic. In other words, the agent can condense and / or encapsulate nucleic acid cargo such as ceDNA but has little or no fusogenic activity. Without wishing to be bound by theory, a condensing agent may have some fusogenic activity when not condensing / encapsulating nucleic acids such as ceDNA, but the nucleic acid encapsulated in the lipid nanoparticles formed with such a condensing agent can be non-fusogenic.

[0146] Cationic lipids are typically used to condense nucleic acid cargo, such as ceDNA, at low pH and to drive membrane association and membrane fusogenicity. Generally, a cationic lipid is a lipid that is positively charged or contains at least one amino group that is protonated under acidic conditions, such as at a pH of 6.5 or less. A cationic lipid can also be an ionizable lipid, such as an ionizable cationic lipid. "Non-fusogenic cationic lipid" means a cationic lipid that can condense and / or encapsulate nucleic acid cargo such as ceDNA but has little or no fusogenic activity.

[0147] In one embodiment, the non-cationic lipid can comprise from 20 to 90% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). For example, the molar content of the cationic lipid can be from 20 to 70% (mol), from 30 to 60% (mol), from 40 to 60% (mol), from 40 to 55% (mol), or from 45 to 55% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the cationic lipid comprises from about 50 mol% to about 90 mol% of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0148] In one embodiment, the SS-cleavable lipid is not MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3). DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the content of which is incorporated herein by reference in its entirety. The structure of D-Lin-MC3-DMA (MC3) is shown below as Formula X. [Chemical Formula]

[0149] In one embodiment, the cleavable lipid is not lipid ATX-002. Lipid ATX-002 is described in WO2015 / 074085, the content of which is incorporated herein by reference in its entirety. In one embodiment, the cleavable lipid is not (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32). Compound 32 is described in WO2012 / 040184, the content of which is incorporated herein by reference in its entirety. In one embodiment, the cleavable lipid is not Compound 6 or Compound 22. Compounds 6 and 22 are described in WO2015 / 199952, the content of which is incorporated herein by reference in its entirety.

[0150] In one embodiment, the lipid particles (e.g., lipid nanoparticles) can further comprise a non-cationic lipid. The non-cationic lipid can help enhance fusion and the stability of the forming LNPs. Examples of non-ionic lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipid can be a neutral non-charged, zwitterionic, or anionic lipid. Non-cationic lipids are typically used to enhance membrane fusion.

[0151] Exemplary non-cationic lipids include distearoyl-sn-glycero-phosphoethanolamine, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dieleoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, or mixtures thereof, but not limited thereto. It should be understood that other diacyl phosphatidylcholine and diacyl phosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably C, 10 ~C 24 acyl groups derived from fatty acids having carbon chains, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0152] Other examples of non-cationic lipids suitable for use in lipid particles (e.g., lipid nanoparticles) include, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amide, dioctadecyldimethylammonium bromide, ceramides, non-phospholipids such as sphingomyelin, etc.

[0153] In one embodiment, the non-cationic lipid is a phospholipid. In one embodiment, the non-cationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the non-cationic lipid is DSPC. In other embodiments, the non-cationic lipid is DOPC. In other embodiments, the non-cationic lipid is DOPE.

[0154] In some embodiments, the non-cationic lipid can constitute 0 to 20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the non-cationic lipid content is 0.5 to 15% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the non-cationic lipid content is 5 to 12% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the non-cationic lipid content is 5 to 10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 6% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 7.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 7.5% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 8.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 9.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the non-cationic lipid content is about 10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the non-cationic lipid content is about 11% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0155] Exemplary non-cationic lipids are described in PCT Publication No. WO 2017 / 099823 and U.S. Patent Publication No. US 2018 / 0028664, the contents of both of which are incorporated herein by reference in their entirety.

[0156] Non-limiting examples of cationic lipids include SS-cleavable and pH-activated lipid-like substances - OP (ss-OP, Formula I), SS-cleavable and pH-activated lipid-like substances - M (SS-M, Formula V), SS-cleavable and pH-activated lipid-like substances - E (SS-E, Formula VI), SS-cleavable and pH-activated lipid-like substances - EC (SS-EC, Formula VII), SS-cleavable and pH-activated lipid-like substances - LC (SS-LC, Formula VIII), SS-cleavable and pH-activated lipid-like substances - OC (SS-OC, Formula IX), polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimer, lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[l-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3b-[N-(N’,N’-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleyl-oxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide (DDAB). Nucleic acids (e.g., ceDNA or CELiD) can also be complexed with, for example, poly(L-lysine) or avidin, and the lipid may or may not be included in this mixture, such as sterol-poly(L-lysine).

[0157] In one embodiment, the cationic lipid is ss-OP of Formula I. In another embodiment, the cationic lipid is SS-PAZ of Formula II.

[0158] In one embodiment, the ceDNA vectors disclosed herein are delivered using the cationic lipids described in U.S. Patent No. 8,158,601 or the lipids described in U.S. Patent No. 8,034,376.

[0159] In one embodiment, the lipid particles (e.g., lipid nanoparticles) can further include components such as sterols to provide membrane integrity and stability of the lipid particles. In one embodiment, exemplary sterols that can be used in the lipid particles are cholesterol or its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholesteryl hemisuccinate (CHEMS).

[0160] Exemplary cholesterol derivatives are described in PCT Publication No. 2009 / 127060 and U.S. Patent Publication No. 2010 / 0130588, the contents of both of which are incorporated herein by reference in their entirety.

[0161] In one embodiment, the component providing membrane integration such as sterol may comprise from 0 to 50% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, such a component is 20 to 50% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such a component is 30 to 40% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such a component is 35 to 45% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles). In some embodiments, such a component is 38 to 42% (mol) of the total lipid content of the lipid particles (e.g., lipid nanoparticles).

[0162] In one embodiment, the lipid particles (e.g., lipid nanoparticles) may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. Generally, these are used to inhibit aggregation of the lipid particles (e.g., lipid nanoparticles) and / or to provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxypolyethylene glycol)-conjugated lipid. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, PEG 2000 -DMG (dimyristoyl glycerol).

[0163] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinic acid diacylglycerol (PEGS-DAG) (4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropyl carbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5,885,613, US6,287,591, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904, the entire contents of all of which are hereby incorporated by reference in their entirety.

[0164] In one embodiment, the PEG-DAA conjugate can be, for example, PEG-dilauryl oxypropyl, PEG-dimyristyl oxypropyl, PEG-dipalmityl oxypropyl, or PEG-distearyl oxypropyl. The PEG-lipids can be PEG-DMG, PEG-dilauryl glycerol, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauryl glycamide, PEG-dimyristyl glycamide, PEG-dipalmitoyl glycamide, PEG-distearyl glycamide, PEG-cholesterol (1-[8’-(cholesta-5-en-3[beta]-oxy) carboxamido-3’,6’-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[omega]-methyl-poly(ethylene glycol) ether), and one or more of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG lipid can be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0165] In one embodiment, lipids conjugated to molecules other than PEG can also be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to PEG-lipids. Exemplary conjugated lipids, namely, PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids are described in PCT Patent Application Publication Nos. WO 96 / 10392, WO 98 / 51278, WO 02 / 87541, WO 05 / 26372, WO 08 / 147438, WO 09 / 086558, WO 12 / 000104, WO 17 / 117528, WO 17 / 099823, WO 15 / 199952, WO 17 / 004143, WO 15 / 095346, WO 12 / 000104, WO 12 / 000104, and WO 10 / 006282, U.S. Patent Application Publication Nos. 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0376224, 2016 / 0317458, 2013 / 0303587, 2013 / 0303587, and 2011 / 0123453, and U.S. Pat. Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, the contents of all of which are hereby incorporated by reference in their entirety.

[0166] In some embodiments, the PEG or conjugated lipid may constitute 0 to 20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the PEG or conjugated lipid content is 2 to 10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the PEG or conjugated lipid content is 2 to 5% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the PEG or conjugated lipid content is 2 to 3% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment, the PEG or conjugated lipid content is about 2.5% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments, the PEG or conjugated lipid content is about 3% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles).

[0167] It is understood that the molar ratios of the cationic lipid, for example, an ionizable cationic lipid, and the non-cationic lipid, sterol, and PEG / conjugated lipid can be varied as needed. For example, the lipid particles (e.g., lipid nanoparticles) can contain 30 - 70% cationic lipid by mole or total weight of the composition, 0 - 60% cholesterol by mole or total weight of the composition, 0 - 30% non-cationic lipid by mole or total weight of the composition, and 1 - 10% PEG or conjugated lipid by mole or total weight of the composition. In one embodiment, the composition, the lipid particles (e.g., lipid nanoparticles) contain 40 - 60% cationic lipid by mole or total weight of the composition, 30 - 50% cholesterol by mole or total weight of the composition, 5 - 15% non-cationic lipid by mole or total weight of the composition, and 1 - 5% PEG or conjugated lipid by mole or total weight of the composition. In one embodiment, the composition, the lipid particles (e.g., lipid nanoparticles) are 40 - 60% cationic lipid by mole or total weight of the composition, 30 - 40% cholesterol by mole or total weight of the composition, 5 - 10% non-cationic lipid by mole or total weight of the composition, and 1 - 5% PEG or conjugated lipid by mole or total weight of the composition. The composition can contain lipid particles (e.g., lipid nanoparticles) that are 60 - 70% cationic lipid by mole or total weight of the composition, 25 - 35% cholesterol by mole or total weight of the composition, 5 - 10% non-cationic lipid by mole or total weight of the composition, and 0 - 5% PEG or conjugated lipid by mole or total weight of the composition. The composition can also contain lipid particles (e.g., lipid nanoparticles) that are up to 45 - 55% cationic lipid by mole or total weight of the composition, 35 - 45% cholesterol by mole or total weight of the composition, 2 - 15% non-cationic lipid by mole or total weight of the composition, and 1 - 5% PEG or conjugated lipid by mole or total weight of the composition.The formulation may also be, for example, 8 - 30% cationic lipid by mole or total weight of the composition, 5 - 15% non - cationic lipid by mole or total weight of the composition, and 0 - 40% cholesterol by mole or total weight of the composition; 4 - 25% cationic lipid by mole or total weight of the composition, 4 - 25% non - cationic lipid by mole or total weight of the composition, 2 - 25% cholesterol by mole or total weight of the composition, 10 - 35% complex lipid by mole or total weight of the composition, and 5% cholesterol by mole or total weight of the composition; or 2 - 30% cationic lipid by mole or total weight of the composition, 2 - 30% non - cationic lipid by mole or total weight of the composition, 1 - 15% cholesterol by mole or total weight of the composition, 2 - 35% PEG or complex lipid by mole or total weight of the composition, and 1 - 20% cholesterol by mole or total weight of the composition; or further, up to 90% cationic lipid by mole or total weight of the composition, and 2 - 10% non - cationic lipid by mole or total weight of the composition; or further, 100% cationic lipid by mole or total weight of the composition, and may be lipid nanoparticles. In some embodiments, the lipid particle formulation comprises cationic lipid, non - cationic phospholipid, cholesterol, and PEGylated lipid (conjugated lipid) in a molar ratio of 50:10:38.5:1.5.

[0168] In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation comprises cationic lipid, non - cationic phospholipid, cholesterol, and PEGylated lipid (conjugated lipid) in a molar ratio of about 50:7:40:3.

[0169] In one embodiment, the lipid particles (e.g., lipid nanoparticles) include a cationic lipid, a non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid (conjugated lipid), and the molar ratios of the lipids are in the range of 20 to 70 mole percent for the cationic lipid (target 30 to 60), the mole percent of the non-cationic lipid is in the range of 0 to 30 (target 0 to 15), the mole percent of the sterol is in the range of 20 to 70 (target 30 to 50), and the mole percent of the PEGylated lipid (conjugated lipid) is in the range of 1 to 6 (target 2 to 5).

[0170] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Application No. 2018 / 050042, filed on September 7, 2018, which is hereby incorporated by reference in its entirety and is contemplated for use in the methods and compositions disclosed herein.

[0171] The pKa of the formulated cationic lipid can correlate with the efficacy of the LNP for nucleic acid delivery (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529 - 8533, Semple et al., Nature Biotechnology 28, 172 - 176 (2010), both of which are incorporated herein by reference in their entirety). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS). Lipid nanoparticles consisting of cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipid can be prepared using the inline process described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. Vesicles can be diluted to 24 mM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, and the pH ranges from 2.5 to 11. An aliquot of the TNS solution can be added to a final concentration of 1 mM, followed by measuring the fluorescence intensity by vortex mixing at room temperature in an SLM Aminco Series 2 luminescence spectrophotometer using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. Sigmoidal best-fit analysis can be applied to the fluorescence data, and the pKa is measured as the pH that yields half-maximal fluorescence intensity.

[0172] In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity is compared at doses of 0.3 and 1.0 mg ceDNA / kg and is expressed as ng of luciferase per gram of liver measured 4 hours after administration.

[0173] Without limitation, the lipid particles (e.g., lipid nanoparticles) of the present invention can include lipid formulations that can be used to deliver a capsid-free non-viral DNA vector to a target site of interest (e.g., cells, tissues, organs, etc.). Generally, the lipid particles (e.g., lipid nanoparticles) include a capsid-free non-viral DNA vector and a cationic lipid or a salt thereof.

