Synthetic DNA vectors and their use

Abstract

The problem that this invention aims to solve is to provide a versatile and efficient method for enhancing the long-term persistence of gene expression, such as that provided by rAAV, while enabling large payloads and reducing the risk of adverse effects (e.g., inflammation). [Solution] The present invention relates to an isolated DNA vector containing heterologous genes, which lacks bacterial plasmid DNA and / or bacterial signatures that can suppress in vivo persistence.

Description

Technical Field

[0001] Sequence Listing This application is electronically submitted in ASCII format and includes a sequence listing that is hereby incorporated by reference in its entirety. The ASCII copy created on March 13, 2019, is named 51219-012WO4_Sequence_Listing_03.13.19_ST25 and is 18,483 bytes in size.

[0002] Field of the Invention Generally, the present invention features a synthetic DNA vector.

Background Art

[0003] Background Gene therapy involves introducing a heterologous gene into target cells to correct a genetic defect underlying a disorder in a subject. Over the past few decades, various transduction approaches have been developed for use in gene therapy. For example, conventional bacterial plasmid DNA vectors are versatile tools in gene delivery, but may exhibit limitations due to their bacterial origin. Plasmid DNA vectors contain bacterial genes such as antibiotic resistance genes and origins of replication. Additionally, plasmid DNA vectors contain bacterial signatures such as CpG motifs. Moreover, the use of bacterial expression systems for generating plasmid DNA vectors carries the risk of introducing contaminating impurities from the bacterial host, such as endotoxins or bacterial genomic DNA and RNA, which can lead to loss of gene expression in vivo, for example, by transcriptional silencing.

[0004] Recombinant adeno-associated virus (rAAV) vectors have a proven track record of highly efficient gene transfer in various model systems and are currently being tested as a therapeutic mode for a wide range of human diseases. The genome of rAAV vectors can persist in vivo (e.g., in post-division cells) as a circular episome. After infection, single-stranded rAAV DNA is converted to double-stranded circular DNA in the cell nucleus and persists in episomal form for the lifespan of the cell. Thus, the substantial advantage of AAV vector systems is their ability to persist for long periods within target cells. On the other hand, AAV vectors may have additional disadvantages, such as a limited packaging capacity of approximately 4.5 Kb, immunogenicity of the viral protein, and difficulties in manufacturing.

[0005] Therefore, there is a need in the art for a versatile and efficient method to enhance the long-term persistence of gene expression, such as that provided by rAAV, while enabling large payloads and reducing the risk of adverse effects (e.g., inflammation). [Overview of the project]

[0006] overview In one aspect of the present invention, a nonviral, isolated circular DNA vector is provided that replicates the in vivo persistence of an rAAV vector. The DNA vector provided herein is nonimmunogenic and is not limited to an AAV packaging capacity of approximately 4.5 kb. The present invention also features a method for generating a circular DNA vector (e.g., in vitro, in the absence of a bacterial expression system), a pharmaceutical composition comprising the circular DNA vector, and a method for using the vector described herein, for example, to induce persistent episomal expression of a heterologous gene and to treat a disease associated with a defective gene.

[0007] In one aspect, the present invention provides an isolated circular DNA vector comprising one or more heterogenes, which lacks a replication origin (e.g., a bacterial replication origin) and / or a drug resistance gene (e.g., as part of a bacterial plasmid). For example, an isolated circular DNA vector comprising one or more heterogenes may lack a replication origin (e.g., a bacterial replication origin). Additionally or alternatively, an isolated circular DNA vector comprising one or more heterogenes may lack a drug resistance gene (e.g., as part of a bacterial plasmid). In some embodiments, an isolated circular DNA vector comprising one or more heterogenes may lack a replication origin (e.g., a bacterial replication origin) and a drug resistance gene (e.g., as part of a bacterial plasmid). In some embodiments, the DNA molecule lacks bacterial plasmid DNA. In some embodiments, the DNA vector lacks an immunogenic bacterial signature (e.g., one or more bacterial-associated CpG motifs, e.g., unmethylated CpG motifs, e.g., CpG islands). In some embodiments, DNA vectors lack RNA polymerase arrest sites (e.g., RNA polymerase II (RNAPII) arrest sites).

[0008] In some embodiments, the isolated circular DNA vector comprises one or more heterogenes encoding therapeutic proteins configured to treat Mendelian retinal dystrophy (e.g., Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome). For example, one or more heterogenes may be ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1.

[0009] In another aspect, the present invention provides an isolated circular DNA vector having one or more heterologous genes selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1, and lacking replication origins and / or drug resistance genes. In some embodiments, one or more heterologous genes encode a therapeutic protein configured to treat retinal dystrophy (e.g., retinal dystrophy selected from the group consisting of Mendelian hereditary retinal dystrophy, e.g., LCA, Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome).

[0010] In another aspect, the Specified Provisions provide an isolated circular DNA vector having one or more heterologous genes encoding a therapeutic protein (e.g., an antibody or a part thereof, a growth factor, an interleukin, an interferon, an anti-apoptotic factor, a cytokine, or an anti-diabetic factor), which lacks replication origins and / or drug resistance genes.

[0011] In another aspect, the present invention provides an isolated circular DNA vector having one or more heterologous genes including a trans-splicing molecule, and lacking replication origins and / or drug resistance genes.

[0012] In another aspect, the present invention provides an isolated circular DNA vector comprising one or more heterologous genes encoding a therapeutic protein secreted from the liver, the DNA vector lacking replication origins and / or drug resistance genes. In some embodiments, the therapeutic protein is secreted into the bloodstream.

[0013] In another aspect, the present invention provides an isolated circular DNA vector comprising one or more heterologous genes, wherein (a) it comprises terminal repeat sequences; and (b) it lacks replication origins and / or drug resistance genes.

[0014] In yet another aspect, the present invention provides an isolated linear DNA molecule having a plurality of identical amplicons, each of which comprises a heterogene encoding a therapeutic protein (e.g., a therapeutic protein configured to treat retinal dystrophy, e.g., Mendelian retinal dystrophy), wherein the DNA molecule lacks (a) replication origins and / or drug resistance genes; and (b) recombination sites. In some embodiments, the retinal dystrophy is selected from the group consisting of LCA, Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, age-related macular degeneration (AMD), Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome. In some embodiments, one or more heterologous genes are selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1.

[0015] In another aspect, the present invention provides an isolated linear DNA molecule having a plurality of identical amplicons, each of which contains a heterogene selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1, wherein the DNA molecule lacks (a) replication start sites and / or drug resistance genes; and (b) recombination sites. In some embodiments, heterologous genes encode therapeutic proteins configured to treat retinal dystrophy (e.g., Mendelian hereditary retinal dystrophy, e.g., LCA, Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, AMD, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome).

[0016] In another aspect, the DNA molecule provided herein is an isolated linear DNA molecule having a plurality of identical amplicons, each of which contains heterogeneous genes encoding antibodies or a portion thereof, coagulation factors, growth factors, hormones, interleukins, interferons, anti-apoptotic factors, antitumor factors, cytokines, and antidiabetic factors, wherein the DNA molecule lacks (a) replication start sites and / or drug resistance genes; and (b) recombination sites.

[0017] In yet another aspect, the present invention is characterized by an isolated linear DNA molecule having a plurality of identical amplicons, each of which contains a heterogene including a trans-splicing molecule, and the DNA molecule lacking (a) replication origins and / or drug resistance genes; and (b) recombination sites.

[0018] In another aspect, the present invention provides an isolated linear DNA molecule having a plurality of identical amplicons, each of which contains a heterogene encoding a therapeutic protein secreted from the liver (e.g., a therapeutic protein secreted into the blood), and the DNA molecule lacking replication origins and / or drug resistance genes.

[0019] In some embodiments of the aforementioned aspects, the circular DNA vector or linear DNA molecule further comprises one or more terminal repeat sequences (e.g., one or more inverted end repeat (ITR) sequences (e.g., two ITR sequences) or a portion thereof (e.g., two A elements, B elements, C elements, or D elements), or long end repeat (LTR) sequences (e.g., two LTR sequences)). In some aspects, terminal repeat sequences are at least 10 base pairs (bp) long (e.g., 10 bp to 500 bp, 12 bp to 400 bp, 14 bp to 300 bp, 16 bp to 250 bp, 18 bp to 200 bp, 20 bp to 180 bp, 25 bp to 170 bp, 30 bp to 160 bp, or 50 bp to 150 bp, e.g., 10 bp to 15 bp, 15 bp to 20 bp, 20 bp to 25 bp, 25 bp to 30 bp, 30 bp to 35 bp, 35 bp to 40 bp, 40 bp to 45 bp, 45 bp to 50 bp, 50 bp to 55 bp, 55 bp to 60 bp, 60 bp to 65 bp, 65 bp to 70 bp, bp~80 bp, 80 bp~90 bp, 90 bp~100 bp, 100 bp~150 bp, 150 bp~200 bp, 200 bp~300 bp, 300 bp~400 bp, or 400 bp~500 bp, for example, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39 bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49 bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, 67 bp, 68 bp, 69 bp, 70 bp, 71 bp, 72 bp, 73 bp, 74bp, 75 bp, 76 bp, 77 bp, 78 bp, 79 bp, 80 bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86 bp, 87 bp, 88 bp, 89 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108 bp, 109 bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117 bp, 118 bp, 119 These are 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126 bp, 127 bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp, 133 bp, 134 bp, 135 bp, 136 bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp, 142 bp, 143 bp, 144 bp, 145 bp, 146 bp, 147 bp, 148 bp, 149 bp, 150 bp, or more. In some cases, the DNA vector contains a DD element.

[0020] In another aspect, the present invention is characterized by an isolated linear DNA molecule comprising a plurality of identical amplicons, each of which comprises heterogeneous genes, and the DNA molecule (a) comprises terminal repeat sequences (e.g., any of the aforementioned terminal repeat sequences); and (b) lacks replication origins and / or drug resistance genes.

[0021] In some embodiments, the circular DNA vector further comprises heterogenes (e.g., one or more heterogenes). In some embodiments, one or more heterologous genes are longer than 4.5 kb (for example, one or more heterologous genes together or individually, 4.5 kb to 25 kb, 4.6 kb to 24 kb, 4.7 kb to 23 kb, 4.8 kb to 22 kb, 4.9 kb to 21 kb, 5.0 kb to 20 kb, 5.5 kb to 18 kb, 6.0 kb to 17 kb, 6.5 kb to 16 kb, 7.0 kb to 15 kb, 7.5 kb to 14 kb, 8.0 kb to 13 kb, 8.5 kb to 12.5 kb, 9.0 kb to 12.0 kb, 9.5 kb to 11.5 kb, or 10.0 kb to 11.0 kb, for example, 4.5 kb to 8 kb, 8 kb to 10 kb, 10 Kb~15 Kb, 15 Kb~20 Kb long, or longer, such as 4.5 Kb~5.0 Kb, 5.0 Kb~5.5 Kb, 5.5 Kb~6.0 Kb, 6.0 Kb~6.5 Kb, 6.5 Kb~7.0 Kb, 7.0 Kb~7.5 Kb, 7.5 Kb~8.0 Kb, 8.0 Kb~8.5 Kb, 8.5 Kb~9.0 Kb, 9.0 Kb~9.5 Kb, 9.5 Kb~10 Kb, 10 Kb~10.5 Kb, 10.5 Kb~11 Kb, 11 Kb~11.5 Kb, 11.5 Kb~12 Kb, 12 Kb~12.5 Kb, 12.5 Kb~13 Kb, 13 Kb~13.5 Kb, 13.5 Kb~14 Kb, 14 Kb~14.5 Kb, 14.5 Kb~15 Kb, 15 Kb~15.5 Kb, 15.5 Kb~16 Kb, 16 Kb~16.5 Kb, 16.5 Kb~17 Kb, 17 Kb~17.5 Kb, 17.5 Kb~18 Kb, 18 Kb~18.5 Kb, 18.5 Kb~19 Kb, 19 Kb~19.5 Kb, 19.5 Kb~20 Kb, 20 Kb~21 Kb, 21 Kb~22 Kb, 22 Kb~23 Kb, 23 Kb~24 Kb, 24 Kb~25 Kb long, or longer, such as about 4.5 Kb, about 5.0 Kb, about 5.5 Kb, approx. 6.0 Kb, approx. 6.5 Kb, about 7.0 Kb, about 7.5 Kb, about 8.0 Kb, about 8.5 Kb, about 9.0 Kb, about 9.(Lengths of 5 Kb, approximately 10 Kb, approximately 11 Kb, approximately 12 Kb, approximately 13 Kb, approximately 14 Kb, approximately 15 Kb, approximately 16 Kb, approximately 17 Kb, approximately 18 Kb, approximately 19 Kb, approximately 20 Kb, or more).