[0174] In one embodiment, the lipid particles (e.g., lipid nanoparticles) include a cationic lipid / non-cationic lipid / sterol / conjugated lipid in a molar ratio of 50:10:38.5:1.5. In another embodiment, the lipid particles (e.g., lipid nanoparticles) include a cationic lipid / non-cationic lipid / sterol / conjugated lipid in a molar ratio of 50:10:37.5:2.5. In one embodiment, the present disclosure provides a lipid particle formulation including phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0175] III. Therapeutic Nucleic Acids Nucleic acids are large, highly charged, rapidly degraded and excreted from the body, and generally have low pharmacological properties. This is because they are recognized as foreign substances by the body and become targets of the innate immune response. Thus, certain therapeutic nucleic acids ("TNAs") (e.g., antisense oligonucleotides or viral vectors) often elicit an immune response in vivo. The present disclosure provides pharmaceutical compositions and methods that can improve, reduce or eliminate such immune responses and enhance the efficacy of therapeutic nucleic acids by increasing the expression level through maximizing the persistence of the therapeutic nucleic acid in a reduced immune response state of the target recipient. Thereby, potential adverse events that can lead to organ damage or other toxicities during the process of gene therapy can be minimized.

[0176] Exemplary therapeutic nucleic acids of the present disclosure include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), ribozymes, closed-ended double-stranded DNAs (e.g., ceDNA, CELiD, linear covalently closed DNA (“miniring”), doggybone (trademark), telomere closed-ended DNA, or dumbbell linear DNA), dicer substrates dsRNA, small hairpin RNAs (shRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), mRNAs, tRNAs, rRNAs, and DNA viral vectors, viral RNA vectors, and any combination thereof.

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

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

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

[0180] According to some embodiments, the therapeutic nucleic acid is a closed-ended double-stranded DNA, such as ceDNA. According to some embodiments, the expression and / or production of a therapeutic protein in a cell is derived from a non-viral DNA vector, such as a ceDNA vector. A distinct advantage of the ceDNA vector over conventional AAV vectors and even lentiviral vectors for the expression of therapeutic proteins is that there are no size constraints on the heterologous nucleic acid sequence encoding the desired protein. Thus, even large therapeutic proteins can be expressed from a single ceDNA vector. Thus, a ceDNA vector can be used to express a therapeutic protein in a subject in need thereof.

[0181] Generally, the ceDNA vectors for the expression of the therapeutic proteins disclosed herein comprise, 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 described herein), and a second AAV ITR. The ITR sequences are selected from any of (i) at least one wild-type (WT) ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., asymmetric modified ITRs) having different three-dimensional spatial configurations relative to each other, (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration.

[0182] IV. Closed-ended DNA (ceDNA) vectors Aspects of the present disclosure generally provide capsid-free non-viral closed-ended DNA vectors and lipid particles (e.g., lipid nanoparticles) comprising lipids.

[0183] Embodiments of the present disclosure are based on methods and compositions comprising closed-ended linear double-stranded (ceDNA) vectors capable of expressing a transgene (e.g., a therapeutic nucleic acid). CeDNA vectors as described herein do not have the packaging constraints imposed by the limited space within a viral capsid. CeDNA vectors represent a variable eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors, in contrast to the encapsulated AAV genome. This permits the insertion of regulatory elements, such as regulatory switches, large transgenes, multiple transgenes, etc., disclosed herein.

[0184] There are many structural features of the ceDNA vector that are different from plasmid-based expression vectors. The ceDNA vector has one or more of the following characteristics: deletion of the original (i.e., uninserted) bacterial DNA, deletion of the prokaryotic origin of replication, self-replicating (i.e., it does not require any sequences other than the two ITRs containing the Rep binding and terminal resolution sites (RBS and TRS) and the exogenous sequences between the ITRs), the presence of ITR sequences that form hairpins of eukaryotic origin (i.e., they are produced in eukaryotic cells), and the absence of bacterial-type DNA methylation or any other methylation that is actually considered abnormal by mammalian hosts. In general, it is preferred that the vector does not contain any prokaryotic cell DNA, although it is contemplated that some prokaryotic cell DNA may be inserted as an exogenous sequence, as a non-limiting example, into a promoter or enhancer region. Another important feature that distinguishes the ceDNA vector from the plasmid expression vector is that the ceDNA vector is single-stranded linear DNA with a closed end, while the plasmid is always double-stranded DNA.

[0185] There are several advantages to using the ceDNA vectors described herein over plasmid-based expression vectors, such advantages including, but not limited to, the following: 1) Plasmids contain bacterial DNA sequences and are subject to prokaryotic cell-specific methylation, e.g., 6-methyladenosine and 5-methylcytosine methylation, whereas the capsid-free AAV vector sequences are of eukaryotic cell origin and are not subject to prokaryotic cell-specific methylation. As a result, capsid-free AAV vectors are less likely to induce inflammation and immune responses compared to plasmids. 2) Plasmids require the presence of resistance genes during the production process, while ceDNA vectors do not. 3) Circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, whereas ceDNA vectors contain viral cis elements, i.e., ITRs, that confer resistance to nucleases and can be designed to be targeted and delivered to the nucleus. The minimum defined elements essential for ITR function are hypothesized to be the Rep binding site (RBS, 5’-GCGCGCTCGCTCGCTC-3’ (SEQ ID NO: 1) in the case of AAV2) and the terminal resolution site (TRS, 5’-AGTTGG-3’ in the case of AAV2), in addition to variable palindromic sequences that allow hairpin formation. 4) ceDNA vectors do not have the overpresentation of CpG dinucleotides, which are often found in prokaryotic cell-derived plasmids that bind to members of the Toll-like family of receptors and induce T cell-mediated immune responses, as reported. In contrast, transduction with the capsid-free AAV vectors disclosed herein can efficiently target cell and tissue types that are difficult to transduce with conventional AAV virions using various delivery reagents.

[0186] The ceDNA vector preferably has a linear and continuous structure rather than a discontinuous structure. The linear and continuous structure is considered to be more stable against attack by cellular endonucleases and at the same time less likely to be recombined and cause mutagenesis. Therefore, the ceDNA vector with a linear and continuous structure is a preferred embodiment. The intraduplex ceDNA vector of a continuous linear single-stranded molecule may be covalently bound at the ends without a sequence encoding an AAV capsid protein. These ceDNA vectors are structurally different from plasmids, which are circular double-stranded nucleic acid molecules of bacterial origin (including the ceDNA plasmids described herein). The complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules. In contrast, the ceDNA vector has complementary strands but is a single DNA molecule and thus remains a single molecule even when denatured. In some embodiments, the ceDNA vector, unlike a plasmid, can be produced without prokaryotic-type DNA base methylation. Therefore, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (particularly linear vs. circular) and the methods used to produce and purify these different objects, and also in terms of their DNA methylation, which is of the prokaryotic cell type in the case of ceDNA-plasmids and of the eukaryotic cell type in the case of ceDNA vectors.

[0187] Provided herein are non-viral capsid-free circular episomal DNA molecules (ceDNAs) having covalently closed ends. These non-viral capsid-free ceDNA molecules can be produced in a permissive host cell from an expression construct (e.g., a ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus or integrated cell line) containing a heterologous gene (e.g., a transgene, particularly a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, wherein the ITRs are different from each other. In some embodiments, one of the ITRs is modified by deletions, insertions, and / or substitutions compared to a wild-type ITR sequence (e.g., an AAV ITR), and at least one of the ITRs contains a functional terminal resolution site (trs) and a Rep binding site. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vector has a covalently closed end and is thus resistant to, for example, exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37° C. for 1 hour or more.

[0188] In one aspect, 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 described herein), and a second AAV ITR. In one embodiment, the first ITR (5' ITR) and the second ITR (3' ITR) are asymmetric with respect to each other. That is, they have different three-dimensional spatial configurations. As an exemplary embodiment, the first ITR can be a wild-type ITR, the second ITR can be a mutant or modified ITR, or vice versa, the first ITR can be a mutant or modified ITR, and the second ITR can be a wild-type ITR. In one embodiment, both the first ITR and the second ITR are modified, but have different sequences, or different modifications, or have different three-dimensional spatial configurations rather than being the same modified ITR. In other words, a ceDNA vector using asymmetric ITRs has ITRs where any change to one ITR relative to the WT-ITR is not reflected in the other ITR, or, if the asymmetric ITRs have modified asymmetric ITR pairs, can have different sequences and different three-dimensional shapes with respect to each other.

[0189] In one embodiment, 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 described herein), and a second AAV ITR, wherein the first ITR (5’ ITR) and the second ITR (3’ ITR) are symmetric or substantially symmetric with respect to each other, i.e., the ceDNA vector may comprise ITR sequences having a symmetric three-dimensional spatial configuration such that their structures are the same shape in geometric space or have the same A, C-C’, B-B’ loops in three-dimensional space. In such embodiments, the symmetric ITR pair, or substantially symmetric ITR pair, can be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair can have one or more modifications from the wild-type ITR and can have the same sequences that are reverse complementary (inverted) to each other. In one embodiment, the modified ITR pair is substantially symmetric as defined herein, i.e., the modified ITR pair can have different sequences but can have corresponding or the same symmetric three-dimensional shapes. In some embodiments, the symmetric ITR, or substantially symmetric ITR, can be a wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but they do not necessarily have to be WT-ITRs of the same AAV serotype. In one embodiment, one WT-ITR can be derived from one AAV serotype and the other WT-ITR can be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they can have one or more conservative nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration.

[0190] The ITR sequences provided herein, which are wild-type, mutant, or modified by other means, represent DNA sequences contained in expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmid, ceDNA-baculovirus) for the production of ceDNA vectors. Thus, the ITR sequences actually contained in ceDNA vectors produced from ceDNA-plasmids or other expression constructs may or may not be identical to the ITR sequences provided herein as a result of naturally occurring changes (e.g., replication errors) that occur during the production process.

[0191] In one embodiment, the ceDNA vectors described herein that contain an expression cassette having a transgene, which is a therapeutic nucleic acid sequence, can be operably linked to one or more regulatory sequences that enable or control the expression of the transgene. In one embodiment, the polynucleotide contains a first ITR sequence and a second ITR sequence, the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetric or symmetric with respect to each other.

[0192] In one embodiment, the expression cassette is located between two ITRs and contains, in this order, a promoter, a post-transcriptional regulatory element, and one or more of a polyadenylation and termination signal, which are operably linked to the transgene. In one embodiment, the promoter is regulatable - inducible or repressible. The promoter can be any sequence that promotes the transcription of the transgene. In one embodiment, the promoter is the CAG promoter or a variant thereof. The post-transcriptional regulatory element is a sequence that modulates the expression of the transgene and, as a non-limiting example, can be any sequence that creates a tertiary structure that enhances the expression of the transgene, which is a therapeutic nucleic acid sequence.

[0193] In one embodiment, the post-transcriptional regulatory element includes WPRE. In one embodiment, the polyadenylation and termination signal includes BGH polyA. Any cis-regulatory element known in the art, or combinations thereof, such as the SV40 late polyA signal upstream enhancer sequence (UES) or other post-transcriptional processing elements (including but not limited to the thymidine kinase genes of herpes simplex virus or hepatitis B virus (HBV)) can be used as an addition. In one embodiment, the 5' to 3' oriented expression cassette length exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0194] In one embodiment, the expression cassette can include more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range from about 4000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette can include a transgene that is a therapeutic nucleic acid sequence in the range of 500 to 50,000 nucleotides in length. In one embodiment, the expression cassette can include a transgene that is a therapeutic nucleic acid sequence in the range of 500 to 75,000 nucleotides in length. In one embodiment, the expression cassette can include a transgene that is a therapeutic nucleic acid sequence in the range of 500 to 10,000 nucleotides in length. In one embodiment, the expression cassette can include a transgene that is a therapeutic nucleic acid sequence in the range of 1000 to 10,000 nucleotides in length. In one embodiment, the expression cassette can include a transgene that is a therapeutic nucleic acid sequence in the range of 500 to 5,000 nucleotides in length. Since the ceDNA vector has no size limit for encapsidated AAV vectors, it is possible to deliver large-sized expression cassettes to the host. In one embodiment, the ceDNA vector lacks prokaryotic cell-specific methylation.

[0195] In one embodiment, the expression cassette may also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments, the ITR can act as a promoter for the transgene. In some embodiments, the ceDNA vector includes additional components for regulating the expression of the transgene, such as a regulatory switch for controlling and regulating the expression of the transgene, and optionally, a regulatory switch that is a kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0196] In one embodiment, the ceDNA vector is capsid-free and can be obtained from a plasmid encoding a first ITR, an expressible transgene cassette, and a second ITR in this order, and at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence.

[0197] In one embodiment, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary use) or for immunogenic polypeptides.

[0198] The expression cassette can include any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector includes any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0199] In one embodiment, the ceDNA expression cassette can include an expressible exogenous sequence (e.g., an open reading frame) that encodes a protein that is inactive, or has insufficient activity, in the recipient subject, or a gene that encodes a protein having a desired biological or therapeutic effect. In one embodiment, an exogenous sequence such as a donor sequence can encode a gene product that can function to correct the expression of a defective gene or transcript. In one embodiment, the expression cassette can also encode a corrective DNA strand and can encode a polypeptide, a sense or antisense oligonucleotide, or an RNA (coding or non-coding; e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). In one embodiment, the expression cassette can include an exogenous sequence that encodes a reporter protein used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0200] Accordingly, the expression cassette can include any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that provides a therapeutic effect when overexpression is considered within the scope of the present disclosure. The ceDNA vector can include a template or donor nucleotide sequence that is used as a corrective DNA strand inserted after a double-strand break (or nick) provided by a nuclease. The ceDNA vector can include a template nucleotide sequence that is used as a corrective DNA strand inserted after a double-strand break (or nick) provided by an inducible RNA nuclease, a meganuclease, or a zinc finger nuclease.

[0201] Preferably, there is no uninserted bacterial DNA, and preferably, there is no bacterial DNA in the ceDNA composition provided herein. In some examples, the protein can change codons without nicks.

[0202] In one embodiment, the sequences provided by the expression cassette, expression construct, or donor sequence of the ceDNA vector described herein can be codon-optimized for the host cell. As used herein, the terms "optimized codon" or "codon optimization" refer to the process of modifying a nucleic acid sequence by replacing codons of at least one, two or more, or a significant number of non-native sequences (e.g., prokaryotic cell sequences) with codons that are more frequently or most frequently used in the genes of that vertebrate for enhanced expression in cells of the vertebrate of interest, e.g., mouse or human cells. Different species exhibit specific biases towards certain codons for a particular amino acid.

[0203] Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another public database.

[0204] Many organisms exhibit a bias in using specific codons to encode the insertion of specific amino acids in the growing peptide chain. Codon preference or codon bias, which is the difference in codon usage frequencies among organisms, is brought about by the degeneracy of the genetic code and is well-documented among many organisms. Codon bias often correlates with the translational efficiency of messenger RNA (mRNA) and is thought to depend particularly on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be adjusted for optimal gene expression in a given organism.

[0205] Given the large number of gene sequences available in a wide variety of animal, plant, and microbial species, it is possible to calculate the relative frequencies of codon usage (Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000)).

[0206] There are many structural features of the ceDNA vector that are different from plasmid-based expression vectors. The ceDNA vector can have one or more of the following features: deletion of the original (i.e., uninserted) bacterial DNA, deletion of the prokaryotic origin of replication, being self-sufficient (i.e., it does not require any sequences other than the two ITRs including the Rep binding and terminal resolution sites (RBS and TRS) and the exogenous sequences between the ITRs), the presence of ITR sequences that form hairpins of eukaryotic origin (i.e., they are produced in eukaryotic cells), and the absence of bacterial-type DNA methylation or any other methylation that is actually considered abnormal by the mammalian host. Generally, it is preferred that the vector does not contain any prokaryotic cell DNA, although it is contemplated that some prokaryotic cell DNA may be inserted as an exogenous sequence, as a non-limiting example, into a promoter or enhancer region. Another important feature that distinguishes the ceDNA vector from plasmid expression vectors is that the ceDNA vector is single-stranded linear DNA with a closed end, while plasmids are always double-stranded DNA.

[0207] In one embodiment, the ceDNA vector produced by the method provided herein preferably has a linear and continuous structure rather than a discontinuous structure. The linear and continuous structure is considered to be more stable against attack by cellular endonucleases and at the same time less likely to be recombined to cause mutagenesis. Thus, a ceDNA vector having a linear and continuous structure is a preferred embodiment. The continuous linear single-stranded intramolecular double-stranded ceDNA vector may be covalently bound to the ends without a sequence encoding an AAV capsid protein. These ceDNA vectors are structurally different from plasmids, which are circular double-stranded nucleic acid molecules of bacterial origin (including the ceDNA plasmids described herein). The complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules. In contrast, a ceDNA vector has complementary strands but is a single DNA molecule and thus remains a single molecule even when denatured. In some embodiments, the ceDNA vectors described herein, unlike plasmids, can be produced without prokaryotic-type DNA base methylation. Thus, ceDNA vectors and ceDNA-plasmids differ with respect to both their structure (notably linear versus circular) and the methods used to produce and purify these different objects, and also with respect to their DNA methylation, which is of the prokaryotic cell type for ceDNA-plasmids and of the eukaryotic cell type for ceDNA vectors.

[0208] Example 1. According to some embodiments, synthetic ceDNA is produced via excision from double-stranded DNA molecules. The synthetic production of ceDNA vectors is described in Examples 2-6 of International Application No. 19 / 14122, filed January 18, 2019, which is hereby incorporated by reference in its entirety. One exemplary method of producing ceDNA vectors using a synthetic method involving excision of double-stranded DNA molecules. Briefly, ceDNA vectors can be generated using double-stranded DNA constructs. See, for example, FIGS. 7A-8E of PCT / US19 / 14122. In some embodiments, the double-stranded DNA construct is a ceDNA plasmid; see, for example, FIG. 6 of International Patent Application No. 2018 / 064242, filed December 6, 2018).

[0209] In some embodiments, the construct for making a ceDNA vector (e.g., a synthetic AAV vector) includes additional components for regulating the expression of the transgene, such as a regulatory switch for regulating the expression of the transgene, or a kill switch that can kill the cells containing the vector.

[0210] A molecular regulatory switch is one that generates a measurable change in state in response to a signal. Such regulatory switches can usefully be combined with the ceDNA vectors described herein to control the output of transgene expression. In some embodiments, the ceDNA vector includes a regulatory switch that helps to finely tune the expression of the transgene. For example, it can serve as a biological containment function of the ceDNA vector. In some embodiments, the switch is an “on / off” switch designed to initiate or stop (i.e., shut down) the controllable and regulatable expression of the gene of interest in the ceDNA vector. In some embodiments, the switch can include a “kill switch” that, once activated, can instruct the cells containing the synthetic ceDNA vector to undergo programmed cell death. Exemplary regulatory switches encompassed for use with the ceDNA vector can be used to regulate the expression of a transgene and are more fully discussed in International Application No. 18 / 49996, which is hereby incorporated by reference in its entirety and described herein.

[0211] Another exemplary method of producing a ceDNA vector using a synthetic method involving the assembly of various oligonucleotides is provided in Example 3 of PCT / US19 / 14122, where the ceDNA vector is produced by synthesizing 5′ oligonucleotides and 3′ ITR oligonucleotides and ligating the ITR oligonucleotides to a double-stranded polynucleotide containing an expression cassette. FIG. 11B of PCT / US19 / 14122, which is hereby incorporated by reference in its entirety, shows an exemplary method of ligating 5′ ITR oligonucleotides and 3′ ITR oligonucleotides to a double-stranded polynucleotide containing an expression cassette.

[0212] Exemplary methods of producing a ceDNA vector using synthetic methods are provided in Example 4 of PCT / US19 / 14122, which is hereby incorporated by reference in its entirety, and use single-stranded linear DNA comprising two sense ITRs covalently linked to two antisense ITRs adjacent to the sense expression cassette sequence and adjacent to the antisense expression cassette, and then ligating the ends of this single-stranded linear DNA to form a closed-ended single-stranded molecule. A non-limiting example is to synthesize and / or produce a single-stranded DNA molecule, anneal a portion of the molecule to form a single linear DNA molecule having one or more base pairing regions of a secondary structure, and then ligate the free 5' and 3' ends to each other to form a closed circular single-stranded molecule.

[0213] In yet another aspect, the invention provides host cell lines that stably incorporate into their own genomes the DNA vector polynucleotide expression templates (ceDNA templates) described herein for use in the production of non-viral DNA vectors. Methods of producing such cell lines are described in Lee, L. et al. (2013) Plos One 8(8):e69879, which is hereby incorporated by reference in its entirety. Preferably, the Rep protein (e.g., as described in Example 1) is added to the host cells at an MOI of 3. In one embodiment, the host cell line is an invertebrate cell line, preferably an insect Sf9 cell. Where the host cell line is a mammalian cell line, preferably a 293 cell, the cell line may have a stably incorporated polynucleotide vector template, and a second vector such as a herpes virus can be used to introduce the Rep protein into the cells, enabling excision and amplification of the ceDNA in the presence of Rep.

[0214] Any promoter can be operably linked to a heterologous nucleic acid (e.g., a reporter nucleic acid or a therapeutic transgene) of the vector polynucleotide. The expression cassette can contain a synthetic regulatory element such as the CAG promoter. The CAG promoter contains (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and the first intron of the chicken beta-actin gene, and (ii) a splice acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette can contain an alpha-1-antitrypsin (AAT) promoter, a liver-specific (LP1) promoter, or a human elongation factor-1 alpha (EF1-α) promoter. In some embodiments, the expression cassette contains one or more constitutive promoters, e.g., a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), a cytomegalovirus (CMV) immediate early promoter (optionally having a CMV enhancer). Alternatively, inducible or repressible promoters, native promoters of the transgene, tissue-specific promoters, or various promoters known in the art can be used. Suitable transgenes for gene therapy are well known to those skilled in the art.

[0215] The capsid-free ceDNA vector can also be produced from a vector polynucleotide expression construct that further includes a cis-regulatory element, or a combination of cis-regulatory elements. Non-limiting examples include the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and BGH polyA, or, for example, beta-globin polyA. Other post-transcriptional processing elements include, for example, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV). The expression cassette can include any polyadenylation sequence known in the art, such as natural or synthetic, isolated from bovine BGHpA or viral SV40pA, or a modification thereof. Some expression cassettes can also include the SV40 late polyA signal upstream enhancer (USE) sequence. The USE can be used in combination with SV40pA or a heterologous polyA signal.

[0216] The time for harvesting and collecting the DNA vectors described herein from cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvesting time can be selected considering cell viability, cell morphology, cell proliferation, etc. In one embodiment, the cells are grown under sufficient conditions and harvested after sufficient time has elapsed from baculovirus infection to produce the DNA vector, but before the majority of the cells begin to die due to virus toxicity. The DNA-vector can be isolated using a plasmid purification kit such as the Qiagen Endo-Free plasmid kit. Other methods developed for plasmid isolation can also be adapted for DNA vectors. Generally, any nucleic acid purification method can be employed.

[0217] The DNA vector can be purified by any means known to those skilled in the art for DNA purification. In one embodiment, the ceDNA vector is purified as a DNA molecule. In another embodiment, the ceDNA vector is purified as exosomes or microparticles.

[0218] In one embodiment, the capsid-free non-viral DNA vector comprises or is obtained from a plasmid containing a polynucleotide template that includes a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette for exogenous DNA), and a modified AAV ITR in this order, and the template nucleic acid molecule lacks the AAV capsid protein coding. In a further embodiment, the nucleic acid template of the present invention lacks a viral capsid protein coding sequence (i.e., lacks not only the AAV capsid gene but also the capsid genes of other viruses). Additionally, in certain embodiments, the template nucleic acid molecule also lacks the AAV Rep protein coding sequence. Thus, in a preferred embodiment, the nucleic acid molecule of the present invention lacks both a functional AAV cap and the AAV rep gene.

[0219] In one embodiment, the ceDNA vector can include an ITR structure mutated with respect to the wild-type AAV2 ITR disclosed herein, but still retains operable RBE, TRS, and RBE' moieties.

[0220] Inverted terminal repeat (ITR) As described herein, in one embodiment, the ceDNA vector is a capsid-free linear double-stranded DNA molecule formed from continuous strands of complementary DNA having covalently linked ends (a linear and continuous non-capsid structure), and includes 5' inverted terminal repeat (ITR) sequences and 3' ITR sequences that are different or asymmetric with respect to each other. At least one of the ITRs includes a functional terminal resolution site and a replication protein binding site (RPS) (also sometimes referred to as a replication protein binding site), e.g., a Rep binding site. Generally, the ceDNA vector contains at least one modified AAV inverted terminal repeat sequence (ITR), i.e., deletions, insertions, and / or substitutions relative to other ITRs, as well as an expressible transgene.

[0221] In one embodiment, at least one of the ITRs is an AAV ITR, for example, a wild-type AAV ITR. In one embodiment, at least one of the ITRs is a modified ITR relative to the other ITRs, i.e., the ceDNA contains ITRs that are asymmetric with respect to each other. In one embodiment, at least one of the ITRs is a non-functional ITR.

[0222] In one embodiment, the ceDNA vector comprises: (1) an expression cassette comprising a cis-regulatory element, a promoter, and at least one transgene; (2) a promoter operably linked to the at least one transgene; and (3) two self-complementary sequences flanking the expression cassette, for example, ITRs, and the ceDNA vector is not associated with a capsid protein. In some embodiments, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, at least one of which comprises an operative Rep-binding element (RBE) and the terminal resolution site (trs) of AAV or a functional variant of the RBE, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments, the ceDNA vector comprises additional components for regulating the expression of the transgene, for example, a regulatory switch for controlling and regulating the expression of the transgene, and may comprise a regulatory switch that is a kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0223] In one embodiment, the two self-complementary sequences can be ITR sequences from any known parvovirus, such as an adeno-associated virus (e.g., AAV1 - AAV12), a dependovirus. In addition to the variable palindromic sequences that allow for hairpin secondary structure formation, any AAV serotype can be used, including but not limited to a modified AAV2 ITR sequence that retains a Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 1) and a terminal resolution site (trs). In some embodiments, the ITR can be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITR does not contain an AAV-based sequence. In yet another embodiment, the synthetic ITR preserves the above ITR structure but has few or no AAV source sequences. In some aspects, the synthetic ITR can preferentially interact with wild-type Rep or Rep of a specific serotype, or in some cases, is not recognized by wild-type Rep and is only recognized by a mutant Rep. In some embodiments, the ITR is a synthetic ITR sequence that retains a functional Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 1) and a terminal resolution site (TRS) in addition to the variable palindromic sequences that allow for hairpin secondary structure formation. In some examples, the modified ITR sequence retains the structure and position of the Rep binding element that forms the terminal loop portion of one of the ITR hairpin secondary structures from the sequences of the RBS, trs, and the corresponding sequence of the wild-type AAV2 ITR. Exemplary ITR sequences for use in the ceDNA vector are disclosed in Tables 2 - 9, 10A and 10B, SEQ ID NOs: 2, 52, 101 - 449 and 545 - 547, and partial ITR sequences are shown in FIGS. 26A - 26B of PCT Application No. 18 / 49996 filed on September 7, 2018, the contents of each of which are hereby incorporated by reference in their entirety.In some embodiments, the ceDNA vector can include an ITR with a modification in the ITR corresponding to any one or more of the modifications in the ITR sequences or ITR subsequences shown in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A and 10B, PCT Application No. 18 / 49996 filed on September 7, 2018.