[0022] In embodiments of circular DNA vectors having two or more heterogeneous genes, the heterogeneous genes may be the same gene or different genes (for example, they may encode peptides that interact functionally (e.g., as part of a signaling pathway) or structurally (e.g., via dimerization, e.g., the heavy and light chains of an antibody or its fragments)).

[0023] In some embodiments, the heterogeneous gene in the circular DNA vector contains one or more trans-splicing molecules.

[0024] In some embodiments, circular DNA vectors are monomeric circular vectors, dimeric circular vectors, trimeric circular vectors, etc. In some embodiments, the DNA vector is a monomeric circular vector. In some embodiments, the circular DNA vector (e.g., monomeric circular vector) is double-stranded. In some embodiments, the circular DNA vector is supercoiled (e.g., monomeric supercoiled).

[0025] In some embodiments, a circular DNA vector includes a promoter sequence upstream of one or more heterogenes. Additionally or alternatively, a circular DNA vector may include a polyadenylation site downstream of one or more heterogenes. Thus, in some embodiments, a circular DNA vector includes the following elements functionally linked from 5' to 3' or 3' to 5': (i) a promoter sequence; (ii) one or more heterogenes; (iii) a polyadenylation site; and (iv) terminal repeat sequences (e.g., one or more terminal repeat sequences (e.g., one or more inverted end repeat (ITR) sequences (e.g., two ITR sequences) or long end repeat (LTR) sequences (e.g., two LTR sequences))).

[0026] In another aspect, the present invention is characterized by a method for generating an isolated circular DNA vector (for example, any of the circular DNA vectors described herein). This method comprises the steps of: (i) providing a sample containing a circular DNA molecule containing an AAV genome (e.g., recombinant AAV (rAAV) genome, e.g., AAV episome) containing heterogeneous genes and terminal repeat sequences (e.g., one or more terminal repeat sequences (e.g., one or more inverted end repeat (ITR) sequences (e.g., two ITR sequences)) or long end repeat (LTR) sequences (e.g., two LTR sequences))); (ii) amplifying the AAV genome using polymerase (e.g., phage polymerase)-mediated rolling circle amplification (e.g., isothermal polymerase (e.g., phage polymerase)-mediated rolling circle amplification) to produce a linear concatemer; (iii) digesting the concatemer using restriction enzymes to produce an AAV genome; and (iv) autoligating the AAV genome to generate an isolated DNA vector containing heterogeneous genes and terminal repeat sequences. In some embodiments, the method further includes the step of purifying the isolated DNA vector into supercoiled DNA by column purification of the isolated DNA vector. The supercoiled DNA may be monomeric supercoiled DNA. In some embodiments, relaxed open-circular DNA may be separated from the supercoiled DNA during column purification and discarded.In some embodiments, the heterogene is any of the heterogenes described in any previous context, for example, retinal dystrophy (e.g., Mendelian hereditary retinal dystrophy, LCA, Stargardt disease, pseudoxanthoma elasticum, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, age-related macular degeneration (AMD), Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2. Heterogenes encoding therapeutic proteins configured to treat retinal dystrophy (selected from the group consisting of Usher syndrome and Wagner syndrome); heterogenes including one or more of the following: ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1; heterogenes encoding antibodies or parts thereof, coagulation factors, growth factors, hormones, interleukins, interferons, anti-apoptotic factors, antitumor factors, cytokines, and antidiabetic factors; and / or heterogenes that are trans-splicing molecules.

[0027] The polymerase may be a thermophilic polymerase or a polymerase with high processing capacity due to GC-rich residues (e.g., compared to a reference polymerase). In some embodiments, the polymerase is a phage polymerase. In some embodiments, the phage polymerase is a Phi29 DNA polymerase.

[0028] In another aspect, the invention provides a method for generating an isolated circular DNA vector, comprising the following steps: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome (e.g., an AAV episome) comprising a heterologous gene and a DD element; (ii) amplifying the AAV genome using a first polymerase-mediated rolling circle amplification (e.g., isothermal polymerase-mediated rolling circle amplification) to generate a first linear concatemer; (iii) digesting the first linear concatemer with a restriction enzyme to generate a first AAV genome; (iv) cloning the first AAV genome into a plasmid vector; (v) identifying a plasmid clone comprising terminal repeats (e.g., one or more terminal repeats (e.g., one or more inverted terminal repeat (ITR) sequences (e.g., two ITR sequences) or long terminal repeat (LTR) sequences (e.g., two LTR sequences))); (vi) digesting the plasmid clone comprising terminal repeats to generate a second AAV genome; (vii) self-ligating the second AAV genome to generate a circular DNA template; (viii) amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification (e.g., isothermal polymerase-mediated rolling circle amplification) to generate a second linear concatemer; (ix) digesting the second linear concatemer with a restriction enzyme to generate a third AAV genome; and (x) self-ligating the third AAV genome to generate an isolated DNA vector comprising a heterologous gene and terminal repeats. In some embodiments, the polymerase used in the method for generating a circular DNA vector is a phage polymerase (e.g., Phi29 DNA polymerase).

[0029] In another aspect, the present invention features an in vitro method for generating a therapeutic circular DNA vector, comprising the following steps: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome (e.g., recombinant AAV (rAAV) genome, e.g., AAV episome) comprising heterogeneous genes and terminal repeat sequences (e.g., one or more terminal repeat sequences (e.g., one or more inverted end repeat (ITR) sequences (e.g., two ITR sequences)) or long end repeat (LTR) sequences (e.g., two LTR sequences))); (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification (e.g., isothermal polymerase-mediated rolling circle amplification) to produce a linear concatemer; (iii) digesting the concatemer using restriction enzymes to produce an AAV genome; and (iv) autoligating the AAV genome to generate an isolated circular DNA vector comprising heterogeneous genes and terminal repeat sequences. In some embodiments, the polymerase is a phage polymerase (e.g., Phi29 DNA polymerase). In some embodiments, the method further includes the step of purifying the supercoiled DNA from the isolated DNA vector by column purification. The supercoiled DNA may be monomeric supercoiled DNA. In some embodiments, relaxed open-circular DNA may be separated from the supercoiled DNA during column purification and discarded.

[0030] In another aspect, pharmaceutical compositions comprising any one or more of the aforementioned circular DNA vectors and a pharmaceutically acceptable carrier are provided herein. In some embodiments, the pharmaceutical composition is non-immunogenic (e.g., substantially lacking bacterial components such as bacterial signatures, such as CpG motifs). In some embodiments, the pharmaceutical composition is substantially lacking viral particles.

[0031] In another aspect, the present invention is characterized by a method for inducing heterologous gene expression (e.g., episomal expression) in a subject requiring such expression, comprising the step of administering a pharmaceutical composition (e.g., a non-immunogenic pharmaceutical composition) comprising one of the aforementioned circular DNA vectors and a pharmaceutically acceptable carrier to the subject.

[0032] In yet another aspect, the present invention is characterized by a method of treatment using the circular DNA vectors and compositions described herein (e.g., either of the circular DNA vectors or compositions of the aforementioned aspects). The present invention includes a method of treating a disorder in a subject (e.g., an eye disorder, e.g., retinal dystrophy, e.g., Mendelian hereditary retinal dystrophy), comprising the step of administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions of the aforementioned aspects. In some embodiments, the pharmaceutical composition is administered repeatedly (e.g., about twice a day, about once a day, about five times a week, about four times a week, about three times a week, about twice a week, about once a week, about twice a month, about once a month, about once every six weeks, about once every two months, about once every three months, about once every four months, about twice a year, about once a year, or less).

[0033] In some embodiments, the pharmaceutical composition is administered topically (e.g., intraocularly (e.g., intravitreally), intrahepatically, intracerebrally, intramuscularly, intradermally, transdermally, or subcutaneously by aerosolization). In some embodiments, the subject is being treated for Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome.

[0034] In another aspect, the present invention features a nonviral isolated DNA vector that replicates the in vivo persistence of an rAAV vector by containing a double D (DD) element in a DNA molecule lacking bacterial plasmid DNA. Therefore, the DNA vectors provided herein are non-immunogenic and are not limited to an AAV packaging capacity of approximately 4.5 Kb. The present invention also features a method for generating a DD-containing DNA vector, a pharmaceutical composition comprising a DD-containing DNA vector, and a method for using the vectors described herein, for example, to induce the episomal expression of heterologous genes and to treat diseases associated with defective genes.

[0035] In one aspect, the present invention provides an isolated DNA vector comprising a DD element that lacks a replication origin (e.g., a bacterial replication origin) and / or a drug resistance gene (e.g., as part of a bacterial plasmid). For example, an isolated DNA vector comprising a DD element may lack a replication origin (e.g., a bacterial replication origin). Additionally or alternatively, an isolated DNA vector comprising a DD element may lack a drug resistance gene (e.g., as part of a bacterial plasmid). In some embodiments, an isolated DNA vector comprising a DD element may lack a replication origin (e.g., a bacterial replication origin) and a drug resistance gene (e.g., as part of a bacterial plasmid). In some embodiments, the DNA molecule lacks bacterial plasmid DNA. In some embodiments, the DNA vector lacks an immunogenic bacterial signature (e.g., one or more bacterial-associated CpG motifs, e.g., a non-methylated CpG motif). In some embodiments, the DNA vector lacks an RNA polymerase stop site (e.g., an RNA polymerase II (RNAPII) stop site).

[0036] In another aspect, the present invention is characterized by an isolated DNA vector comprising a DD element and a bacterial replication origin and / or drug resistance gene (e.g., as part of a bacterial plasmid).

[0037] In some aspects of the preceding scenarios, the DNA vector further comprises heterogenes (e.g., one or more heterogenes). In some embodiments, one or more heterologous genes are longer than 4.5 kb (for example, one or more heterologous genes together or individually, 4.5 kb to 25 kb, 4.6 kb to 24 kb, 4.7 kb to 23 kb, 4.8 kb to 22 kb, 4.9 kb to 21 kb, 5.0 kb to 20 kb, 5.5 kb to 18 kb, 6.0 kb to 17 kb, 6.5 kb to 16 kb, 7.0 kb to 15 kb, 7.5 kb to 14 kb, 8.0 kb to 13 kb, 8.5 kb to 12.5 kb, 9.0 kb to 12.0 kb, 9.5 kb to 11.5 kb, or 10.0 kb to 11.0 kb, for example, 4.5 kb to 8 kb, 8 kb to 10 kb, 10 Kb~15 Kb, 15 Kb~20 Kb long, or longer, such as 4.5 Kb~5.0 Kb, 5.0 Kb~5.5 Kb, 5.5 Kb~6.0 Kb, 6.0 Kb~6.5 Kb, 6.5 Kb~7.0 Kb, 7.0 Kb~7.5 Kb, 7.5 Kb~8.0 Kb, 8.0 Kb~8.5 Kb, 8.5 Kb~9.0 Kb, 9.0 Kb~9.5 Kb, 9.5 Kb~10 Kb, 10 Kb~10.5 Kb, 10.5 Kb~11 Kb, 11 Kb~11.5 Kb, 11.5 Kb~12 Kb, 12 Kb~12.5 Kb, 12.5 Kb~13 Kb, 13 Kb~13.5 Kb, 13.5 Kb~14 Kb, 14 Kb~14.5 Kb, 14.5 Kb~15 Kb, 15 Kb~15.5 Kb, 15.5 Kb~16 Kb, 16 Kb~16.5 Kb, 16.5 Kb~17 Kb, 17 Kb~17.5 Kb, 17.5 Kb~18 Kb, 18 Kb~18.5 Kb, 18.5 Kb~19 Kb, 19 Kb~19.5 Kb, 19.5 Kb~20 Kb, 20 Kb~21 Kb, 21 Kb~22 Kb, 22 Kb~23 Kb, 23 Kb~24 Kb, 24 Kb~25 Kb long, or longer, such as about 4.5 Kb, about 5.0 Kb, about 5.5 Kb, approx. 6.0 Kb, approx. 6.5 Kb, about 7.0 Kb, about 7.5 Kb, about 8.0 Kb, about 8.(Lengths of 5 Kb, approximately 9.0 Kb, approximately 9.5 Kb, approximately 10 Kb, approximately 11 Kb, approximately 12 Kb, approximately 13 Kb, approximately 14 Kb, approximately 15 Kb, approximately 16 Kb, approximately 17 Kb, approximately 18 Kb, approximately 19 Kb, approximately 20 Kb, or more).