[0224] In one embodiment, the ceDNA vector can be produced from an expression construct that further includes a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir-regulatory elements, post-transcriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments, the ITR can act as a promoter for the transgene. In some embodiments, the ceDNA vector includes additional components for regulating the expression of the transgene, such as the regulatory switch described in PCT Application No. 18 / 49996 filed on September 7, 2018, and regulates the expression of a kill switch that can kill the transgene or cells containing the ceDNA vector.

[0225] In one embodiment, the expression cassette can also include post-transcriptional elements to increase the expression of the transgene. In one embodiment, the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase the expression of the transgene. Other post-transcriptional processing elements, such as those from the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV), can be used. The secretion sequence can be ligated to the transgene, such as the VH-02 and VK-A26 sequences. The expression cassette can include a polyadenylation sequence or a variant thereof known in the art, such as a natural sequence isolated from bovine BGHpA or viral SV40pA, or a synthetic sequence. Some expression cassettes can also include the SV40 late polyA signal upstream enhancer (USE) sequence. The USE can be used in combination with SV40pA or a heterologous polyA signal.

[0226] Figures 1A - 1C of International Application No. 2018 / 050042, filed on September 7, 2018, and incorporated herein by reference in its entirety, show schematic diagrams of non - limiting exemplary ceDNA vectors, or corresponding sequences of ceDNA plasmids. The ceDNA vector is capsid - free and can be obtained from a plasmid encoding a first ITR, an expressible transgene cassette, and a second ITR in this order, wherein at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild - type AAV2 ITR sequence. The expressible transgene cassette preferably contains one or more of an enhancer / promoter, an ORF reporter (transgene), a post - transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA) in this order.

[0227] Promoter: Suitable promoters, including those described above, can be of viral origin and thus can be referred to as viral promoters, or they can be from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6, e.g., (Miyagishi el al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), the human H1 promoter (H1), the CAG promoter, the human α1-antitrypsin (HAAT) promoter (e.g., etc.), but are not limited to these. In one embodiment, these promoters are modified at their downstream intron-containing ends to include one or more nuclease cleavage sites. In one embodiment, the DNA containing the nuclease cleavage site is foreign to the promoter DNA.

[0228] In one embodiment, the promoter can further enhance expression and / or alter its spatial and / or temporal expression by including one or more specific transcriptional regulatory sequences. The promoter can also include distal enhancer or repressor elements that can be located thousands of base pairs away from the transcription start site. The promoter can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter can regulate the expression of gene components constitutively or differentially in response to the cell, tissue, or organ in which expression occurs, or the developmental stage at which expression occurs, or external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter and the CMV IE promoter, and the promoters listed below. Such promoters and / or enhancers can be used for the expression of any gene of interest, such as a therapeutic protein. For example, the vector can include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein. In one embodiment, the promoter operably linked to the therapeutic protein coding sequence can be a promoter derived from simian virus 40 (SV40), the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) promoter, such as the long terminal repeat (LTR) promoter of the bovine immunodeficiency virus (BIV), the Moloney virus promoter, the avian leukosis virus (ALV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter, the Epstein-Barr virus (EBV) promoter, or the Rous sarcoma virus (RSV) promoter.In one embodiment, the promoter may also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a natural or synthetic, tissue-specific promoter, for example, a liver-specific promoter such as human alpha1-antitrypsin (HAAT). In one embodiment, delivery to the liver can be achieved using the endogenous ApoE-specific targeting of the composition containing the ceDNA vector to hepatocytes via the low density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0229] In one embodiment, the promoter used is the native promoter of the gene encoding the therapeutic protein. The promoter and other regulatory sequences of each gene encoding a therapeutic protein are known and characterized. The promoter region used may further comprise one or more additional regulatory sequences (e.g., native), such as an enhancer.

[0230] Non-limiting examples of suitable promoters for use according to the present invention include, for example, the CAG promoter, the HAAT promoter, the human EF1-α promoter, or fragments of the EF1-α promoter and the rat EF1-α promoter.

[0231] Polyadenylation sequence: The sequence encoding the polyadenylation sequence can be included in the ceDNA vector to stabilize the mRNA expressed from the ceDNA vector and to assist in nuclear transport and translation. In one embodiment, the ceDNA vector does not contain a polyadenylation sequence. In other embodiments, the vector contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenine nucleotides. In some embodiments, the polyadenylation sequence includes about 43 nucleotides, about 40 - 50 nucleotides, about 40 - 55 nucleotides, about 45 - 50 nucleotides, about 35 - 50 nucleotides, or any range therebetween.

[0232] In one embodiment, the ceDNA is obtainable from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two different inverted terminal repeats (ITRs) (e.g., AAV ITRs), wherein at least one of the ITRs includes a terminal resolution site and a replication protein binding site (RPS), e.g., a Rep binding site (e.g., wt AAV ITR), and one of the ITRs includes a deletion, insertion, or substitution with respect to the other ITR, e.g., a functional ITR.

[0233] In one embodiment, the host cell does not express a viral capsid protein and the polynucleotide vector template lacks any viral capsid coding sequence. In one embodiment, the polynucleotide vector template lacks the AAV capsid gene and also lacks the capsid genes of other viruses. In one embodiment, the nucleic acid molecule also lacks the AAV Rep protein coding sequence. Thus, in some embodiments, the nucleic acid molecule of the present invention lacks both a functional AAV cap and AAV rep genes.

[0234] In one embodiment, the ceDNA vector does not have a modified ITR.

[0235] In one embodiment, the ceDNA vector includes a regulatory switch as disclosed herein (or in PCT Application No. PCT / US18 / 49996 filed Sep. 7, 2018).

[0236] V. Production of ceDNA Vectors Methods for the production of the ceDNA vectors described herein, including asymmetric ITR pairs or symmetric ITR pairs as defined herein, are described in Section IV of PCT / US18 / 49996 filed Sep. 7, 2018, which is hereby incorporated by reference in its entirety. As described herein, ceDNA vectors can be obtained, for example, by a process comprising: a) incubating a population of host cells (e.g., insect cells) containing a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus) in the presence of a Rep protein under conditions effective to induce production of the ceDNA vector in the host cell and for a time sufficient therefor, and lacking a viral capsid coding sequence, wherein the host cells are incubated without 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 to produce the ceDNA vector in the host cell.

[0237] However, viral particles (e.g., AAV virions) are not expressed. Thus, there are no size limitations such as those imposed naturally in AAV or other virus-based vectors.

[0238] The presence of the ceDNA vector isolated from the host cells can be confirmed by digesting the DNA isolated from the host cells 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 linear and continuous DNA bands as compared to linear and discontinuous DNA.

[0239] In one embodiment, the present invention provides for the use of host cell lines that stably integrate a DNA vector polynucleotide expression template (ceDNA template) into their own genomes in the production of non-viral DNA vectors, as described, for example, in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cells at an MOI of about 3. When the host cell line is a mammalian cell line, e.g., HEK293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector such as a herpes virus can be used to introduce the Rep protein into the cells, enabling excision and amplification of the ceDNA in the presence of Rep and the helper virus.

[0240] In one embodiment, the host cells used to generate the ceDNA vectors described herein are insect cells, and baculovirus is used to deliver both a polynucleotide encoding the Rep protein and a non-viral DNA vector polynucleotide expression construct template of the ceDNA. In some embodiments, the host cells are engineered to express the Rep protein.

[0241] The ceDNA vector is then harvested and isolated from the host cells. The time for harvesting the ceDNA vector described herein from the cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvest time can be selected considering cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are grown under sufficient conditions and harvested after sufficient time has elapsed from baculovirus infection to produce the ceDNA vector, but before the majority of the cells begin to die due to the toxicity of the baculovirus. The DNA-vector can be isolated using a plasmid purification kit such as the Qiagen Endo-Free plasmid kit. Other methods developed for plasmid isolation can also be adapted for the DNA-vector. Generally, any nucleic acid purification method can be employed.

[0242] The DNA vector can be purified by any means known to those skilled in the art for DNA purification. In one embodiment, the ceDNA vector is purified as a DNA molecule. In one embodiment, the ceDNA vector is purified as exosomes or microparticles. The presence of the ceDNA vector can be confirmed by digesting the vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector and using gel electrophoresis to analyze both the digested DNA material and the undigested DNA material to confirm the presence of characteristic linear and continuous DNA as compared to linear and discontinuous DNA.

[0243] ceDNA plasmid The ceDNA-plasmid is a plasmid used for the late production of the ceDNA vector. In one embodiment, the ceDNA-plasmid can be constructed using known techniques that provide at least (1) a modified 5’ ITR sequence, (2) an expression cassette containing cis-regulatory elements, such as a promoter, inducible promoter, regulatory switch, enhancer, etc., and (3) a modified 3’ ITR sequence (the 3’ ITR sequence is asymmetric with respect to the 5’ ITR sequence) as operably linked components in the transcription direction. In some embodiments, the expression cassette flanked by the ITRs contains a cloning site for introducing exogenous sequences. The expression cassette replaces the rep and cap coding regions of the AAV genome.

[0244] In one embodiment, the ceDNA vector is herein referred to as a "ceDNA-plasmid" that encodes a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette containing a transgene, and a mutant or modified AAV ITR in this order, and the ceDNA-plasmid lacks the AAV capsid protein coding sequence. In an alternative embodiment, the ceDNA-plasmid encodes a first (or 5') modified or mutant AAV ITR, an expression cassette containing a transgene, and a second (or 3') modified AAV ITR in this order, the ceDNA-plasmid lacks the AAV capsid protein coding sequence, and the 5' and 3' ITRs are symmetric with respect to each other. In an alternative embodiment, the ceDNA-plasmid encodes a first (or 5') modified or mutant AAV ITR, an expression cassette containing a transgene, and a second (or 3') mutant or modified AAV ITR in this order, the ceDNA-plasmid lacks the AAV capsid protein coding sequence, and the 5' and 3' modified ITRs have the same modification (i.e., they are reverse complementary or symmetric with respect to each other).

[0245] In one embodiment, the ceDNA-plasmid system lacks the viral capsid protein coding sequence (i.e., lacks not only the AAV capsid gene but also the capsid genes of other viruses). Additionally, in certain embodiments, the ceDNA-plasmid also lacks the AAV Rep protein coding sequence. Thus, in a preferred embodiment, the ceDNA-plasmid lacks the variable palindromic sequence that enables hairpin formation in addition to the functional AAV cap and AAV rep genes (GG-3' in the case of AAV2). In one embodiment, the ceDNA-plasmid of the present disclosure can be generated using the native nucleotide sequence of the genome of any AAV serotype well-known in the art. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV 10, AAV 11, AAV 12, AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8 genomes, for example, NCBI: NC 002077, NC 001401, NC001729, NC001829, NC006152, NC 006260, NC 006261, Kotin and Smith, The Springer Index of Viruses, available at the URL managed by Springer. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV2 genome. In one embodiment, the ceDNA-plasmid backbone is a synthetic backbone genetically engineered to be included in the 5' and 3' ITRs derived from one of these AAV genomes.

[0246] In one embodiment, the ceDNA-plasmid may optionally include a selectable or selection marker for use in the establishment of a ceDNA vector-producing cell line. In one embodiment, the selection marker may be inserted downstream (i.e., 3') of the 3' ITR sequence. In another embodiment, the selection marker may be inserted upstream (i.e., 5') of the 5' ITR sequence. Suitable selection markers include, for example, those that confer drug resistance. The selection marker can be, for example, a blasticidin S resistance gene, kanamycin, geneticin, etc. In a preferred embodiment, the drug selection marker is a blasticidin S resistance gene.

[0247] In one embodiment, an exemplary ceDNA (e.g., rAAVO) is produced from an rAAV plasmid. A method for the production of an rAAV vector comprises: (a) providing an rAAV plasmid as described above to a host cell, wherein both the host cell and the plasmid lack a capsid protein coding gene; (b) culturing the host cell under conditions that allow for the production of the ceDNA genome; and (c) harvesting the cells and isolating the AAV genome produced from the cells.

[0248] Exemplary method for generating a ceDNA vector from a ceDNA plasmid In one embodiment, methods for generating a capsid-free ceDNA vector, particularly methods having a high enough yield to provide sufficient vectors for in vivo experiments, are also provided herein.

[0249] In one embodiment, the method for producing a ceDNA vector includes: (1) introducing a nucleic acid construct containing an expression cassette and two symmetric ITR sequences into a host cell (e.g., Sf9 cells); (2) optionally, establishing a clonal cell line by using a selectable marker, for example, present on a plasmid; (3) introducing a Rep coding gene into the insect cells (either by transfection or infection with a baculovirus carrying the gene); and (4) harvesting the cells and purifying the ceDNA vector. The nucleic acid construct containing the above-mentioned expression cassette and two ITR sequences for the production of the ceDNA vector can be in the form of a ceDNA-plasmid, or a bacmid or baculovirus generated from the ceDNA plasmid as described below. The nucleic acid construct can be introduced into the host cell by transfection, viral transduction, stable integration, or other methods known in the art.

[0250] Cell line In one embodiment, the host cell line used in the production of the ceDNA vector can include insect cell lines derived from Spodoptera frugiperda (Sf9, Sf21, etc.) or Trichoplusia ni cells, or other eukaryotic cell lines including other invertebrate, vertebrate, or mammalian cells. Other cell lines known to those skilled in the art, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1180, monocytes, and mature and immature dendritic cells, can also be used. The host cell line can be transfected for stable expression of the ceDNA-plasmid for high-yield production of the ceDNA vector.

[0251] In one embodiment, the ceDNA-plasmid can be introduced into Sf9 cells by transient transfection using reagents known in the art (e.g., liposomes, calcium phosphate) or physical means (e.g., electroporation). Alternatively, stable Sf9 cell lines that stably integrate the ceDNA-plasmid into their genomes can be established. Such stable cell lines can be established by incorporating a selectable marker into the above ceDNA-plasmid. When the ceDNA-plasmid used to transfect the cell line contains a selectable marker such as an antibiotic, cells transfected with the ceDNA-plasmid and incorporating the ceDNA-plasmid DNA into their genomes can be selected by the addition of the antibiotic to the cell growth medium. Next, resistant clones of the cells can be isolated and propagated by single cell dilution or colony transfer techniques.

[0252] Isolation and purification of ceDNA vectors Examples of processes for obtaining and isolating ceDNA vectors (e.g., gene editing) are described in FIGS. 4A-4E of International Application No. 2018 / 064242, filed Dec. 6, 2018, the content of which is hereby incorporated by reference in its entirety. In one embodiment, the ceDNA-vector is obtained from producer cells expressing the AAV Rep protein and can be further transformed with a ceDNA-plasmid, a ceDNA-bacmid, or a ceDNA-baculovirus. Plasmids useful for the production of ceDNA vectors include the plasmids shown in FIGS. 6A (useful for Rep BIIC production) and 6B (plasmids used to obtain ceDNA vectors) of International Application No. 2018 / 064242.

[0253] In one embodiment, the polynucleotide encodes an AAV Rep protein (Rep78 or 68) delivered to producer cells in a plasmid (Rep-plasmid), a bacmid (Rep-bacmid), or a baculovirus (Rep-baculovirus). Rep-plasmids, Rep-bacmids, and Rep-baculoviruses can be generated by the methods described above.

[0254] Methods for producing a ceDNA vector, which is an exemplary ceDNA vector, are described herein. Expression constructs used to generate the ceDNA vectors of the present invention can be plasmids (e.g., ceDNA-plasmids), bacmids (e.g., ceDNA-bacmids), and / or baculoviruses (e.g., ceDNA-baculoviruses). By way of example only, ceDNA vectors can be generated from cells co-infected with ceDNA-baculovirus and Rep-baculovirus. Rep proteins produced from Rep-baculovirus replicate the ceDNA-baculovirus to generate ceDNA vectors. Alternatively, ceDNA vectors can be generated from cells stably transfected with a construct containing a sequence encoding an AAV Rep protein (Rep78 / 52) delivered in a Rep-plasmid, Rep-bacmid, or Rep-baculovirus. ceDNA-baculovirus can be transiently transfected into cells and replicated by Rep proteins to produce ceDNA vectors.

[0255] Bacmids (e.g., ceDNA-bacmids) can be transfected into permissive insect cells such as Sf9, Sf21, Tni (Trichoplusia ni) cells, High Five cells, etc., to generate a ceDNA-baculovirus, which is a recombinant baculovirus containing sequences including symmetric ITRs and an expression cassette. The ceDNA-baculovirus can be reinfected into insect cells to obtain a next-generation recombinant baculovirus. Optionally, this step can be repeated once or multiple times to produce a greater amount of recombinant baculovirus.

[0256] The time for harvesting and collecting the ceDNA vector described in this specification from cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvesting time can be selected considering cell viability, cell morphology, cell proliferation, etc. Generally, cells can be harvested after sufficient time has elapsed since baculovirus infection for producing the ceDNA vector (e.g., the ceDNA vector), but before the majority of the cells begin to die due to the toxicity of the virus. The ceDNA vector can be isolated from Sf9 cells using a plasmid purification kit such as the Qiagen ENDO-FREE PLASMID® kit. Other methods developed for plasmid isolation can also be adapted for the ceDNA vector. Generally, any nucleic acid purification method known in the art, as well as commercially available DNA extraction kits, can be employed.

[0257] Alternatively, purification can be implemented by subjecting the cell pellet to an alkaline lysis process, centrifuging the resulting lysate, and performing chromatographic separation. As one non-limiting example, this process can be carried out by loading the supernatant onto an ion exchange column that retains nucleic acids (e.g., SARTOBIND Q®), then eluting (e.g., with a 1.2 M NaCl solution), and performing further chromatographic purification on a gel filtration column (e.g., 6 High Load GE). The capsid-free AAV vector is then recovered, for example, by precipitation.

[0258] In one embodiment, the ceDNA vector can also be purified in the form of exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins but also complex protein / nucleic acid cargos via the shedding of membrane microvesicles (Cocucci et al, 2009, EP10306226.1). Such vesicles include microvesicles (also referred to as microparticles) and exosomes (also referred to as nanovesicles), both of which contain proteins and RNA as cargos. Microvesicles are generated from the direct budding of the plasma membrane, and exosomes are released into the extracellular environment upon fusion of multivesicular endosomes with the plasma membrane. Thus, microvesicles and / or exosomes containing the ceDNA vector can be isolated from ceDNA plasmids, or cells transfected with bacmids or baculoviruses generated from ceDNA plasmids.

[0259] In one embodiment, microvesicles can be isolated by filtering or ultracentrifuging the culture medium at 20,000×g and subjecting it to exosomes at 100,000×g. The optimal period of ultracentrifugation can be determined experimentally and will depend on the specific cell type from which the vesicles are isolated. Preferably, the culture medium is first cleared by low-speed centrifugation (e.g., 2000×g for 5-20 minutes) and then subjected to spin concentration, for example, using an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified via FACS or MACS by using specific antibodies that recognize specific surface antigens present on the microvesicles and exosomes. Other microvesicle and exosome purification methods include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. During purification, the vesicles are washed, for example, with phosphate-buffered saline. One advantage of using microvesicles or exosomes to deliver ceDNA-containing vesicles is that these vesicles can be targeted to various cell types by including them on their membrane proteins that are recognized by specific receptors on each cell type. (See also EP10306226, which is hereby incorporated by reference in its entirety).

[0260] Another aspect of the invention relates to a method for purifying a ceDNA vector from a host cell line that stably integrates the ceDNA construct into its own genome. In one embodiment, the ceDNA vector is purified as a DNA molecule. In another embodiment, the ceDNA vector is purified as exosomes or microparticles.

[0261] Figure 5 of PCT / US18 / 49996 shows a gel confirming the production of ceDNA from multiple ceDNA plasmid constructs using the method described in the examples.

[0262] VI. Preparation of Lipid Particles Lipid particles (e.g., lipid nanoparticles) can be spontaneously formed upon mixing of ceDNA and lipids. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane (e.g., 100 nrn cut-off) using a thermobarrel extruder such as a Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. In one embodiment, the lipid nanoparticles are formed as described in Example 6 herein.

[0263] Generally, lipid particles (e.g., lipid nanoparticles) can be formed by any method known in the art. For example, lipid particles (e.g., lipid nanoparticles) can be prepared by the methods described in, for example, US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129 and US2010 / 0130588, the contents of each of which are hereby incorporated by reference in their entirety. In some embodiments, lipid particles (e.g., lipid nanoparticles) can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. The processes and apparatus for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007 / 0042031, the content of which is hereby incorporated by reference in its entirety. The processes and apparatus for preparing lipid nanoparticles using a stepwise dilution process are described in US2004 / 0142025, the content of which is hereby incorporated by reference in its entirety.

[0264] In one embodiment, lipid particles (e.g., lipid nanoparticles) can be prepared by a collision jet process. Generally, the particles are formed by mixing lipid dissolved in an alcohol (e.g., ethanol) with ceDNA dissolved in a buffer solution, 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 mixing ratio of lipid to ceDNA can be about 45 - 55% lipid and about 65 - 45% ceDNA.

[0265] The lipid solution can contain a cationic lipid (e.g., an ionizable cationic lipid), a non-cationic lipid (e.g., phospholipids such as DSPC, DOPE, and DOPC), a PEG or PEG-conjugated molecule (e.g., PEG-lipid), and a sterol (e.g., cholesterol) in an alcohol, such as ethanol, at a total lipid concentration of 5 - 30 mg / mL, more likely 5 - 15 mg / mL, and most likely 9 - 12 mg / mL. In the lipid solution, the molar ratio of lipids can be in the range of about 25 - 98%, preferably about 35 - 65% for the cationic lipid, about 0 - 15%, preferably about 0 - 12% for the non-ionic lipid, about 0 - 15%, preferably about 1 - 6% for the PEG or PEG-conjugated lipid molecule, and about 0 - 75%, preferably about 30 - 50% for the sterol.

[0266] The ceDNA solution can contain ceDNA at a concentration range of 0.3 - 1.0 mg / mL, preferably 0.3 - 0.9 mg / mL, in a buffer solution having a pH in the range of 3.5 - 5.

[0267] To form LNP, in one exemplary but non-limiting embodiment, the two liquids are heated to a temperature in the range of about 15 - 40 °C, preferably about 30 - 40 °C, and then mixed, for example, in a collision jet mixer to immediately form LNP. The mixing flow rate can be in the range of 10 - 600 mL / min. The tube ID can range from 0.25 - 1.0 mm and the total flow rate can be 10 - 600 mL / min. The combination of flow rate and tube ID can have the effect of controlling the particle size of the LNP to 30 - 200 nm. The solution can then be mixed with a buffer solution at a higher pH in a mixing ratio in the range of 1:1 - 1:3 vol:vol, preferably about 1:2 vol:vol. Optionally, this buffer solution can be at a temperature in the range of 15 - 40 °C or 30 - 40 °C. The mixed LNP can then undergo an anion exchange filtration step. Before anion exchange, the mixed LNP can be incubated for a period of time, for example, 30 minutes to 2 hours. The temperature during incubation can be in the range of 15 - 40 °C or 30 - 40 °C. After incubation, the solution is filtered through a filter such as a 0.8 μm filter including an anion exchange separation step. In this process, tube IDs in the range of 1 mm ID to 5 mm ID and flow rates of 10 - 2000 mL / min can be used.

[0268] After formation, the LNP can be concentrated and ultrafiltered through an ultrafiltration process in which alcohol is removed and the buffer is exchanged with a final buffer solution, for example, phosphate buffered saline (PBS) at about pH 7, such as about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0269] In the diafiltration process, a tangential flow filtration format (TFF) with a membrane nominal molecular weight cut-off range of 30 to 500 kD can be used. The membrane format is a hollow fiber or a flat sheet cassette. In a TFF process with an appropriate molecular weight cut-off, the LNP can be retained in the retentate, and the filtrate or permeate contains alcohol, citrate buffer, and waste of the final buffer. The TFF process is a multi-step process with an initial ceDNA concentration of 1 to 3 mg / mL. After concentration, the LNP solution is ultrafiltered against the final buffer at a volume of 10 to 20, and alcohol is removed to perform buffer exchange. Then, the material can be further concentrated 1 to 3 times. The concentrated LNP solution can be sterile filtered.

[0270] VII. Pharmaceutical Compositions and Formulations Also provided herein are pharmaceutical compositions comprising ceDNA lipid particles and a pharmaceutically acceptable carrier or excipient.

[0271] In one embodiment, the ceDNA lipid particles (e.g., lipid nanoparticles) are provided with complete encapsulation, partial encapsulation of the therapeutic nucleic acid. In one embodiment, the nucleic acid therapeutic agent is completely encapsulated in lipid particles (e.g., lipid nanoparticles) to form a nucleic acid-containing lipid particle. In one embodiment, the nucleic acid can be encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0272] In one embodiment, the lipid particles have an average diameter of about 20 nm to about 100 nm, 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm to ensure effective delivery. Nucleic acid-containing lipid particles (e.g., lipid nanoparticles) and methods for their preparation are disclosed, for example, in PCT / US18 / 50042, US Patent Publication Nos. 2004 / 0142025 and 2007 / 0042031, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. In one embodiment, the lipid particle (e.g., lipid nanoparticle) size can be determined by quasi-elastic light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, UK) system.

[0273] Generally, the lipid particles (e.g., lipid nanoparticles) of the present invention have an average diameter selected to provide the intended therapeutic effect.

[0274] Depending on the intended use of the lipid particles (e.g., lipid nanoparticles), the ratio of the components can vary, and the delivery efficiency of a particular formulation can be measured, for example, using an endosomal release parameter (ERP) assay.