[0038] In embodiments having two or more heterogeneous genes, the heterogeneous genes may be the same gene or different genes (for example, they may encode peptides that interact functionally (e.g., as part of a signaling pathway) or structurally (e.g., via dimerization, e.g., the heavy and light chains of an antibody or a fragment thereof)).

[0039] In some embodiments, heterologous genes include one or more trans-splicing molecules.

[0040] In some embodiments, DNA vectors are circular vectors (e.g., monomeric circular vectors, dimeric circular vectors, trimeric circular vectors, etc.).

[0041] In some embodiments, the DNA vector includes a promoter sequence upstream of one or more heterogenes. Additionally or alternatively, the DNA vector may include a polyadenylation site downstream of one or more heterogenes. Thus, in some embodiments, the DNA vector includes the following elements functionally linked from 5' to 3' or 3' to 5': (i) a promoter sequence; (ii) one or more heterogenes; (iii) a polyadenylation site; and (iv) a DD element.

[0042] In another aspect, the present invention features a method for generating an isolated DNA vector (e.g., any of the DNA vectors described herein), comprising the following steps: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome (e.g., recombinant AAV (rAAV) genome, e.g., AAV episome) comprising heterogeneous genes and DD elements; (ii) amplifying the AAV genome using polymerase (e.g., phage polymerase)-mediated rolling circle amplification (e.g., isothermal polymerase (e.g., phage polymerase)-mediated rolling circle amplification) to produce a linear concatemer; (iii) digesting the concatemer using restriction enzymes to produce an AAV genome; and (iv) autoligating the AAV genome to generate an isolated DNA vector comprising heterogeneous genes and DD elements. The polymerase may be a thermophilic polymerase or a polymerase having high processing capacity due to GC-rich residues (e.g., compared to a reference polymerase). In some embodiments, the polymerase is a phage polymerase. In some embodiments, phage polymerase is Phi29 DNA polymerase.

[0043] In another aspect, the present invention provides a method for generating an isolated DNA vector, comprising the following steps: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome (e.g., an AAV episome) comprising heterogeneous genes and DD elements; (ii) amplifying the AAV genome using a first polymerase-mediated rolling circle amplification (e.g., isothermal polymerase-mediated rolling circle amplification) to produce a first linear concatemer; (iii) digesting the first linear concatemer using restriction enzymes to produce a first AAV genome; (iv) cloning the first AAV genome into a plasmid vector; (v) identifying a plasmid clone containing DD elements; (vi) digesting the plasmid clone containing DD elements to produce a second AAV genome; (vii) autoligating the second AAV genome to generate a circular DNA template; (viii) (ix) amplified a circular DNA template using a second polymerase-mediated rolling circle amplification (e.g., isothermal polymerase-mediated rolling circle amplification) to produce a second linear concatemer; (ix) digested the second linear concatemer using restriction enzymes to produce a third AAV genome; and (x) autoligated the third AAV genome to generate an isolated DNA vector containing heterologous genes and DD elements. In some embodiments, the polymerase is a phage polymerase (e.g., Phi29 DNA polymerase).

[0044] In another aspect, the present invention features an in vitro method for generating a therapeutic DNA vector, comprising the following steps: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome (e.g., recombinant AAV (rAAV) genome, e.g., AAV episome) comprising heterogeneous genes and DD elements; (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification (e.g., isothermal polymerase-mediated rolling circle amplification) to produce a linear concatemer; (iii) digesting the concatemer using restriction enzymes to produce an AAV genome; and (iv) autoligating the AAV genome to generate an isolated DNA vector comprising heterogeneous genes and DD elements. In some embodiments, the polymerase is a phage polymerase (e.g., Phi29 DNA polymerase).

[0045] In another aspect, a pharmaceutical composition comprising a DNA vector and a pharmaceutically acceptable carrier as described above is provided herein. In some embodiments, the pharmaceutical composition is non-immunogenic (substantially lacking immunogenic components such as bacterial signatures, e.g., CpG motifs). In some embodiments, the pharmaceutical composition is substantially lacking viral particles.

[0046] In another aspect, the present invention features a method for inducing heterologous gene expression (e.g., episomal expression) in a subject requiring such expression, comprising the step of administering to the subject a pharmaceutical composition (e.g., a non-immunogenic pharmaceutical composition) comprising one of the DNA vectors described in the preceding aspects and a pharmaceutically acceptable carrier. In some embodiments, the expression is induced in the liver of the subject. The liver may secrete therapeutic proteins encoded by the heterologous gene (e.g., into the bloodstream).

[0047] In yet another aspect, the present invention is characterized by a method of treatment using the DNA vectors and compositions described herein (e.g., either of the vectors or compositions in the preceding aspects). The present invention includes a method of treating a disorder in a subject (e.g., an eye disorder, e.g., retinal dystrophy, e.g., Mendelian hereditary retinal dystrophy), comprising the step of administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions in the preceding aspects. In some embodiments, the pharmaceutical composition is administered repeatedly (e.g., about twice a day, about once a day, about five times a week, about four times a week, about three times a week, about twice a week, about once a week, about twice a month, about once a month, about once every six weeks, about once every two months, about once every three months, about once every four months, about twice a year, about once a year, or less).

[0048] In some embodiments, the pharmaceutical composition is administered topically (e.g., intraocularly (e.g., intravitreally), intrahepatically, intracerebrally, intramuscularly, intradermally, transdermally, or subcutaneously by aerosolization). In other embodiments, the pharmaceutical composition is administered systemically (e.g., intravenously). In some embodiments, the subject is being treated for Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome. [Brief explanation of the drawing]