[0275] In one embodiment, the lipid particles (e.g., lipid nanoparticles) can be conjugated to other moieties to prevent aggregation. Such lipid conjugates include, for example, PEG conjugated to dialkyloxypropyl (e.g., PEG-DAA conjugate), PEG conjugated to diacylglycerol (e.g., PEG-DAG conjugate), PEG conjugated to cholesterol, PEG conjugated to phosphatidylethanolamine, and PEG conjugated to ceramide (see, e.g., U.S. Patent No. 5,885,613), such as PEG-lipid conjugates, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugate, see, e.g., U.S. Provisional Application No. 61 / 294,828, filed January 13, 2010, and U.S. Provisional Application No. 61 / 295,140, filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugate), and mixtures thereof, but are not limited thereto. Additional examples of POZ-lipid conjugates are described in PCT Publication No. WO 2010 / 006282. PEG or POZ can be conjugated directly to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for conjugating PEG or POZ to the lipid can be used, including non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties such as amide or carbamate are used. The disclosures of each of the above patent documents are hereby incorporated by reference in their entirety for all purposes.

[0276] In one embodiment, the ceDNA can be complexed with the lipid portion of the particle or encapsulated at the lipid position of a lipid particle (e.g., a lipid nanoparticle). In one embodiment, the ceDNA can be completely encapsulated at the lipid position of a lipid particle (e.g., a lipid nanoparticle), thereby protecting it from degradation by nucleases, for example, in an aqueous solution. In one embodiment, the ceDNA in a lipid particle (e.g., a lipid nanoparticle) is not substantially degraded after exposure of the lipid particle (e.g., a lipid nanoparticle) to nucleases at 37 °C for at least about 20, 30, 45, or 60 minutes. In some embodiments, the ceDNA in a lipid particle (e.g., a lipid nanoparticle) is not substantially degraded after incubation of the particle in serum at 37 °C for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0277] In one embodiment, the lipid particle (e.g., a lipid nanoparticle) is substantially non-toxic to a subject, such as a mammal, e.g., a human.

[0278] In one embodiment, a pharmaceutical composition comprising a therapeutic nucleic acid of the present disclosure can be formulated into lipid particles (e.g., lipid nanoparticles). In some embodiments, the lipid particles comprising the therapeutic nucleic acid can be formed from cationic lipids. In some other embodiments, the lipid particles comprising the therapeutic nucleic acid can be formed from non-cationic lipids. In a preferred embodiment, the lipid particles of the present invention are lipid particles containing nucleic acid, which are mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), minicircle DNA, minigene, viral DNA (e.g., lentiviral or AAV genome) or non-viral synthetic DNA vector, closed-ended linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, doggybone (trademark) DNA vector, minimally immunologically defined gene expression (MIDGE) vector, non-viral minicircle DNA vector (linear covalently closed DNA vector), or dumbbell-shaped DNA minimal vector ("dumbbell DNA") selected from the group consisting of therapeutic nucleic acids formed from cationic lipids.

[0279] In another preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles, which are formed from non-cationic lipids and optionally conjugated lipids that prevent aggregation of the particles.

[0280] In one embodiment, the lipid particle formulation is an aqueous solution. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation is a lyophilized powder.

[0281] According to some aspects, the present disclosure provides a lipid particle formulation further comprising one or more pharmaceutical excipients. In one embodiment, the lipid particle (e.g., lipid nanoparticle) formulation further comprises sucrose, tris, trehalose, and / or glycine.

[0282] In one embodiment, the lipid particles (e.g., lipid nanoparticles) disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to the subject's cells, tissues, or organs. Typically, the pharmaceutical composition comprises a ceDNA lipid particle (e.g., lipid nanoparticle) disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the ceDNA lipid particles (e.g., lipid nanoparticles) of the present disclosure can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transfection via high-pressure intravenous or intra-arterial injection, as well as intracellular injection such as nuclear microinjection or cytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. A sterile injectable solution can be prepared by incorporating the required amount of the ceDNA vector compound in a suitable buffer, optionally in combination with one or more of the ingredients listed above, and filtering to sterilize.

[0283] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retrobulbar, intraretinal, subretinal, choroidal, subchoroidal, interstitial, intraocular, and vitreal), intravestibular, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transfection via high-pressure intravenous or intra-arterial injection, as well as intracellular injection such as nuclear microinjection or cytoplasmic injection, are also contemplated.

[0284] A pharmaceutically active composition comprising ceDNA lipid particles (e.g., lipid nanoparticles) can be formulated to deliver a transgene in the nucleic acid to recipient cells, resulting in therapeutic expression of the transgene therein. The composition can also comprise a pharmaceutically acceptable carrier.

[0285] Pharmaceutical compositions for therapeutic purposes are typically sterile and must be 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 vector concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compound in a suitable buffer, optionally with one or a combination of the ingredients listed above, and filtering sterilization.

[0286] In one embodiment, the lipid particles (e.g., lipid nanoparticles) are solid core particles having at least one lipid bilayer. In one embodiment, the lipid particles (e.g., lipid nanoparticles) have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) form. Without limitation, examples of non-bilayer forms can include, for example, three-dimensional tubes, rods, cubic symmetries, etc. The non-lamellar form (i.e., non-bilayer structure) of lipid particles (e.g., lipid nanoparticles) is known to those skilled in the art and can be determined using analytical techniques used by those skilled in the art. Such techniques include, but are not limited to, cryogenic transmission electron microscopy ("Cryo-TEM"), differential scanning calorimetry ("DSC"), X-ray diffraction, etc. For example, the morphology (lamellar vs. non-lamellar) of lipid particles can be readily evaluated and characterized using the Cryo-TEM analysis described in US2010 / 0130588, the content of which is hereby incorporated by reference in its entirety.

[0287] In one embodiment, the lipid particles (e.g., lipid nanoparticles) having a non-lamellar form have a high electron density.

[0288] In one embodiment, the present disclosure provides lipid particles (e.g., lipid nanoparticles) that are either a single lamellar structure or a multi-lamellar structure. In some aspects, the present disclosure provides lipid particle (e.g., lipid nanoparticle) formulations comprising polyplex particles and / or foamed-based particles. By controlling the composition and concentration of the lipid components, the rate at which lipid conjugates exchange outside the lipid particles (lipid nanoparticles) and, in turn, the rate at which the lipid nanoparticles become membrane fusogenic can be controlled. Additionally, other variables, such as, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which lipid particles (e.g., lipid nanoparticles) become membrane fusogenic. Other methods that can be used to control the rate at which lipid particles (e.g., lipid nanoparticles) become membrane fusogenic will be apparent to those skilled in the art based on the present disclosure. It will also be apparent that the lipid particle size can be controlled by controlling the composition and concentration of the lipid conjugates.

[0289] In one embodiment, the pKa of the formulated cationic lipid can correlate with the efficacy of the LNP for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533, Semple et al, Nature Biotechnology 28, 172-176 (2010). Both of these are incorporated herein by reference in their entirety). In one embodiment, a preferred range of pKa is from about 5 to about 7. In one embodiment, the pKa of the cationic lipid can be determined in lipid particles (e.g., lipid nanoparticles) using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS).

[0290] In one embodiment, encapsulation of ceDNA in lipid particles (e.g., lipid nanoparticles) can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or the PicoGreen® assay, that uses a dye that enhances fluorescence when associated with nucleic acids. Generally, encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic surfactant. Surfactant-mediated disruption of the lipid bilayer releases the encapsulated ceDNA and allows it to interact with the membrane-impermeable dye. Encapsulation of ceDNA can be calculated as E = (Io − I) / Io, where I and Io refer to the fluorescence intensities before and after addition of the surfactant.

[0291] Unit dosage In one embodiment, the pharmaceutical composition can be presented in unit dosage form. The unit dosage form will typically be adapted to one or more routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by an inhaler. In some embodiments, the unit dosage form is adapted for administration by a nebulizer. In some embodiments, the unit dosage form is adapted for administration by an aerosolizer. In some embodiments, the unit dosage form is adapted for oral, buccal, or sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intraventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect.

[0292] VIII. Methods of treatment The ceDNA vectors (e.g., ceDNA vector lipid particles described herein) and compositions described herein can be used to introduce nucleic acid sequences (e.g., therapeutic nucleic acid sequences) into host cells. In one embodiment, the introduction of a nucleic acid sequence into a host cell using a ceDNA vector (e.g., ceDNA vector lipid particles described herein) can be monitored with an appropriate biomarker from the treated patient to evaluate gene expression.

[0293] The compositions and vectors provided herein can be used to deliver transgenes (nucleic acid sequences) for various purposes. In one embodiment, a ceDNA vector (e.g., ceDNA vector lipid particles described herein) can be used in a variety of ways, including, for example, ex situ, in vitro, and in vivo applications, methodologies, diagnostic procedures, and / or gene therapy regimens.

[0294] Provided herein is a method of treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of a ceDNA vector (e.g., ceDNA vector lipid particles described herein), optionally together with a pharmaceutically acceptable carrier, into target cells (e.g., muscle cells or tissues, or other diseased cell types) of the subject in need of treatment. The ceDNA vector (e.g., ceDNA vector lipid particles described herein) can be introduced in the presence of a carrier, although such a carrier is not required. The implemented ceDNA vector (e.g., ceDNA vector lipid particles described herein) contains a nucleotide sequence useful for treating the disease. In particular, the ceDNA vector can contain a desired exogenous DNA sequence operably 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 ceDNA vector (e.g., ceDNA vector lipid particles described herein) can be administered via any suitable route described herein and known in the art. In one embodiment, the target cells are in a human subject.

[0295] Provided herein is a method for providing a diagnostically or therapeutically effective amount of a ceDNA vector (e.g., a ceDNA vector lipid particle as described herein) to a subject in need thereof, the method comprising providing an amount of a ceDNA vector (e.g., a ceDNA vector lipid particle as described herein) to the cells, tissues, or organs of a subject in need thereof for a time effective to permit expression of the transgene from the ceDNA vector, thereby providing to the subject a protein, peptide, or nucleic acid expressed by a diagnostically or therapeutically effective amount of a ceDNA vector (e.g., a ceDNA vector lipid particle as described herein). In one embodiment, the subject is human.

[0296] Provided herein is a method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, disorder, dysfunction, injury, abnormality, or trauma in a subject. Generally, the method comprises at least the step of administering one or more ceDNA vectors (e.g., a ceDNA vector lipid particle as described herein) to a subject in need thereof in an amount and for a time sufficient to diagnose, prevent, treat, or ameliorate one or more symptoms of the subject's disease, disorder, dysfunction, injury, abnormality, or trauma. In one embodiment, the subject is human.

[0297] Provided herein is a method that includes using a ceDNA vector as a tool for treating or reducing one or more symptoms of a disease or disease state. There are several genetic diseases in which the defective gene is known, and typically they are classified into two classes: a deficiency state of an enzyme that is usually inherited in a generally recessive manner, and a disproportion state that may be involved in regulatory or structural proteins but is not typically always inherited in a dominant manner. In the case of diseases in the deficiency state, a ceDNA vector (e.g., the ceDNA vector lipid particles described herein) is used to deliver a transgene to carry a normal gene into the affected tissue for replacement therapy, and in some embodiments, antisense mutations can be used to create an animal model of the disease. In the case of the disproportion disease state, a ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be used to create a disease state in a model system, which can then be used to attempt to counteract that disease state. Thus, the ceDNA vectors (e.g., the ceDNA vector lipid particles described herein) and methods disclosed herein enable the treatment of genetic diseases. As used herein, a disease state is treated by partially or wholly remedying a deficiency or disproportion that causes or exacerbates the disease.

[0298] Generally, any transgene can be delivered using a ceDNA vector (e.g., a ceDNA vector lipid particle as described herein) according to the above description to treat, prevent, or ameliorate symptoms associated with any disorder related to gene expression. Exemplary disease states include 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, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondrial myopathy (e.g., Leber's hereditary optic neuropathy (LHON), Leigh syndrome, and subacute sclerosing panencephalitis), myopathy (e.g., facioscapulohumeral muscular dystrophy (FSHD) and cardiomyopathy), diseases of solid organs (e.g., brain, liver, kidney, heart), etc., but are not limited thereto. In some embodiments, a ceDNA vector as disclosed herein can be advantageously used in the treatment of individuals having a metabolic disorder (e.g., ornithine transcarbamylase deficiency).

[0299] In one embodiment, the ceDNA vectors described herein can be used to treat, ameliorate, and / or prevent diseases or disorders caused by mutations in genes or gene products. Exemplary diseases or disorders that can be treated with ceDNA vectors (e.g., the ceDNA vector lipid particles described herein, such as lipid nanoparticles) include metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage diseases); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII, Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (e.g., hemophilia (A and B), thalassemia, and anemia); cancer and tumors, and genetic diseases or disorders (e.g., cystic fibrosis), but are not limited thereto.

[0300] In one embodiment, in situations where it is desirable to regulate the expression level of a transgene (e.g., a transgene encoding a hormone or growth factor as described herein), a heterologous nucleotide sequence can be delivered using a ceDNA vector (e.g., the ceDNA vector lipid particles described herein).

[0301] In one embodiment, a ceDNA vector (e.g., a ceDNA vector lipid particle as described herein) can be used to correct abnormal levels and / or functions of gene products (e.g., absence or deficiency in a protein) that result in a disease or disorder. The ceDNA vector (e.g., a ceDNA vector lipid particle as described herein) can produce a functional protein and / or modify the level of a protein to alleviate or reduce symptoms resulting from a particular disease or disorder caused by an absence or deficiency in a protein, or to confer a benefit. For example, treatment of OTC deficiency can be achieved by producing a functional OTC enzyme. Treatment of hemophilia A and B can be achieved by modifying the levels of factor VIII, factor IX, and factor X. Treatment of PKU can be achieved by modifying the level of the phenylalanine hydroxylase enzyme. Treatment of Fabry disease or Gaucher disease can be achieved by producing a functional α-galactosidase or β-glucocerebrosidase, respectively. Treatment of MFD or MPSII can be achieved by producing a functional arylsulfatase A or iduronate-2-sulfatase, respectively. Treatment of cystic fibrosis can be achieved by producing a functional cystic fibrosis transmembrane conductance regulator. Treatment of glycogen storage disease can be achieved by restoring the function of the functional G6Pase enzyme. Treatment of PFIC can be achieved by producing a functional ATP8B1, ABCB11, ABCB4, or TJP2 gene.

[0302] In one embodiment, a ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be used to provide an RNA-based therapeutic agent to cells in vitro or in vivo. Examples of RNA-based therapeutic agents include, but are not limited to, mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). For example, in one embodiment, a ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be used to provide an antisense nucleic acid to cells in vitro or in vivo. For example, when the transgene is an RNAi molecule, the expression of the antisense nucleic acid or RNAi in the target cells reduces the expression of a specific protein by the cells. Thus, a transgene that is an RNAi molecule or an antisense nucleic acid can be administered to reduce the expression of a specific protein in a subject in need thereof. The antisense nucleic acid can also be administered to cells in vitro to regulate cell physiology, e.g., to optimize a cell or tissue culture system.

[0303] In one embodiment, a ceDNA vector (e.g., the ceDNA vector lipid particles described herein) 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, minicircle DNA, minigenes, viral DNA (e.g., lentiviral or AAV genomes) or non-viral synthetic DNA vectors, closed-ended linear double-stranded DNA (ceDNA / CELiD), plasmids, bacteriophages, doggybone™ DNA vectors, minimally immunologically defined gene expression (MIDGE)-vectors, non-viral miniring DNA vectors (linearly covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors (“dumbbell DNA”). For example, in one embodiment, a ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be used to deliver minicircles to cells in vitro or in vivo. For example, when the transgene is minicircle DNA, expression of the minicircle DNA in the target cells reduces the expression of a specific protein by the cells. Thus, administration of a transgene that is minicircle DNA can reduce the expression of a specific protein in a subject in need thereof. Minicircle DNA can also be administered to cells in vitro to modulate cell physiology, e.g., to optimize a cell or tissue culture system.

[0304] In one embodiment, exemplary transgenes encoded by the ceDNA vector include X, lysosomal enzymes (e.g., hexosaminidase A associated with Tay-Sachs disease, or iduronate sulfatase associated with Hunter syndrome / MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, and cytokines (e.g., interferon, beta-interferon, interferon-g, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophin-3 and 4, brain-derived neurotrophic factor (BDNF), glial-derived growth factor (GDNF), transforming growth factors-a and -b, etc.), receptors (e.g., tumor necrosis factor receptor), but are not limited thereto. In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, two or more transgenes are encoded by the ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments, the transgene encodes an antibody, including a full-length antibody or an antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as defined herein. Other exemplary transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, and tumor suppressor gene products.

[0305] Administration In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) can be administered to an organism for in vivo cell transfection. In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) can be administered to an organism for ex vivo cell transfection.

[0306] Generally, administration is by any of the routes commonly used to ultimately contact the molecule with blood or tissue cells. Suitable methods for administering such nucleic acids are available and well known to those of skill in the art, and two or more routes may be used to administer a particular composition, but a particular route is often more immediate and may provide a more effective response than another route. Exemplary modes of administration of the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intradermal, intrauterine (or intraovum), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to skeletal, diaphragm, and / or cardiac muscle), intrapleural, intracerebral, and intraarterial), topical (e.g., to the skin and mucosal surfaces including the airway surface, and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., to the liver, eye, skeletal muscle, cardiac muscle, diaphragm, muscle, or brain).

[0307] Administration of the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) can be performed on any site of a subject including, but not limited to, sites selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. In one embodiment, administration of the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) can also be to a tumor (e.g., within or near a tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition being treated, ameliorated, and / or prevented, as well as the nature of the particular ceDNA vector (e.g., the ceDNA vector lipid particle described herein) being used. Additionally, the ceDNA enables administration of two or more transgenes in a single vector or multiple ceDNA vectors (e.g., a ceDNA cocktail).

[0308] In one embodiment, administration of the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) to skeletal muscle includes, but is not limited to, administration to the skeletal muscle of the limbs (e.g., upper arm, lower arm, upper extremity, and / or lower extremity), waist, neck, head (e.g., tongue), pharynx, abdomen, pelvis / perineum, and / or fingers. The ceDNA vector (e.g., the ceDNA vector lipid particle described herein) can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal, limb perfusion (optionally, isolated limb perfusion of the leg and / or arm, see, e.g., Arruda et al., (2005) Blood 105:3458-3464), and / or direct intramuscular injection. In certain embodiments, the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) is administered to the limbs (arms and / or legs) of a subject (e.g., a subject having a muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-arterial administration). In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particle described herein) can be administered without using "hydrodynamic" techniques.

[0309] Administration of the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. The ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion. Administration to the diaphragm muscle can be performed by any suitable method including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to smooth muscle can be performed by any suitable method including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment, the administration can be performed on endothelial cells present in, near, and / or on smooth muscle.

[0310] In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) is administered to skeletal muscle, diaphragm muscle, and / or myocardium (e.g., to treat, ameliorate, and / or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure).

[0311] A ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be administered to the CNS (e.g., the brain or eye). A ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (including the cerebral cortex, basal ganglia, hippocampus, and amygdala (porta amygdala), the cerebral hemisphere including the striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. A ceDNA vector (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can also be administered to different regions of the eye such as the retina, cornea, and / or optic nerve. A ceDNA vector (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be delivered to the cerebrospinal fluid (e.g., by lumbar puncture). A ceDNA vector (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can further be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0312] In one embodiment, a ceDNA vector (the ceDNA vector lipid particles described herein) can be administered to a desired region of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intratympanic, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's space) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[0313] In some embodiments, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) is administered in a liquid formulation by direct injection (e.g., stereotactic injection) into a desired region or compartment in the CNS. According to other embodiments, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be provided by topical application to a desired region or by intranasal administration of an aerosol formulation. Administration to the eye may be by topical application of droplets. As a further alternative, the ceDNA vector can be administered as a solid sustained release formulation (see, e.g., U.S. Patent No. 7,201,898, which is incorporated herein by reference in its entirety). In one embodiment, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) is used for retrograde transport to treat, ameliorate, and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, the ceDNA vector (e.g., the ceDNA vector lipid particles described herein) can be delivered to muscle tissue and move from there into neurons.

[0314] In one embodiment, repeated administration of the therapeutic product can be carried out until an appropriate level of expression is achieved. Thus, in one embodiment, the therapeutic nucleic acid can be administered and readministered multiple times. For example, the therapeutic nucleic acid can be administered on day 0. Following the first treatment on day 0, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, about 18 years, about 19 years, about 20 years, about 21 years, about 22 years, about 23 years, about 24 years, about 25 years, about 26 years, about 27 years, about 28 years, about 29 years, about 30 years, about 31 years, about 32 years, about 33 years, about 34 years, about 35 years, about 36 years, about 37 years, about 38 years, about 39 years, about 40 years, about 41 years, about 42 years, about 43 years, about 44 years, about 45 years, about 46 years, about 47 years, about 48 years, about 49 years or about 50 years after the first treatment with the therapeutic nucleic acid, a second dosing (readministration) can be performed.

[0315] In one embodiment, one or more additional compounds may also be included. Those compounds can be administered separately, or the additional compound can be included in the lipid particles (e.g., lipid nanoparticles) of the present invention. In other words, the lipid particles (e.g., lipid nanoparticles) can contain other compounds in addition to the ceDNA, or at least a second ceDNA that is different from the first one. Without limitation, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and their derivatives, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0316] In one embodiment, one or more additional compounds can be therapeutic agents. The therapeutic agent can be selected from any class suitable for therapeutic purposes. Thus, the therapeutic agent can be selected from any class suitable for therapeutic purposes. The therapeutic agent can be selected according to the desired therapeutic purpose and biological effect. For example, in one embodiment, if the ceDNA within the LNP is useful for treating cancer, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates)). In one embodiment, if the LNP containing ceDNA is useful for treating an infectious disease, the additional compound can be an antimicrobial agent (e.g., an antibiotic or an antiviral compound). In one embodiment, if the LNP containing ceDNA is useful for treating an immune disease or disorder, the additional compound can be a compound that modulates the immune response (e.g., an immunosuppressive agent, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In one embodiment, different cocktails of different lipid particles containing different compounds, such as ceDNA encoding different proteins or different compounds (such as therapeutic agents), can be used in the compositions and methods of the present invention. In one embodiment, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressive agent. In some embodiments, the additional compound is immunostimulatory.

Example

[0317] The following examples are provided by way of illustration and not limitation. Those skilled in the art will understand that a ceDNA vector can be constructed from any of the wild-type or modified ITRs described herein, and that the activity of such a ceDNA vector can be constructed and evaluated using the following exemplary methods. These methods are illustrated using a particular ceDNA vector, but they are applicable to any ceDNA vector according to the description.

[0318] Example 1: Constructing a ceDNA Vector Using an Insect Cell-Based Method The production of the ceDNA vector using the polynucleotide construct template is described in Example 1 of PCT / US18 / 49996, which is hereby incorporated by reference in its entirety. For example, the polynucleotide construct template used to generate the ceDNA vector of the present invention can be a ceDNA-plasmid, a ceDNA-bacmid, and / or a ceDNA-baculovirus. Without being limited by theory, in a permissive host cell, e.g., in the presence of Rep, a polynucleotide construct template having two symmetrical ITRs (at least one of the ITRs being modified relative to the wild-type ITR sequence) and an expression construct replicates to produce a ceDNA vector. CeDNA vector production proceeds through two steps: excision ("rescue") of the template from a first, template backbone (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genome, etc.) via the Rep protein, and second, Rep-mediated replication of the excised ceDNA vector.

[0319] Exemplary methods for producing the ceDNA vector are derived from the ceDNA-plasmids described herein. Referring to FIGS. 1A and 1B, the polynucleotide construct template of each ceDNA-plasmid includes both a left-modified ITR and a right-modified ITR, and between the ITR sequences are (i) an enhancer / promoter, (ii) a cloning site for the transgene, (iii) a post-transcriptional response element (e.g., the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)), (iv) a polyadenylation signal (e.g., from the bovine growth hormone gene (BGHpA)). Unique restriction endonuclease recognition sites (R1-R6) (shown in FIGS. 1A and 1B) are also introduced between each component to facilitate the introduction of new gene components to specific sites in the construct. The R3 (PmeI) 5'-GTTTAAAC-3' and R4 (PacI) 5'-TTAATTAA-3' enzyme sites are designed in the cloning site to introduce the open reading frame of the transgene. These sequences were cloned into the pFastBac HT B plasmid obtained from ThermoFisher Scientific.

[0320] Production of ceDNA-bacmid: Transform DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells, Thermo Fisher) with either the test plasmid or the control plasmid according to the protocol provided by the manufacturer. Recombination between the plasmid in DH10Bac cells and the baculovirus shuttle vector was induced to generate recombinant ceDNA-bacmid. Positive selection was screened by blue-white screening in E. coli on bacterial agar plates containing X-gal and IPTG together with the antibiotics selected for the transformation and maintenance of the bacmid and transposase plasmids (the Φ80dlacZΔM15 marker provides α-complementation of the β-galactosidase gene from the bacmid vector). Recombinant bacmids were selected by picking white colonies caused by transpositions that disrupt the β-galactosidase reporter gene and culturing them in 10 mL of medium.

[0321] Isolate the recombinant ceDNA-bacmid from E. coli and transfect it into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adherent Sf9 or Sf21 insect cells were cultured in 50 mL of medium in a T25 flask at 25 °C. After 4 days, the culture medium (containing P0 virus) was removed from the cells, filtered through a 0.45 μm filter to separate the infectious baculovirus particles from the cells or cell debris.

[0322] Optionally, the first-generation baculovirus (P0) was amplified in 50 - 500 mL of medium by infecting naive Sf9 or Sf21 insect cells. Cells in suspension cultures in an orbital shaker incubator were maintained at 130 rpm and 25 °C, monitoring cell diameter, viability, and density of approximately 4.0E+6 cells / mL until the cells reached a diameter of 18 - 19 nm (from a naive diameter of 14 - 15 nm). Between 3 and 8 days post-infection, P1 baculovirus particles in the medium were collected after centrifugation, filtered through a 0.45 μm filter after removing cells and debris.

[0323] The ceDNA-baculovirus containing the test construct was collected, and the infectivity or titer of the baculovirus was determined. Specifically, 4 × 20 mL Sf9 cell cultures at 2.5E+6 cells / mL were treated with P1 baculovirus at the following dilutions: 1 / 1000, 1 / 10,000, 1 / 50,000, 1 / 100,000, and incubated at 25 - 27°C. Infectivity was determined daily over 4 - 5 days by the rate of increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[0324] The "Rep-plasmid" disclosed in Figure 8A of PCT / US18 / 49996, which is hereby incorporated by reference in its entirety, is produced in a pFASTBAC™ dual expression vector (ThermoFisher) containing both Rep78 and Rep52 or Rep68 and Rep40. The Rep-plasmid is transformed into DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ Competent Cells (Thermo Fisher)) according to the protocol provided by the manufacturer. Recombination between the Rep-plasmid and the baculovirus shuttle vector in DH10Bac cells is induced to produce a recombinant bacmid ("Rep-bacmid"). The recombinant bacmid is selected by positive selection including blue-white screening in E. coli on bacterial agar plates containing X-gal and IPTG (the Φ80dlacZΔM15 marker provides α-complementation of the β-galactosidase gene from the bacmid vector). Isolated white colonies are picked and seeded into 10 mL of selective medium (kanamycin, gentamicin, tetracycline in LB broth). The recombinant bacmid (Rep-bacmid) is isolated from E. coli and transfected into Sf9 or Sf21 insect cells to produce infectious baculovirus.