[0049] [Figure 1]Figure 1 is a schematic diagram illustrating the formation of AAV2 terminal repeats (in this case, double D (DD) elements). AAV2 inverted terminal repeats (ITRs) are 145 bp long and located at each end of the AAV genome. ITRs contain inverted sequences (denoted A, B, C, and D) that can pair to form hairpin-like structures. A single ITR contains two "A," "B," and "C" regions, as well as a single "D" region. Two ITRs can recombinate to form a DD element, which is 165 bp long and similar to a single ITR, but this time containing two "D" regions. [Figure 2A] Figures 2A–2I are a series of diagrams illustrating exemplary ITR sequences for various AAV serotypes, showing the positions and sequences of the A, B, C, and D elements within the ITR. Figure 2A is a diagram of the AAV1 ITR. Figure 2B is a diagram of the AAV2 ITR. Figure 2C is a diagram of the AAV3 ITR. Figure 2D is a diagram of the AAV4 ITR. Figure 2E is a diagram of the AAV5 ITR. Figure 2F is a diagram of the AAV6 ITR. Figure 2G is a diagram of the AAV7 ITR. Figure 2H is a diagram of a partial AAV8 ITR. Figure 2I is a diagram of a partial AAV9 ITR. [Figure 2B] Please refer to the explanation in Figure 2A. [Figure 2C] Please refer to the explanation in Figure 2A. [Figure 2D] Please refer to the explanation in Figure 2A. [Figure 2E] Please refer to the explanation in Figure 2A. [Figure 2F] Please refer to the explanation in Figure 2A. [Figure 2G] Please refer to the explanation in Figure 2A. [Figure 2H] Please refer to the explanation in Figure 2A. [Figure 2I] Please refer to the explanation in Figure 2A. [Figure 3A]This flowchart shows exemplary steps in the DD vector generation and characterization process described in the examples. The first step is to construct or obtain a viral rAAV vector containing the expression cassette (e.g., heterologous gene) required for downstream function. The virus is infected into cells in vitro to form a circular double-stranded episome containing the DD element. In the second major step, the circular rAAV genome is cloned from the cells and sequenced to confirm the presence of the DD element. This can then be used to construct a plasmid-based template and generate the DD vector in vitro using rolling circle amplification (steps 3 and 4). The final step is to confirm gene expression of the DD vector in vitro before proceeding to in vivo studies. [Figure 3B] This flowchart shows exemplary steps in the process of generating and characterizing the synthetic circular vector described in the examples. The first step is to construct or obtain a viral rAAV vector containing the expression cassette (e.g., heterologous gene) required for downstream function. The virus is infected into cells in vitro to form a circular double-stranded episome with terminal repeat sequences (in this case, DD elements). In the second major step, the circular rAAV genome is cloned from the cells and sequenced to confirm the presence of DD elements. This can then be used to create a plasmid-based template and generate the DD vector in vitro using rolling circle amplification (steps 3 and 4). The final step is to confirm gene expression of the DD vector in vitro before proceeding to in vivo studies. [Figure 4] Figure 4 is a schematic diagram illustrating the process for constructing a circular rAAV genome in vitro. A plasmid containing the rAAV genome of interest is transfected with an additional AAV-producing plasmid (triple transfection) to generate an rAAV virus vector (serotype 2) containing the packaged genome. When the resulting virus infects HEK293T cells, a circular rAAV genome is generated within them. [Figure 5]Figure 5 is a schematic diagram showing the rolling circle amplification reaction for the detection of the rAAV circular genome. Whole cell DNA was digested with a restriction enzyme (in this case, AVrII) that does not cleave within the AAV genome. The DNA was then treated with Plasmid-Safe DNase, which degrades linear fragments but preserves the circular double-stranded DNA. The digested product served as a template for linear rolling circle amplification using random primers and Phi29 DNA polymerase. After amplification of the circular AAV episome, a large linear concatemer array was generated. The linear array was then digested into unit-length monomeric AAV genomes by restriction enzyme digestion with EcoRI, which cleaves the AAV genome at a single point. The unit-length AAV genomes were then cloned into a pBlueScript vector for further sequencing analysis. [Figure 6A]Figures 6A–6J are a series of diagrams illustrating exemplary sequences of various AAV2 terminal repeat sequences (in this case, DD elements). Figure 6A is a diagram of a standard DD element (SEQ ID NO: 9) functionally linked in a 5'→3' configuration, containing a 5'D element, a 5'A element, a 5'C element, a 3'C element, a 5'B element, a 3'B element, a 3'A element, and a 3'D element. Figure 6B is a diagram of a standard DD element (SEQ ID NO: 10) functionally linked in a 5'→3' configuration, containing a 5'D element, a 5'A element, a 5'B element, a 3'B element, a 5'C element, a 3'C element, a 3'A element, and a 3'D element. Figure 6C illustrates a DD element (SEQ ID NO: 11) without a B element, containing 5'D, 5'A, 5'C, 3'C, 3'A, and 3'D elements functionally linked in a 5'→3' configuration. Figure 6D illustrates a DD element (SEQ ID NO: 12) without a C element, containing 5'D, 5'A, 5'B, 3'B, 3'A, and 3'D elements functionally linked in a 5'→3' configuration. Figure 6E illustrates a DD element (SEQ ID NO: 13) without B and C elements, containing 5'D, 5'A, 3'A, and 3'D elements functionally linked in a 5'→3' configuration. Figure 6F illustrates a DD element (SEQ ID NO: 14) functionally linked in a 5'→3' configuration, containing 5'D and 3'D elements, but lacking A, B, and C elements. Figure 6G illustrates a DD element (SEQ ID NO: 15) functionally linked in a 5'→3' configuration, containing a 5'D element, a 5'A element, a 5'C element, a nucleic acid replacing the 3'A element, and a 3'D element. Figure 6H illustrates a DD element (SEQ ID NO: 16) functionally linked in a 5'→3' configuration, containing a 5'D element, a 5'A element, duplicate 5'C and 3'A elements, and a 3'D element.Figure 6I illustrates a DD element (SEQ ID NO: 17) functionally linked in a 5'→3' configuration, containing a 5'D element, a partial 5'A element, a partial 3'A element, and a 3'D element. Figure 6J illustrates a DD element (SEQ ID NO: 18) functionally linked in a 5'→3' configuration, containing a 5'D element, a 5'A element, a partial 3'A element, and a 3'D element. [Figure 6B] Please refer to the explanation in Figure 6A. [Figure 6C] Please refer to the explanation in Figure 6A. [Figure 6D] Please refer to the explanation in Figure 6A. [Figure 6E] Please refer to the explanation in Figure 6A. [Figure 6F] Please refer to the explanation in Figure 6A. [Figure 6G] Please refer to the explanation in Figure 6A. [Figure 6H] Please refer to the explanation in Figure 6A. [Figure 6I] Please refer to the explanation in Figure 6A. [Figure 6J] Please refer to the explanation in Figure 6A. [Figure 7] Figure 7 is a schematic diagram illustrating the preparation of a plasmid-derived circular template. Plasmid TG-18 is first digested with EcoRI to release a linear rAAV genome containing terminal repeat sequences (DD elements; represented as ribbons). The ends of the linear fragments are ligated together to form a double-stranded circular structure. [Figure 8]Figure 8 is a photograph of an agarose gel containing DNA bands at different stages of the template formation process. Lane 1 is a linear DNA fragment released from the pBlueScript vector. This fragment contains the CMV promoter, eGFP cDNA, BGHpA, and terminal repeat sequences (DD elements). Lane 2 is the result of self-ligation of the linear fragment from lane 1. Multiple DNA morphologies are present, including circular and linear DNA of various sizes resulting from the ligation of one or more DNA fragments. Lane 3 shows the DNA remaining after treatment with a plasmid-safe DNase that degrades linear DNA but not circular DNA. [Figure 9] Figure 9 is a schematic diagram illustrating the process for analyzing Phi29 fidelity during amplification of terminal repeat sequences (DD elements). A bacterial circular DD vector serves as a template for linear rolling-circle amplification using random primers and Phi29 DNA polymerase. After amplification of the circular AAV episome, a large linear concatemer array is generated. The linear array is then digested by restriction enzyme digestion to assess the presence of DD elements. The SwaI enzyme cleaves both sides of the DD element, producing 244-bp fragments. The AhdI enzyme cleaves once within the DD element, digesting the concatemer into 2.1 Kb unit-length fragments. [Figure 10] Figure 10 is a photograph of an agarose gel showing the results of Swat digestion of amplified DNA. Digestion of DNA amplified from either 1 ng or 6 ng of TG-18 plasmid template with SwaI yielded 244-bp fragments (lanes 2 and 3, arrows). This is the same size as the fragment released from the original TG-18 plasmid vector (lane 1). Also included is DNA amplified from a plasmid template lacking the DD element (TG-dDD), generated by removing the DD element from TG-18 using SwaI digestion (lanes 4 and 5). [Figure 11]Figure 11 is a photograph of an agarose gel showing AhdI digestion of amplified DNA. AhdI cleaves once within the DD element. Digestion of DNA amplified from either 1 ng or 6 ng of TG-18 plasmid template with AhdI yielded 2.1-kb fragments (lanes 1 and 2, arrows). DNA amplified from a plasmid template lacking the DD element (TG-dDD) is also included (lanes 3 and 4). This DNA should not be digested by AhdI because it does not contain the DD element. [Figure 12A] Figure 12A is a schematic diagram showing the self-ligation of a bacterial plasmid template. A plasmid containing a terminal repeat sequence vector (in this case, a DD element-containing vector) is first digested with EcoRI to release a linear rAAV genome containing terminal repeat sequences (DD elements) represented as ribbons within the gene sequence. The ends of the linear fragments are ligated together to form a double-stranded ring. [Figure 12B] Figure 12B is a photograph of an agarose gel showing DNA at different stages of the template formation process. Lane 1 is a linear DNA fragment released from the pBlueScript vector. This fragment contains the CMV promoter, eGFP cDNA, BGHpA, and DD elements. Lane 2 is the result of self-ligation of the linear fragment from lane 1. Multiple DNA morphologies are present, including circular and linear DNA of various sizes resulting from the ligation of one or more DNA fragments. Lane 3 shows the DNA remaining after treatment with a plasmid-safe DNase that degrades linear DNA but not circular DNA. [Figure 13A] Figure 13A is a schematic diagram illustrating the generation of linear concatemers by Phi29 polymerase. The bacterial templates shown in Figures 11A and 11B functioned as templates for linear RCAs using random primers and Phi29 DNA polymerase. After amplification of circular AAV episomes, a large linear concatemer array was generated. The linear array was then digested into unit-length monomeric AAV genomes by restriction enzyme digestion with EcoRI. [Figure 13B]Figure 13B is a photograph of an agarose gel showing size-fractionated digested DNA. [Figure 14] Figure 14A is a schematic diagram of an in vitro-derived rAAV genome self-ligated from a linear form to a circular product. Figure 14B is a photograph of an agarose gel showing the resulting monomeric circular DNA vector illustrated in Figure 14A. The majority of the DNA is monomeric supercoiled circular DNA. [Figure 15] Figure 15A is a micrograph showing GFP fluorescence in cells transfected with the synthetic vector characterized in Figure 14B. Fluorescence was detected using a Spectramax MiniMax300 imaging cytometer. Figure 15B is a micrograph showing GFP fluorescence in cells transfected with the original plasmid containing the rAAV genome. Fluorescence was detected using a Spectramax MiniMax300 imaging cytometer. [Figure 16] Figure 16 is a Western blot image showing GFP expression in cells transfected with pBS alone (lane 1), an in vitro generated TG-18-derived DD vector (lane 2), an in vitro generated TG-18-derived vector without a DD element (lane 3), a plasmid-derived TG-18-derived DD vector (lane 4), and a plasmid-derived TG-18-derived vector without a DD element (lane 5). A band showing antitubulin staining is shown as a control. [Figure 17] Figure 17 is a schematic diagram illustrating an exemplary process for generating a synthetic DNA vector using rolling circle amplification. This process includes column purification to separate the open circular DNA molecule from the supercoiled DNA monomer. [Modes for carrying out the invention]

[0050] Detailed explanation The present invention features a nonviral DNA vector that provides long-term transduction of quiescent cells (e.g., post-division cells) in a manner similar to that of AAV vectors. The present invention is partly based on the development of an in vitro cell-free system for the synthetic generation of circular AAV-like DNA vectors (e.g., DNA vectors containing terminal repeat sequences such as DD elements) by isothermal rolling circle amplification and ligation-mediated circularization (in contrast to, for example, bacterial expression and site-directed recombination). The methods of the present invention enable scalability and improved manufacturing efficiency in the generation of circular AAV-like DNA vectors. Furthermore, vectors produced by these methods are designed to overcome many of the problems associated with plasmid DNA vectors, e.g., those discussed in Lu et al., Mol. Ther. 2017, 25(5): 1187-98, which is incorporated herein by reference in whole. For example, transcriptional silencing can be reduced or eliminated by eliminating or reducing the presence of bacterial plasmid DNA sequences such as CpG islands and / or RNAPII arrest sites, thereby increasing the persistence of heterologous genes. Furthermore, eliminating the presence of immunogenic components (e.g., bacterial endotoxins, DNA, RNA, or bacterial signatures such as CpG motifs) reduces the risk of stimulating the host immune system. Such advantages are particularly beneficial in the treatment of certain disorders, such as retinal dystrophy (e.g., Mendelian retinal dystrophy).

[0051] Accordingly, the vectors of the present invention include (i) substantially lacking bacterial plasmid DNA sequences (e.g., RNAPII arrest sites, replication origins, and / or resistance genes) and other bacterial signatures (e.g., immunogenic CpG motifs); and / or (ii) synthetic DNA vectors that can be entirely synthesized and amplified in vitro (e.g., replication within bacteria is not required, e.g., bacterial replication origins and bacterial resistance genes are not required). In some embodiments, the vectors include DD elements specific to AAV vectors. The present invention makes it possible to transduce target cells (e.g., retinal cells) with a heterologous DNA vector that behaves like AAV viral DNA (e.g., with low and enhanced transcriptional silencing) without requiring the virus itself.

[0052] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains and by reference to published textbooks, which provide general guidance to those skilled in the art for many of the terms used herein. In the event of any conflict between the definitions set forth herein and the definitions in the referenced publications, the definitions set forth herein shall prevail.

[0053] As used herein, the terms “circular vector” or “circular DNA vector” refer to a circular nucleic acid molecule. Such circular molecules can typically be amplified by rolling circle amplification to form concatemers. Linear double-stranded nucleic acids having strands joined at their ends (e.g., a covalently bonded skeleton by a hairpin loop or other structure) are not considered circular vectors as used herein.

[0054] As used herein, “Mendelian retinal dystrophy” refers to a disorder of the retina that follows a Mendelian inheritance pattern with variable penetrance (i.e., complete or incomplete penetrance). Mendelian retinal dystrophy may result from (a) a single mutation in one allele (as in the case of a dominant disorder) or (b) a single mutation in each allele (as in the case of a recessive disorder). The mutation may be a point mutation, insertion, deletion, or splice variant. Exemplary Mendelian retinal dystrophy includes Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome. Mendelian hereditary retinal dystrophy does not include multifactorial disorders that have multiple genetic associations with the potential to develop a disease, such as age-related macular degeneration (AMD).

[0055] As used herein, the term “terminal repeat sequence” refers to a portion of a nucleic acid molecule having a sequence of nucleotides that is repeated in adjacent parts of the nucleic acid molecule. The sequence may be repeated in the same direction or in opposite directions (e.g., ABCDABCD or ABCDDCBA, respectively). In some embodiments, for example, a terminal repeat sequence may be an inverted terminal repeat sequence (ITR) or a long terminal repeat sequence (LTR), or may be derived from them (e.g., the product of their ligation). A terminal repeat sequence derived from an ITR may have repeated A, B, C, and / or D elements (A, B, C, and D elements are defined by SEQ ID NO: 31-37, shown in Figures 2A-2H). For example, each of Figures 6A-6J is a terminal repeat sequence, and all DD elements (e.g., SEQ ID NO: 9 or 10) are examples of terminal repeat sequences. A terminal repeat sequence may have structures resulting from homologous recombination (e.g., intermolecular or intramolecular homologous recombination).