[0325] Sf9 or Sf21 insect cells are cultured in 50 mL of medium for 4 days, and infectious recombinant baculovirus ("Rep-baculovirus") is isolated from the culture. Optionally, the first-generation Rep-baculovirus (P0) is amplified by infecting naive Sf9 or Sf21 insect cells and cultured in 50 - 500 mL of medium. 3 to 8 days after infection, the P1 baculovirus particles in the medium are collected either by separating the cells by centrifugation or by filtration or any other fractionation process. The Rep-baculovirus is collected and the infectious activity of the baculovirus is determined. Specifically, 4 × 20 mL Sf9 cell cultures at 2.5×10 6 cells / mL are treated with P1 baculovirus at the following dilutions 1 / 1000, 1 / 10,000, 1 / 50,000, 1 / 100,000 and incubated. Infectivity is determined daily over 4 - 5 days by the rate of increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[0326] ceDNA vector generation and characterization Referring to Figure 4B, next, either (1) a sample containing ceDNA-bacmid or ceDNA-baculovirus, and (2) Sf9 insect cell culture medium containing any of the above Rep-baculovirus are added to fresh cultures of Sf9 cells (2.5E+6 cells / mL, 20 ml) at ratios of 1:1000 and 1:10,000, respectively. The cells are then cultured at 25°C, 130 rpm. 4 - 5 days after co-infection, the cell diameter and viability are detected. When the viability reaches about 70 - 80% and the cell diameter reaches 18 - 20 nm, the cell culture is centrifuged, the medium is removed, and the cell pellet is collected. First, the cell pellet is resuspended in an appropriate amount of aqueous medium (either water or buffer). The ceDNA vector is isolated and purified from the cells using the Qiagen MIDI PLUS™ purification protocol (Qiagen, 0.2 mg of processed cell pellet mass per column).

[0327] The yield of the ceDNA vector produced and purified from Sf9 insect cells was initially determined based on the UV absorbance at 260 nm.

[0328] The ceDNA vector can be evaluated by identifying it by agarose gel electrophoresis under native or denaturing conditions as illustrated in Figure 4D. (a) The presence of characteristic bands migrating at twice the size on the denaturing gel relative to the native gel after restriction endonuclease digestion and gel electrophoresis analysis, and (b) the presence of monomer and dimer (2x) bands on the denaturing gel of the uncut material are specific to the presence of the ceDNA vector.

[0329] The structure of the isolated ceDNA vector was further analyzed by digesting the DNA obtained from co-infected Sf9 cells (as described herein) with a restriction endonuclease for a) the presence of only a single cleavage site within the ceDNA vector, and b) the resulting fragments being large enough to be clearly visible when fractionated on a 0.8% denaturing agarose gel (>800 bp). As shown in Figures 4D and 4E, a linear DNA vector with a discontinuous structure and a ceDNA vector with a linear and continuous structure can be distinguished by the size of their reaction products. For example, a DNA vector with a discontinuous structure is expected to produce 1 kb and 2 kb fragments, while a non-capsidated vector with a continuous structure is expected to produce 2 kb and 4 kb fragments.

[0330] Thus, to qualitatively demonstrate that the isolated ceDNA vector is covalently closed-ended, as required by the definition, the sample is digested with a restriction endonuclease identified to have a single restriction site in the context of the specific DNA vector sequence, preferably resulting in two cleavage products of unequal sizes (e.g., 1000 bp and 2000 bp). Following digestion and electrophoresis on a denaturing gel (which separates the two complementary DNA strands), linear non-covalently closed DNA will resolve at sizes of 1000 bp and 2000 bp if the two DNA strands are ligated and unfolded to twice the length (but are single-stranded), while covalently closed DNA (i.e., the ceDNA vector) will resolve at twice the size (2000 bp and 4000 bp). Furthermore, digestion of monomeric, dimeric, and n-mer forms of the DNA vector will all resolve as fragments of the same size due to end-to-end ligation of the multimeric DNA vector (see Figure 4D).

[0331] As used herein, the phrase "assay for the identification of DNA vectors by agarose gel electrophoresis under native and denaturing conditions" refers to an assay for assessing the closed-ended nature of ceDNA by performing an electrophoretic evaluation of the digestion products following restriction endonuclease digestion. One such exemplary assay is shown below, but those skilled in the art will understand that many variations known in the art for this example are possible. A restriction endonuclease is selected such that it is a single-cut enzyme for the ceDNA vector of interest that will produce products approximately 1 / 3 and 2 / 3 the length of the DNA vector. This will resolve bands on both native and denaturing gels. It is important to remove buffer from the samples prior to denaturation. The Qiagen PCR Cleanup Kit or a desalting "spin column", e.g., the GE HEALTHCARE ILUSTRA™ MICROSPIN™ G-25 column are some options known in the art for endonuclease digestion. The assay includes, for example, i) digesting the DNA with an appropriate restriction endonuclease, ii) applying it, for example, to the Qiagen PCR Cleanup Kit and eluting with distilled water, iii) adding 10X denaturing solution (10X = 0.5 M NaOH, 10 mM EDTA), adding 10X dye, without buffering, and adding the 10X denaturing solution four-fold with a DNA ladder prepared by incubating previously with 1 mM EDTA and 200 mM NaOH on a 0.8 - 1.0% gel, analyzing together to ensure that the NaOH concentration is uniform in the gel and gel box and flowing the gel in the presence of 1X denaturing solution (50 mM NaOH, 1 mM EDTA). Those skilled in the art will understand the voltage to use to perform electrophoresis based on the size and desired timing of the results. After electrophoresis, the gel is discharged, neutralized in 1X TBE or TAE, and transferred to distilled water or 1X TBE / TAE containing 1X SYBR Gold.Next, for example, bands can be visualized using Thermo Fisher's SYBR® Gold nucleic acid gel stain (10,000X concentrate in DMSO) and an epi-fluorescent light (blue) or UV (312 nm).

[0332] The purity of the generated ceDNA vector can be evaluated using any method known in the art. As one exemplary, non-limiting method, the contribution of the ceDNA-plasmid to the overall UV absorbance of the sample can be estimated by comparing the fluorescence intensity of the ceDNA vector to a standard. For example, based on UV absorbance, if 4 μg of the ceDNA vector is loaded onto a gel and the ceDNA vector fluorescence intensity corresponds to a 2 kb band known to be 1 μg, then 1 μg of ceDNA vector is present and the ceDNA vector is 25% of the total UV-absorbing material. The band intensity on the gel is then plotted against the calculated input represented by the band. For example, if the total ceDNA vector is 8 kb and the excised comparison band is 2 kb, the band intensity is plotted as 25% of the total input, which in this case is 0.25 μg for 1....

Claims

**Claim 1** A pharmaceutical composition comprising lipid nanoparticles (LNP), wherein the LNP comprises an SS-cleavable lipid, a sterol, and a closed-ended DNA (ceDNA), and the SS-cleavable lipid has the formula I: 【Chemical 12】 The pharmaceutical composition comprising the ss-OP lipid of. **Claim 2** The pharmaceutical composition according to claim 1, wherein the sterol is cholesterol or β-sitosterol. **Claim 3** The pharmaceutical composition according to any one of claims 1 to 2, wherein the LNP further comprises polyethylene glycol (PEG) or a PEG-lipid conjugate. **Claim 4** The pharmaceutical composition according to claim 3, wherein the PEG or PEG-lipid conjugate is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG) or 1,2-distearoyl-rac-glycerol-3-methyl polyoxyethylene (DSG-PEG2000). **Claim 5**: The LNP is distearoyl-sn-glycero-phosphoethanolamine, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine, dimethyl phosphatidylethanolamine, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dieleoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,The pharmaceutical composition according to any one of claims 1 to 4, further comprising a non-cationic lipid selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof. **Claim 6** The pharmaceutical composition according to claim 5, wherein the non-cationic lipid is selected from the group consisting of dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE). **Claim 7** The pharmaceutical composition according to claim 5, wherein the monomethyl phosphatidylethanolamine is 16-O-methyl PE or the dimethyl phosphatidylethanolamine is 16-O-dimethyl PE. **Claim 8** The PEG or PEG-lipid conjugate is present at about 1.5% to about 3%; The sterol is cholesterol, and the cholesterol is present at a molar percentage of about 20% to about 40% or about 30% to about 50%; and / or The SS-cleavable lipid is present at a molar percentage of about 80% to about 60% or about 50%, The pharmaceutical composition according to claim 3. **Claim 9** The composition comprises cholesterol, PEG or a PEG-lipid conjugate, and a non-cationic lipid, The SS-cleavable lipid is present at a molar percentage of about 42.5% to about 62.5%; The PEG or PEG-lipid conjugate is present at about 1.5% to about 3%; The cholesterol is present at a molar percentage of about 30% to about 50%; and / or The non-cationic lipid is present in a molar percentage of from about 2.5% to about 12.5%. The pharmaceutical composition according to any one of claims 1 to 2.

10. The cholesterol is present in a molar percentage of about 40%, the SS-cleavable lipid is present in a molar percentage of about 52.5%, the non-cationic lipid is present in a molar percentage of about 7.5%, and the PEG or PEG-lipid conjugate is present in about 3%. The pharmaceutical composition according to claim 9.

11. The diameter of the LNP is from about 50 nm to about 110 nm. The pharmaceutical composition according to any one of claims 1 to 10.

12. The diameter of the LNP is less than about 100 nm. The pharmaceutical composition according to any one of claims 1 to 10.

13. The composition has a total lipid to ceDNA ratio of about 15:1, about 30:1, about 40:1 or about 50:

1. The pharmaceutical composition according to any one of claims 1 to 12.

14. The composition further comprises N-acetylgalactosamine (GalNAc). The pharmaceutical composition according to any one of claims 1 to 13.

15. The ceDNA is a closed-ended linear double-stranded DNA, and the ceDNA comprises an expression cassette containing a promoter sequence and a transgene. The pharmaceutical composition according to any one of claims 1 to 14.

16. The ceDNA comprises at least one inverted terminal repeat (ITR) adjacent to either the 5' or 3' end of the expression cassette. The pharmaceutical composition according to claim 15.

17. The expression cassette is flanked by two ITRs, and the two adjacent ITRs comprise one 5' ITR and one 3' ITR. The pharmaceutical composition according to claim 16.

18. At least one of the 5' ITR or the 3' ITR is a wild-type AAV ITR; At least one of the 5' ITR or the 3' ITR is a modified ITR; and / or The 5' ITR and the 3' ITR are symmetric ITRs or asymmetric ITRs. The pharmaceutical composition according to claim 16.

19. The pharmaceutical composition according to claim 16, wherein at least one of the adjacent ITRs is selected from the group consisting of an ITR derived from an AAV serotype, an ITR derived from an ITR of avian virus, an ITR derived from B19 virus ITR, and a wild-type ITR derived from parvovirus.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the ceDNA is selected from the group consisting of CELiD, DNA-based minicircle, MIDGE, ministring DNA, dumbbell-shaped linear double-stranded closed-ended DNA containing two hairpin structures of ITR at the 5' and 3' ends of an expression cassette, and doggybone (trademark) DNA.

21. The pharmaceutical composition according to any one of claims 1 to 20, for use in a method of treating a genetic disorder in a subject.

22. wherein the genetic disorder is melanoma, hemophilia A (factor VIII (FVIII) deficiency), hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR deficiency), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, hereditary liver metabolic disorder, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I type), Scheie syndrome (MPS IS type), Hurler-Scheie syndrome (MPS IH-S type), Hunter syndrome (MPS II type), Sanfilippo A, B, C, and D types (MPS III A, B, C, and D types), Morquio A and B types (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI type), Sly syndrome (MPS VII type), hyaluronidase deficiency (MPSType IX), Niemann-Pick diseases A / B, C1 and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis I, II / III and IV, sialidosis I and II, glycogen storage diseases I and II (Pompe disease), Gaucher diseases I, II and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis (LCA), Stargardt macular dystrophy (ABCA4 deficiency), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, and cathepsin A deficiency, the pharmaceutical composition according to claim 21, selected from the group consisting of.

23. The pharmaceutical composition according to claim 21 or claim 22, wherein the method further comprises administering an immunosuppressant.

24. The subject shows a reduced immune response level to the pharmaceutical composition as compared to the immune response level observed after administration of the pharmaceutical composition with an LNP containing MC3 as the main cationic lipid, and the immune response level to the pharmaceutical composition is at least 50% lower than the level observed with the LNP containing MC3. The pharmaceutical composition according to any one of claims 21 to 23.

25. The LNP containing the SS-cleavable lipid and the closed-ended DNA (ceDNA) shows a phagocytosis level that is at least 50% lower than the phagocytosis level of an LNP containing MC3 as the main cationic lipid administered under the same conditions or not being phagocytosed. The pharmaceutical composition according to any one of claims 21 to 24.

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