[0056] The term “inverted terminal repeat” or “ITR” refers to a sequence of nucleic acids present in AAV and / or recombinant AAV (rAAV) that can form a T-shaped palindromic structure necessary to complete the lytic and latent life cycle of AAV, as described in Muzyczka and Berns, Fields Virology 2001, 2: 2327-2359. The terms “double D element” and “DD element” are used interchangeably herein and refer to a type of terminal repeat sequence that has a 5'D element (i.e., a nucleic acid sequence having at least 80% homology (e.g., 80%, 85%, 90%, 95%, or 100% homology) to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 19, 21, 23, 25, 27, 29, 38, and 40) and a 3'D element (i.e., a nucleic acid sequence having at least 80% homology (e.g., 80%, 85%, 90%, 95%, or 100% homology) to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, 20, 22, 24, 26, 28, 30, 39, and 41) on the same strand of nucleic acid. In some embodiments, the 5'D element is 100% homologous to the nucleic acid sequence of SEQ ID NO: 1, and / or the 3'D element is 100% homologous to the nucleic acid sequence of SEQ ID NO: 8. DD elements can be constructed by ligation, as shown in Figure 1, by linking two AAV inverted terminal repeats (ITRs) from the same molecule (intramolecular recombination) or different molecules (intermolecular recombination). Such ligation can occur between ITRs of any AAV serotype, and exemplary structures are shown in Figures 2A–2I. A DD element comprises two D elements on a single nucleic acid strand and may include additional elements such as one or more A, B, and / or C elements, or a portion thereof, functionally linking the 3' end of the 5'D element to the 5' end of the 3'D element. In some embodiments, there are no heterogenes between the 3' end of the 5'D element and the 5' end of the 3'D element. Exemplary DD element arrays derived from AAV2 are shown in Figures 6A to 6J, respectively.DD elements derived from other AAV serotypes (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9) may also be used. Representative 5' and 3'D elements from AAV serotypes 1-7 are provided below.

[0057] (Table 1) Representative 5' and 3'D elements from AAV serotypes 1-7 [Table 1]

[0058] The term “heterogene” refers to a gene that does not exist naturally as part of a viral genome. For example, a heterogene may be a mammalian gene, e.g., a therapeutic gene, e.g., a mammalian gene encoding a therapeutic protein. In some embodiments, a heterogene encodes a protein or a portion thereof that is defective or absent in the target cell and / or subject. In some embodiments, a heterogene comprises one or more exons encoding a protein that is defective or absent in the target cell and / or subject. For example, in some embodiments, a heterogene comprises one or more trans-splicing molecules, e.g., as described in WO 2017 / 087900, which is incorporated herein by reference in whole. In some embodiments, a heterogene comprises therapeutic nucleic acids, such as therapeutic RNA (e.g., microRNA).

[0059] As used herein, “trans-splicing molecule” has three main elements: (a) a binding domain that confers specificity to the trans-splicing molecule by tethering it to its target gene (e.g., pre-mRNA); (b) a splicing domain (e.g., a splicing domain having a 3' or 5' splice site); and (c) a coding sequence configured to be trans-spliced ​​onto a target gene, which can replace one or more exons in the target gene (e.g., one or more mutant exons).

[0060] The term “promoter” refers to a sequence that regulates the transcription of a heterologous gene functionally linked to a promoter. A promoter can provide a sequence sufficient to direct transcription, as well as a recognition site for RNA polymerase and other transcription factors necessary for efficient transcription, thereby directing cell-specific expression. In addition to a sequence sufficient to direct transcription, the promoter sequences of the present invention may also include sequences of other regulatory elements involved in the regulation of transcription (e.g., enhancers, Kozak sequences, and introns). Examples of promoters known in the art and useful in viral vectors described herein include the CMV promoter, the CBA promoter, the smCBA promoter, and promoters derived from immunoglobulin genes, SV40, or other tissue-specific genes. Standard techniques for creating functional promoters by mixing and fitting known regulatory elements are known in the art. “Cut-type promoters” can also be made from promoter fragments or by mixing and fitting fragments of known regulatory elements; for example, the smCBA promoter is a cut-type of the CBA promoter.

[0061] As used herein, a vector or composition (e.g., a pharmaceutical composition comprising the DNA vector of the present invention) is "substantially lacking" an immunogenic component, such as an immunogenic bacterial signature, if the composition does not induce a measurable inflammatory response (e.g., a phenotype associated with Toll-like receptor signaling) at a therapeutically relevant dose. Methods for screening a composition for the presence of an immunogenic component include in vitro and in vivo animal assays according to methods known in the art. In some embodiments, a vector or composition substantially lacking an immunogenic component is non-immunogenic.

[0062] As used herein, the term “non-immunogenic” means that the vector or composition does not induce a measurable inflammatory response (e.g., a phenotype associated with Toll-like receptor signaling) at therapeutically relevant doses. Methods for screening a composition for the presence of immunogenic components include in vitro and in vivo animal assays according to methods known in the art. For example, a suitable in vitro assay for determining whether a vector or composition is non-immunogenic involves culturing human peripheral blood mononuclear cells (PBMCs) or bone marrow cells (e.g., monocytes) derived from human PBMCs in the presence of the vector or composition and measuring the amounts of IL-1β, IL-6, and / or IL-12 in the culture after 8 hours. If the concentrations of IL-1β, IL-6, and / or IL-12 do not increase in the sample containing the vector or composition compared to a negative control, the vector or composition is non-immunogenic.

[0063] As used herein, “concatemer” typically refers to a nucleic acid molecule containing multiple copies of the same or substantially identical nucleic acid sequence (e.g., subunits) that are linked together in succession.

[0064] As used herein, the term “isolated” means artificially produced. In some embodiments, with respect to DNA vectors, the term “isolated” means (i) amplified in vitro, for example by rolling circle amplification or polymerase chain reaction (PCR); (ii) produced recombinantly by molecular cloning; (iii) purified, for example by restriction endonuclease cleavage and gel electrophoresis fractionation or column chromatography; or (iv) synthesized, for example by chemical synthesis. Isolated DNA vectors are readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector is considered isolated if its 5' and 3' restriction sites are known or polymerase chain reaction (PCR) primer sequences are disclosed, but the nucleic acid sequence present in its native host in its native state is not considered isolated. Isolated DNA vectors may, but do not need to be substantially purified.

[0065] As used herein, “vector” refers to a nucleic acid molecule capable of delivering heterologous genes into target cells, where the heterologous genes can subsequently be replicated, processed, and / or expressed. After the target cell or host cell has processed the vector’s genome (e.g., by creating DD elements), that genome is no longer considered a vector.

[0066] As used herein, “target cell” refers to any cell that expresses a target gene and is infected with or intended to be infected by a vector. A vector can infect target cells present in a subject (in situ) or target cells in culture. In some embodiments, the target cells of the present invention are post-divided cells. Target cells include both vertebrate and invertebrate cells (as well as cell lines of animal origin). Representative examples of vertebrate cells include mammalian cells, e.g., human, rodent (e.g., rat and mouse), and ungulate (e.g., cattle, goat, sheep, and pig). Target cells include ocular cells such as retinal cells. Alternatively, target cells may be stem cells (e.g., pluripotent cells (i.e., cells whose offspring can differentiate into several limited cell types, e.g., hematopoietic stem cells or other stem cells) or totipotent cells (i.e., cells whose offspring can become any cell type in an organism, e.g., embryonic stem cells, and somatic stem cells, e.g., hematopoietic cells)). In yet another embodiment, target cells include oocytes, eggs, embryonic cells, zygotes, spermatids, and somatic (non-stem) mature cells derived from various organs or tissues, such as hepatocytes, nerve cells, muscle cells, and blood cells (e.g., lymphocytes).

[0067] "Host cell" refers to any cell that harbors the DNA vector of interest. A host cell can be used as a recipient of a DNA vector as described herein. The term includes offspring of the transfected original cell. Thus, as used herein, "host cell" may refer to a cell that has been transfected with a heterologous gene (for example, by the DNA vector described herein). It is understood that offspring of a single parent cell may not necessarily be completely identical in morphology or genomic or total DNA complement to the original parent due to natural, accidental, or intentional mutations.

[0068] As used herein, the term “Subject” includes any mammal that requires any treatment or preventive method described herein. In some embodiments, the subject is human. Other mammals requiring such treatment or preventive method include dogs, cats, or other domesticated animals, horses, livestock, laboratory animals including non-human primates, etc. The subject may be male or female. In one embodiment, the subject has a disease or disorder caused by a mutation in the target gene. In another embodiment, the subject is at risk of developing a disease or disorder caused by a mutation in the target gene. In another embodiment, the subject is showing clinical signs of a disease or disorder caused by a mutation in the target gene. The subject may be of any age for which treatment or preventive therapy may be beneficial. For example, in some embodiments, the subject may be 0–5 years, 5–10 years, 10–20 years, 20–30 years, 30–50 years, 50–70 years, or over 70 years.

[0069] Where used herein, “effective amount” or “effective dose” of a vector or composition refers to an amount sufficient to achieve the desired biological and / or pharmacological effect when delivered to cells or organisms according to, for example, a selected form of administration, route, and / or schedule. As will be recognized by those skilled in the art, the absolute amount of a particular effective vector or composition may vary depending on factors such as the desired biological or pharmacological endpoint, the drug to be delivered, the target tissue, etc. Those skilled in the art will further understand that the “effective amount” may be used in a single dose or in multiple doses to contact cells or administer to a subject.

[0070] As used herein, the term “persistence” refers to the period during which a gene can be expressed within a cell. The persistence of a DNA vector, or the persistence of a heterogene within a DNA vector, can be quantified against a reference vector, such as a control vector produced in bacteria (e.g., a circular vector produced in bacteria or having one or more bacterial signatures not present in the vector of the present invention), using any gene expression characterization method known in the Art. In some embodiments, the control vector lacks a DD element. Additionally or alternatively, persistence can be quantified at any given point in time after administration of the vector. For example, in some embodiments, a heterogene in the DNA vector of the present invention persists for at least six months after administration if its expression is detected in situ six months after administration of the vector. In some embodiments, a gene “persists” within a target cell if its transcription is detectable at three, four, five, six, seven, eight, nine, ten, eleven months, one year, two years, or longer after administration. In some embodiments, a gene is said to persist if, after a given period of time following administration (e.g., 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or more), any detectable percentage of the original expression level (e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, or at least 100% of the original expression level) remains.

[0071] As used herein, “mutation” refers to any abnormal nucleic acid sequence that results in a defective (e.g., non-functional, reduced-function, abnormally functional, or lower-than-normal production) protein product. Mutations include base-pair mutations (e.g., single nucleotide polymorphisms), missense mutations, frameshift mutations, deletions, insertions, and splice mutations.

[0072] As used herein, the terms “mutation-related disorder” or “disorder-related mutation” refer to the correlation between the disorder and the mutation. In some embodiments, a mutation-related disorder is known or suspected to be caused, all or partly, directly or indirectly, by the mutation. For example, an object with a mutation may be at risk of developing a disorder, and this risk may depend additionally on other factors such as other (e.g., independent) mutations (e.g., in the same or different genes) or environmental factors.

[0073] As used herein, the term “treatment” or its grammatical derivatives are defined as reducing the progression of a disease, reducing the severity of disease symptoms, slowing the progression of disease symptoms, eliminating disease symptoms, or delaying the onset of a disease.

[0074] As used herein, the term “prevention” of a disorder, or its grammatical derivatives, is defined as reducing the risk of developing a disease, for example, preventive therapy for a subject at risk of developing a mutation-related disorder. A subject can be characterized as “at risk” of developing a disorder by identifying the mutation associated with the disorder in accordance with any suitable method known in the art or described herein. In some embodiments, a subject at risk of developing a disorder has one or more mutations associated with the disorder. Additionally or alternatively, a subject can be characterized as “at risk” of developing a disorder if the subject has a family history of the disorder.

[0075] As used in the methods described herein, the term “administer” or its grammatical derivatives refers to delivering a composition or cells treated ex vivo to a subject that requires it, for example, a subject having a mutation or defect in a target gene. For example, in one embodiment in which ocular cells are targeted, the method involves delivering the composition by subretinal injection into photoreceptor cells or other ocular cells. In another embodiment, intravitreal injection into ocular cells or injection into ocular cells via the eyelid vein may be used. In yet another embodiment, the composition is administered intravenously. Further methods of administration may be selected by those skilled in the art in consideration of this disclosure.

[0076] The term "pharmaceutically acceptable" means safe for administration to mammals, such as humans. In some embodiments, a pharmacopoeia is approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more specifically in humans.

[0077] The term "carrier" refers to a diluent, auxiliary agent, excipient, or medium administered together with the vector or composition of the present invention. For an example of a suitable pharmaceutical carrier, see "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA., 2 nd This is documented in edited, 2005.

[0078] The terms “one (a)” and “one (an)” mean “one or more.” For example, “one gene” is understood to represent one or more such genes. Thus, the terms “one (a)” and “one (an),” “one or more of one (a) (or one (an)),” and “at least one of one (a) (or one (an))” are used interchangeably herein.

[0079] As used herein, the term “approximately” refers to a value within ±10% of the reference value, unless otherwise specified.

[0080] In the event of any inconsistency in definitions among various sources or references, the definitions provided herein shall prevail.

[0081] II. Vectors Synthetic DNA vectors characterized by heterogeneous genes and double D (DD) elements are provided herein. Synthetic DNA vectors having DD elements can persist in cells as episomes (e.g., in quiescent cells such as post-dividing cells) in a manner similar to, for example, AAV vectors. The vectors provided herein may be naked DNA vectors lacking components specific to viral vectors (e.g., viral proteins) and bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG islands or CpG motifs)), or components additionally or otherwise associated with reduced persistence (e.g., CpG islands or CpG motifs).

[0082] Further provided are synthetic circular DNA vectors characterized by heterogeneity and lacking replication origins and / or drug resistance genes, which are referred to herein as circular DNA vectors. The present invention provides circular DNA vectors produced by synthesis.

[0083] Synthetic circular DNA vectors can persist in cells as episomes (e.g., in quiescent cells such as post-dividing cells) in a manner similar to, for example, AAV vectors. The vectors provided herein may be naked DNA vectors lacking components specific to viral vectors (e.g., viral proteins) and bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG motifs)), or components additionally or otherwise associated with reduced persistence (e.g., CpG islands).

[0084] In some embodiments relating to each of the vectors described above, the DNA vector may be persistent in vivo (e.g., circular and non-bacterial (i.e., by in vitro synthesis) are related to the long-term transcription or expression of heterologous genes in the DNA vector). In some embodiments, the persistence of the circular DNA vector is 5% to 50%, 50% to 100%, 1 to 5, or 5 to 10 times higher than a reference vector (e.g., a circular vector produced in bacteria or having one or more bacterial signatures not present in the vectors of the present invention) (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75% higher, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the circular DNA vector of the present invention lasts for 1 to 4 weeks, 1 to 4 months, 4 months to 1 year, 1 to 5 years, 5 to 20 years, or 20 to 50 years (e.g., at least 1 week, at least 2 weeks, at least 1 month, at least 4 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, or at least 50 years). In some embodiments, the DNA vector includes DD elements, which may be associated with increased persistence.

[0085] The DNA vector may be a circular DNA vector. The circular DNA vector may be monomeric, dimeric, trimer, tetramer, pentamer, hexamer, etc. Preferably, the circular DNA vector is monomeric. In another preferred embodiment, the circular DNA vector is a monomeric supercoiled circular DNA molecule. In some embodiments, the DNA vector contains a nick. In some embodiments, the DNA vector is open circular. In some embodiments, the DNA vector is double-stranded circular.

[0086] Additionally or alternatively, DNA vectors may include DD elements. In certain embodiments, a DNA vector (e.g., a circular DNA vector, e.g., a monomeric circular DNA vector) includes (i) a 5'D element, (ii) a heterogene, and (iii) a 3'D element functionally linked in the 5' to 3' direction. In some embodiments, a DNA vector includes (i) a 5'D element, (ii) a promoter, (iii) a heterogene, and (iv) a 3'D element functionally linked in the 5' to 3' direction. In some embodiments, a DNA vector includes (i) a 5'D element, (ii) a promoter, (iii) a heterogene, (iv) a polyadenylation site, and (v) a 3'D element functionally linked in the 5' to 3' direction.

[0087] For example, a DNA vector may contain (i) a 5'A element, (ii) a 5'D element, (iii) a heterogene, (iv) a 3'D element, and (v) a 5'A element functionally linked in the 5' to 3' direction. In some embodiments, a DNA vector contains (i) a 5'A element, (ii) a 5'D element, (iii) a promoter, (iv) a heterogene, (v) a 3'D element, and (vi) a 5'A element in the 5' to 3' direction. In some embodiments, a DNA vector contains (i) a 5'A element, (ii) a 5'D element, (iii) a promoter, (iv) a heterogene, (v) a polyadenylation site, (vi) a 3'D element, and (vii) a 5'A element in the 5' to 3' direction. In some embodiments, the DNA vector comprises (i) a 5'C element, (ii) a 5'A element, (iii) a 5'D element, (iv) a heterogene, (v) a 3'D element, (vi) a 3'A element, and (vii) a 3'B element in the 5' to 3' direction. In some embodiments, the DNA vector comprises (i) a 5'C element, (ii) a 5'A element, (iii) a 5'D element, (iv) a promoter, (v) a heterogene, (vi) a 3'D element, (vii) a 3'A element, and (viii) a 3'B element in the 5' to 3' direction. In some embodiments, the DNA vector comprises (i) a 5'C element, (ii) a 5'A element, (iii) a 5'D element, (iv) a promoter, (v) a heterogene, (vi) a polyadenylation site, (vii) a 3'D element, (viii) a 3'A element, and (ix) a 3'B element in the 5' to 3' direction.

[0088] In some embodiments, the DNA vector includes a DD element having a nucleic acid sequence having at least a 5'D element and a 3'D element on the same nucleic acid (e.g., DNA) strand. For example, in some embodiments, the DNA vector includes (i) a heterogene and (ii) a DD element functionally linked in the 5' to 3' direction. In some embodiments, the DNA vector includes (i) a promoter, (ii) a heterogene, and (iii) a DD element in the 5' to 3' direction. In some embodiments, the DNA vector includes (i) a heterogene, (ii) a polyadenylation site, and (iii) a DD element in the 5' to 3' direction. In some embodiments, the DNA vector includes (i) a promoter, (ii) a heterogene, (iii) a polyadenylation site, and (iv) a DD element in the 5' to 3' direction.

[0089] Terminal repeat sequences In some embodiments of the present invention, the vectors and compositions provided herein include terminal repeat sequences, which may originate from, for example, ITRs, LTRs, or other terminal structures as a result of cyclization. Terminal repeat sequences are at least 10 base pairs (bp) long (e.g., 10 bp-500 bp, 12 bp-400 bp, 14 bp-300 bp, 16 bp-250 bp, 18 bp-200 bp, 20 bp-180 bp, 25 bp-170 bp, 30 bp-160 bp, or 50 bp-150 bp, e.g., 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, 30 bp-35 bp, 35 bp-40 bp, 40 bp-45 bp, 45 bp-50 bp, 50 bp-55 bp, 55 bp-60 bp, 60 bp-65 bp, 65 bp-70 bp, 70 bp~80 bp, 80 bp~90 bp, 90 bp~100 bp, 100 bp~150 bp, 150 bp~200 bp, 200 bp~300 bp, 300 bp~400 bp, or 400 bp~500 bp, for example, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39 bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49 bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, 67 bp, 68 bp, 69 bp, 70 bp, 71 bp, 72 bp, 73 bp, 74 bp, 75 bp, 76 bp, 77 bp, 78 bp, 79 bp, 80 bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86 bp, 87 bp, 88 bp, 89 bp, 90 bp, 91bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108 bp, 109 bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117 bp, 118 bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126 bp, 127 bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp, 133 The values ​​may be 1 bp, 134 bp, 135 bp, 136 bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp, 142 bp, 143 bp, 144 bp, 145 bp, 146 bp, 147 bp, 148 bp, 149 bp, 150 bp, or more.

[0090] In some embodiments of the present invention, the terminal repeat sequence of the synthetic vector may be a DD element (e.g., a DD element derived from and / or containing one or more portions of an ITR). The DD element contains two D elements on a single DNA molecule. In some embodiments, the two D elements are separated by about 125 nucleic acids. The DD elements may be derived from any serotype of AAV, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

[0091] In some embodiments, the DD element includes two D elements directly linked to each other, for example, in the configuration shown in Figure 6F. Thus, in some embodiments, the DD element has the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the DD element is 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 14.

[0092] In another embodiment, the DD element of the present invention has at least one additional element separating the 5'D element from the 3'D element, for example, one or more A elements; one or more B elements; and / or one or more C elements, etc., which may be arranged in any suitable order. For example, in some embodiments, the DD element includes (i) a 5'D element (i.e., a nucleic acid sequence having at least 80% homology (e.g., 80%, 85%, 90%, 95%, or 100% homology) with any one nucleic acid sequence among SEQ ID NO: 1, 19, 21, 23, 25, 27, 29, 38, or 40) functionally linked in a 5'→3' configuration; (ii) one or more internal nucleic acids (e.g., non-heterogeneous nucleic acids); and (iii) a 3'D element (i.e., a nucleic acid sequence having at least 80% homology (e.g., 80%, 85%, 90%, 95%, or 100% homology) with any one nucleic acid sequence among SEQ ID NO: 8, 20, 22, 24, 26, 28, 30, 39, or 41).In some embodiments, one or more nucleic acids of (ii) are 1 to 125 nucleic acids, 2 to 100 nucleic acids, 5 to 80 nucleic acids, or 10 to 50 nucleic acids, for example, 1 to 20 nucleic acids, 20 to 40 nucleic acids, 40 to 60 nucleic acids, 60 to 80 nucleic acids, 80 to 100 nucleic acids, or 100 to 125 nucleic acids, for example, 1 to 5 nucleic acids, 5 to 10 nucleic acids, 10 to 15 nucleic acids, 15 to 20 nucleic acids, 20 to 25 nucleic acids, 25 to 30 nucleic acids, 30 to 35 nucleic acids, 35- 40 nucleic acids, 40-45 nucleic acids, 45-50 nucleic acids, 50-55 nucleic acids, 55-60 nucleic acids, 60-65 nucleic acids, 65-70 nucleic acids, 70-75 nucleic acids, 75-80 nucleic acids, 80-85 nucleic acids, 85-90 nucleic acids, 90-95 nucleic acids, 95-100 nucleic acids, 100-105 nucleic acids, 105-110 nucleic acids, 110-115 nucleic acids, 115-120 nucleic acids, 120-125 nucleic acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, These are 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 nucleic acids.

[0093] In some embodiments, a DD element includes two A elements (e.g., a 5'A element (e.g., SEQ ID NO: 2) and a 3'A element (e.g., SEQ ID NO: 7)), two B elements (e.g., a 5'B element (e.g., SEQ ID NO: 5) and a 3'B element (e.g., SEQ ID NO: 6)), and two C elements, in addition to two D elements (e.g., a 5'D element (e.g., SEQ ID NO: 1, 19, 21, 23, 25, 27, 29, 38, or 40) and a 3'D element (e.g., SEQ ID NO: 8, 20, 22, 24, 26, 28, 30, 39, or 41)), e.g., SEQ ID NO: 1-8. The nucleic acid sequences of SEQ ID NO: 1-8 may be functionally linked sequentially from 5' to 3', as shown, for example, in Figure 6A. Therefore, in some embodiments, the DD element contains the nucleic acid sequence of SEQ ID NO: 9. Alternatively, SEQ ID NOs 1-8 can be functionally concatenated in any suitable order. For example, in some embodiments, the DD element contains the nucleic acid sequence of SEQ ID NO: 10. In certain embodiments, SEQ ID NOs 1 and 8 (i.e., two D elements) are adjacent to the remaining elements and / or nucleic acids inside the D element.

[0094] The elements with SEQ ID NO: 1-8 may be directly linked to each other or indirectly linked (e.g., functionally linked). For example, SEQ ID NO: 1-8 may be functionally linked in the 5' to 3' direction. Alternatively, as shown in Figures 6A and 6B, there may be one or more nucleic acids separating one or more functionally linked elements. In some ways, the DD element is between the 5'D element and the 3'D element (for example, the following pairs of elements: 5'D element and 5'A element, 5'D element and 5'B element, 5'D element and 3'B element, 5'D element and 5'C element, 5'D element and 3'C element, 5'D element and 3'A element, 5'D element and 3'D element, 5'A element and 5'B element, 5'A element and 3'B element, 5'A element and 5'C element, 5'A element and 3'C element, 5'A element and 3'A element, 5'A element and 3'D element, 5'B element and 3'B element, 5'B element and 5'C element, 5'B element and 3'C element) It includes 1 to 100 additional nucleic acids (e.g., 3 to 50 nucleic acids, e.g., 3 to 10 nucleic acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more additional nucleic acids) arranged between one, two, three, four, five, or more of the following: 5'B element and 3'A element, 5'B element and 3'D element, 3'B element and 5'C element and 5'C element and 5'C element and 5'C element and 3'D element, 3'C element and 3'A element and 3'D element.

[0095] Additional nucleic acids can serve as restriction sites, for example, as shown by the AhdI site in Figures 6A and 6B.

[0096] In some embodiments, one or more of elements A, B, or C (e.g., SEQ ID NO: 2-7) are absent. For example, Figure 6C shows a DD element derived from AAV2 without the B element. Thus, in some embodiments, the DD element of the present invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 11. Similarly, Figure 6D shows a DD element without the C element. Thus, in some embodiments, the DD element of the present invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 12. In some embodiments, the DD element does not include either the B or C element, as shown in Figure 6E. Therefore, in some embodiments, the DD element of the present invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 13.

[0097] Alternatively, one or more of elements A, B, or C (e.g., SEQ ID NO: 2-7) may be replaced with different nucleic acid sequences, as shown in Figure 6G, which illustrates a suitable DD element having a different nucleic acid sequence in place of its 3'A element. Thus, in some embodiments, the DD elements include SEQ ID NO: 1-3 and 8. In some embodiments, the DD elements of the present invention may have nucleic acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 15.

[0098] In some embodiments, one or more (e.g., one, two, three, four, five, six, or more) nucleic acids overlap between two adjacent elements. For example, in some embodiments, the overlapping nucleic acids do not need to be repeated, such as when one or more nucleic acids at the 3' end of a first element match one or more nucleic acids at the 5' end of a second element ligated to its 3' end. An example of such a DD element is shown in Figure 6H, in which case the 3' end of the 5'C element overlaps with the 5' end of the 3'A element. Thus, in some embodiments, the DD elements of the present invention may have nucleic acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 16.

[0099] The nucleic acid sequence between the 5'D element and the 3'D element may be any one or more portions of a 5' or 3'A element, a 5' or 3'B element, or a 5' or 3'C element. In certain embodiments, the DD element includes one or more partial A elements, as shown in Figures 6I and 6J. The partial A element may include a nucleic acid sequence having six or more consecutive matching nucleic acids with SEQ ID NO: 2 or 7 (e.g., 6-40, 8-35, 10-30, or 15-25 consecutive matching nucleic acids, e.g., 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, or 40 consecutive matching nucleic acids). In some embodiments, the DD element of the present invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 17. In some embodiments, the DD element of the present invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 18.

[0100] Exemplary nucleic acid sequences of DD elements and their subelements derived from AAV2 are provided in Table 2 below.

[0101] (Table 2) Exemplary nucleic acid sequences of DD elements and their subelements [Table 2]

[0102] heterogeneity Heterogenes can be inserted into target cells using any of the vectors of the present invention (e.g., DNA vectors having a circular structure including a DD element, or both). As disclosed herein, a wide range of heterogenes can be delivered to target cells by the vectors of the present invention. In some embodiments, the heterogene is configured to transfect target cells having disease-related mutations that can be treated by the expression of a gene encoding a therapeutic protein, such as a protein that is defective or absent in the target cell and / or subject. In such examples, the heterogene may encode all or part of ophthalmoproteins such as CEP290, ABCA4, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, C3, IFT172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, SNRNP200, RP1, MYO7A, PRPF8, VCAN, USH2A, and HMCN1 (e.g., as part of a trans-splicing molecule). Other exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptotic factors, cytokines, anti-diabetic factors, anti-apoptotic agents, coagulation factors, and antitumor factors. Therapeutic proteins may include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropins, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10, viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and / or IL-18.

[0103] As part of the vector of the present invention, other heterologous genes encoding the polypeptide of interest may be included, for example, growth hormone or insulin-like growth factor (IGF) for promoting the growth of transgenic animals, α-antitrypsin, erythropoietin (EPO), factors VIII, IX, X, and XI of the blood coagulation system, LDL receptor, GATA-1, etc. Nucleic acid sequences include, for example, suicide genes encoding apoptosis or apoptosis-related enzymes, and AIF, Apaf (e.g., Apaf-1, Apaf-2, or Apaf-3), APO-2 (L), APO-3 (L), apopine, Bad, Bak, Bax, Bcl-2, Bcl-x.sub.L, Bcl-x.sub.S, bik, CAD, calpain, caspases, for example, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, or granzyme B, ced-3, ced-9, ceramide, c-Jun, c-Myc, CPP32, crm A, Cytochrome c, D4-GDP-DI, Daxx, CdR1, DcR1, DD, DED, DISC, DNA-PK.sub.CS, DR3, DR4, DR5, FADD / MORT-1, FAK, Fas, Fas ligand CD95 / fas (receptor), FLICE / MACH, FLIP, Fodrin, fos, G-actin, Gas-2, Gerzolin, Glucocorticoid / Glucocorticoid receptor, Granzyme A / B, hnRNP This may include genes such as C1 / C2, ICAD, ICE, JNK, lamin A / B, MAP, MCL-1, MdM-2, MEKK-1, MORT-1, NEDD, NF-κB, NuMa, p53, PAK-2, PARP, perforin, PITSLRE, PKC-δ, pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, SREBP, herpes simplex-derived thymidine kinase, TNF-α, TNF-α receptor, TRADD, TRAF2, TRAIL-R1, TRAIL-R2, TRAIL-R3, transglutaminase, U1 70 kDa snRNP, YAMA, etc.

[0104] In some embodiments, heterologous genes encode antibodies, or parts, fragments, or variants thereof. Antibodies include fragments capable of binding to antigens, such as Fv, single-chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single-domain antibodies), and (Fab')2 (including chemically linked F(ab')2). Papain digestion of antibodies yields two identical antigen-binding fragments, each having a single antigen-binding site, referred to as "Fab" fragments, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. Antibodies also include chimeric antibodies and humanized antibodies. Furthermore, for all antibody constructs provided herein, variants having sequences from other organisms are also contemplated. Thus, where human versions of antibodies are disclosed, those skilled in the art will understand how to convert antibodies based on human sequences to sequences from mice, rats, cats, dogs, horses, etc. Antibody fragments also include any of the following orientations: single-stranded scFv, tandem di-scFv, diabody, tandem tri-sdcFv, minibody, etc. In some embodiments, such as when the antibody is scFv, a single polynucleotide of a heterologous gene encodes a single polypeptide containing both a linked heavy chain and a light chain. Antibody fragments also include nanobodies (e.g., sdAb, an antibody having a single monomeric domain such as a pair of heavy chain variable domains and lacking a light chain). Multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies, etc.) are known in the art and are intended as heterologous gene expression products of the present invention.

[0105] In some embodiments, heterologous genes include reporter sequences, which may be useful, for example, for verifying the expression of heterologous genes in specific cells and tissues. Reporter sequences that may be provided to a transgene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. If the reporter sequence is associated with a regulatory element that drives their expression, it provides a signal detectable by conventional means, including enzyme assays, radiometric assays, colorimetric assays, fluorescence assays, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemical tests. For example, if the marker sequence is the LacZ gene, the presence of a vector carrying that signal can be detected by an assay for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the vector carrying that signal can be visually measured by the generation of color or light using a luminometer.

[0106] In some embodiments, heterogenes do not contain coding sequences. The vector may include non-coding sequences such as shRNA, promoters, enhancers, sequences for labeling DNA (e.g., for antibody recognition), PCR amplification sites, sequences defining restriction enzyme sites, site-specific recombinase recognition sites, sequences recognized by nucleic acid-binding and / or nucleic acid-modifying proteins, and linkers. In examples where the heterogene is a trans-splicing molecule, the non-coding sequence includes a binding domain that binds to a target intron.

[0107] In some embodiments, the heterologous gene is 0.1 Kb to 100 Kb in length (e.g., the heterologous gene is 0.2 Kb to 90 Kb, 0.5 Kb to 80 Kb, 1.0 Kb to 70 Kb, 1.5 Kb to 60 Kb, 2.0 Kb to 50 Kb, 2.5 Kb to 45 Kb, 3.0 Kb to 40 Kb, 3.5 Kb to 35 Kb, 4.0 Kb to 30 Kb, 4.5 Kb to 25 Kb, 4.6 Kb to 24 Kb, 4.7 Kb to 23 Kb, 4.8 Kb to 22 Kb, 4.9 Kb to 21 Kb, 5.0 Kb to 20 Kb, 5.5 Kb to 18 Kb, 6.0 Kb to 17 Kb, 6.5 Kb to 16 Kb, 7.0 Kb to 15 Kb, 7.5 Kb to 14 Kb, 8.0 Kb to 13 Kb, 8.5 Kb to 12.5 Kb, 9.0 Kb to 12.0 Kb, 9.5 Kb to 11.5 Kb, or 10.0 Kb to 11.0 Kb in length, e.g., 0.1 Kb to 0.5 Kb, 0.5 Kb to 1.0 Kb, 1.0 Kb to 2.5 Kb, 2.5 Kb to 4.5 Kb, 4.5 Kb to 8 Kb, 8 Kb to 10 Kb, 10 Kb to 15 Kb, 15 Kb to 20 Kb in length, or more, e.g., 0.1 Kb to 0.25 Kb, 0.25 Kb to 0.5 Kb, 0.5 Kb to 1.0 Kb, 1.0 Kb to 1.5 Kb, 1.5 Kb to 2.0 Kb, 2.0 Kb to 2.5 Kb, 2.5 Kb to 3.0 Kb, 3.0 Kb to 3.5 Kb, 3.5 Kb to 4.0 Kb, 4.0 Kb to 4.5 Kb, 4.5 Kb to 5.0 Kb, 5.0 Kb to 5.5 Kb, 5.5 Kb to 6.0 Kb, 6.0 Kb to 6.5 Kb, 6.5 Kb to 7.0 Kb, 7.0 Kb to 7.5 Kb, 7.5 Kb to 8.0 Kb, 8.0 Kb to 8.5 Kb, 8.5 Kb to 9.0 Kb, 9.0 Kb to 9.5 Kb, 9.5 Kb to 10 Kb, 10 Kb to 10.5 Kb, 10.5 Kb to 11 Kb, 11 Kb to 11.5 Kb, 11.5 Kb to 12 Kb, 12 Kb to 12.5 Kb, 12.5 Kb to 13 Kb, 13 Kb to 13.5 Kb, 13.5 Kb to 14 Kb, 14 Kb to 14.5 Kb, 14.5 Kb to 15 Kb, 15 Kb to 15.5 Kb, 15.5 Kb to 16 Kb, 16 Kb to 16.5 Kb, 16.5 Kb~17 Kb, 17 Kb~17.5 Kb, 17.5 Kb~18 Kb, 18 Kb~18.5 Kb, 18.5 Kb~19 Kb, 19 Kb~19.5 Kb, 19.5 Kb~20 Kb, 20 Kb~21 Kb, 21 Kb~22 Kb, 22 Kb~23 Kb, 23 Kb~24 Kb, 24 Kb~25 Kb long, or longer, such as about 4.5 Kb, about 5.0 Kb, about 5.5 Kb, about 6.0 Kb, about 6.5 Kb, about 7.0 Kb, about 7.5 Kb, about 8.0 Kb, about 8.5 Kb, about 9.0 Kb, about 9.5 Kb, about 10 Kb, about 11 Kb, about 12 Kb, about 13 Kb, about 14 (Bb, approximately 15 Kb, approximately 16 Kb, approximately 17 Kb, approximately 18 Kb, approximately 19 Kb, length approximately 20 Kb or more).

[0108] control element In addition to terminal repeat sequences (e.g., DD elements) and heterogenes, the DNA vectors of the present invention (e.g., circular DNA vectors described herein) may include necessary conventional regulatory elements functionally linked to heterogenes in a manner that enables transcription, translation, and / or expression in target cells.

[0109] Expression regulatory sequences include appropriate transcription start, stop, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (poly-A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance the secretion of the encoded product. A variety of expression regulatory sequences, including promoters that are innate, constitutive, inducible, and / or tissue-specific, are known in the art and may be used as part of the present invention. A promoter region is functionally linked to a heterologous gene if the promoter region can influence the transcription of that gene, and as a result, the resulting transcript can be translated into a desired protein or polypeptide. Promoters useful as part of DNA vectors described herein include constitutive and inducible promoters. Examples of constitutive promoters include the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the retroviral Roussarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter.

[0110] Inducible promoters allow for the regulation of gene expression, which can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological conditions, such as the acute phase, a specific differentiation state of cells, or only in replicating cells. Inducible promoters and induction systems are available from various commercial sources. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone-inducible mouse mammary cancer virus promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline repressor system, the tetracycline induction system, the RU486 induction system, and the rapamycin induction system. Further types of inducible promoters that may be useful in this context are those regulated by specific physiological conditions, such as temperature, the acute phase, a specific differentiation state of cells, or only in replicating cells.

[0111] In another embodiment, a native promoter of a heterologous gene is used. A native promoter is considered preferable when it is desirable for the expression of the heterologous gene to mimic native expression. A native promoter may be used when the expression of the heterologous gene must be regulated temporally or developmentally, in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression regulatory elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.

[0112] With respect to heterogeneous genes encoding proteins, polyadenylation (pA) sequences may be inserted after the heterogeneous gene and before terminal repeat sequences. The heterogeneous genes useful in this disclosure may also preferably include introns located between the promoter / enhancer sequence and the heterogeneous gene. The selection of introns and other common vector elements is conventional, and many such sequences are available.

[0113] The exact nature of the regulatory sequences required for gene expression in host cells may vary depending on the species, tissue, or cell type, but generally, they shall include, as necessary, 5' untranscribed and 5' untranslated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, enhancer elements, and similar elements. In particular, such 5' untranscribed regulatory sequences shall include a promoter region containing a promoter sequence for transcriptional control of functionally linked genes. The regulatory sequences may also optionally include enhancer sequences or upstream activating sequences. The vectors of this disclosure may optionally include a 5' leader sequence or a signal sequence.

[0114] III. Method of Generation Methods for producing synthetic DNA vectors (e.g., circular DNA vectors described herein, and / or DNA vectors having DD elements) are provided herein. In particular, the methods provided herein involve in vitro synthesis (e.g., in the absence of cells) rather than bacterial cell synthesis. In vitro synthesis of DNA vectors (e.g., circular DNA vectors described herein, and / or DNA vectors having DD elements) relies on effective replication using a polymerase such as a phage polymerase (e.g., Phi29 polymerase). In some embodiments, Phi29 polymerase is particularly useful for processing the replication of terminal repeat sequences such as DD elements. The polymerases used herein may be thermophilic polymerases with high processing capacity due to GC-rich residues. In some embodiments, the polymerase used to replicate (e.g., amplify) the DD elements is Phi29 polymerase. Specific methods for producing the DNA vectors of the present invention are described in detail in the following examples.

[0115] Generally, the generation of the DNA vector of the present invention (e.g., the circular DNA vector described herein) can begin with providing a sample having a circular DNA molecule containing an AAV genome (e.g., an rAAV genome) having heterogeneous genes and terminal repeat sequences (e.g., DD elements). For example, the sample may be a lysate or other preparation from cells (e.g., mammalian cells) infected with an AAV vector (e.g., an rAAV vector). Double-stranded circular DNA can be obtained from cells using standard DNA extraction / isolation techniques. In some embodiments, the circular DNA is purified by specifically degrading linear DNA using, for example, a plasmid-safe DNase.

[0116] Next, double-stranded circular DNA containing the AAV genome can be amplified in vitro in cell-free preparations by incubation of DNA with a polymerase (e.g., phage polymerase, e.g., Phi29 DNA polymerase; TempliPhi kit, GE Healthcare), primers (e.g., random primers), and a nucleotide mixture (e.g., dNTPs, e.g., dATP, dCTP, dGTP, and dTTP). The polymerase (e.g., phage polymerase, e.g., Phi29 polymerase) amplifies the AAV genome (e.g., AAV genome containing intact terminal repeat sequences, e.g., DD elements) by rolling circle amplification (e.g., isothermal rolling circle amplification) to produce linear concatemers containing multiple AAV genome copies. Suitable polymerases include thermophilic polymerases and polymerases characterized by high processing capacity due to GC-rich residues.

[0117] The resulting concatemer can be digested using restriction enzymes to perform a single cleavage within the genome, thereby constructing a unit-length linear AAV genome containing heterogeneous genes and terminal repeat sequences (e.g., DD elements). Self-ligation of this linear DNA molecule yields the circular synthetic DNA vector of the present invention, containing heterogeneous genes and intact terminal repeat sequences (e.g., DD elements). Alternatively, prior to self-ligation, the linear DNA molecule can be cloned into a plasmid vector according to known techniques and characterized prior to self-ligation to form the final DNA vector (e.g., a circular vector as described herein, and / or a DD-containing DNA vector), as illustrated in the following examples.

[0118] Since genome replication and amplification can be carried out using polymerase under cell-free conditions, synthetic DNA vectors can be isolated from the bacterial components of the plasmid from which they were cloned, and bacterial signatures such as bacterial CpG motifs are not present in the isolated vector.

[0119] IV. Pharmaceutical Compositions Pharmaceutical compositions are provided herein that contain, in a pharmaceutically acceptable carrier, one of the DNA vectors described herein (e.g., synthetic DNA vectors) (e.g., DNA vectors containing DD elements, and / or the circular DNA vectors described herein). The pharmaceutical compositions described herein are substantially free of contaminants such as viral particles, viral capsid proteins, or their peptide fragments. In some embodiments, the pharmaceutical compositions provided herein are non-immunogenic. For example, non-immunogenic pharmaceutical compositions may substantially lack pathogen-associated molecular patterns recognizable by cells of the innate immune system. Such pathogen-associated molecular patterns include CpG motifs (e.g., unmethylated CpG motifs or hypomethylated CpG motifs), endotoxins (e.g., lipopolysaccharides (LPS), e.g., bacterial LPS), flagellins, lipoteichoic acid, peptidoglycans, and viral nucleic acid molecules such as double-stranded RNA.

[0120] The pharmaceutical compositions described herein can be evaluated for contamination by conventional methods and formulated into pharmaceutical compositions intended for appropriate routes of administration. Further compositions containing DNA vectors may be similarly formulated with appropriate carriers. Such formulations involve the use of pharmaceutically and / or physiologically acceptable media or carriers, particularly for administration to target cells. In one embodiment, a carrier suitable for administration to target cells includes buffered saline, isotonic sodium chloride solution, or other buffers to maintain pH at an appropriate physiological level, such as HEPES, and optionally other agents, pharmaceutical substances, stabilizers, buffers, carriers, adjuvants, or diluents.

[0121] In some embodiments, the carrier is an injection solution. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free phosphate-buffered saline. Various such known carriers are provided in U.S. Patent No. 7,629,322, incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is an equilibrium salt solution. In one embodiment, the carrier contains tween. If the vector is to be stored for an extended period, it can be frozen in the presence of glycerol or Tween20.

[0122] In other embodiments, compositions comprising the vectors described herein include a surfactant. Useful surfactants such as Pluronic F68 (also known as Poloxamer 188, LUTROL® F68) may be included to prevent the AAV from adhering to inert surfaces and thus ensure delivery of the desired dose. The carrier is an isotonic sodium chloride solution comprising the surfactant Pluronic F68.

[0123] To introduce the compositions of this disclosure into suitable host cells, delivery media such as liposomes, nanoparticles, microparticles, microspheres, lipid particles, vesicles, and the like can be used. In particular, DNA vectors can be formulated for delivery by encapsulating them in lipid particles, liposomes, vesicles, or nanoparticles. In some embodiments, DNA vectors are compounded with delivery media such as poloxamers and / or polycationic substances.

[0124] A pharmaceutical composition having any of the DNA vectors of the present invention (e.g., circular DNA vectors described herein, and / or DNA vectors containing DD elements) contains 10 μg to 10 mg of DNA (e.g., 25 μg to 5.0 mg, 50 μg to 2.0 mg, or 100 μg to 1.0 mg of DNA, e.g., 10 μg to 20 μg, 20 μg to 30 μg, 30 μg to 40 μg, 40 μg to 50 μg, 50 μg to 75 μg, 75 μg to 100 μg, 100 μg to 200 μg, 200 μg to 300 μg, 300 μg to 400 μg, 400 μg to 500 μg, 500 μg to 1.0 mg, 1.0 mg to 5.0 mg, or 5.0 mg to 10 mg) A unit dose may contain an amount of mg of DNA, for example, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 750 μg, about 1.0 mg, about 2.0 mg, about 2.5 mg, about 5.0 mg, about 7.5 mg, or about 10 mg of DNA.

[0125] In some embodiments, the pharmaceutical composition contains at least about 0.01% by weight of a DNA vector. For example, the pharmaceutical composition may contain 0.01% to 80% by weight of a DNA vector (e.g., 0.05% to 50% by weight, 0.1% to 10% by weight, 0.5% to 5% by weight, or 1% to 2.5% by weight of a DNA vector, e.g., 0.01% to 0.05% by weight, 0.05% to 0.1% by weight, 0.1% to 0.5% by weight, 0.5% to 1.0% by weight, 1.0% to 2% by weight, 2% to 3% by weight, 3% to 5% by weight, 5% to 10% by weight, 10% to 20% by weight, or 20% to 50% by weight of a DNA vector).

[0126] The pharmaceutical compositions of the present invention may contain any of the synthetic circular DNA vectors described herein in monomeric form (for example, monomers with more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 98%, or more than 99%). In some embodiments, 70% to 99.99% of the synthetic circular DNA vector molecules in a pharmaceutical composition are monomers (e.g., 70% to 99.9%, 70% to 99.5%, 70% to 99%, 75% to 99.9%, 75% to 99.5%, 75% to 99%, 80% to 99.9%, 80% to 99.5%, 80% to 99%, 85% to 99.9%, 85% to 99.5%) , 85%~99%, 90%~99.9%, 90%~99.5%, 90%~99%, 95%~99.9%, 95%~99.5%, or 95%~99% are monomers (for example, about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% are monomers in synthetic cyclic DNA vector molecules in pharmaceutical compositions).

[0127] V. How to use This specification provides a method for inducing heterogeneous gene expression (e.g., episomal expression) in a subject requiring it (e.g., as part of a gene therapy regimen) by administering any of the DNA vectors described herein (e.g., circular DNA vectors and / or DNA vectors containing DD elements) or their pharmaceutically acceptable compositions to a subject. Subject cells containing heterogeneous genes can be characterized by assaying the presence of heterogeneous genes contained in the vector by examining the nucleic acid sequence (e.g., RNA sequence, e.g., mRNA sequence) of the host cell, such as by Southern blotting or PCR analysis. Alternatively, heterogeneous gene expression in a subject can be characterized (e.g., quantitatively or qualitatively) by monitoring the progression of a disease associated with a defect or mutation in the target gene corresponding to the heterogeneous gene. In some embodiments, heterogeneous gene expression (e.g., episomal expression) is confirmed by observing a reduction in one or more disease-related symptoms.

[0128] Accordingly, the present invention provides a method for treating a disease in a subject associated with a defect in a target gene (e.g., a gene corresponding to a heterologous gene) by administering any of the DNA vectors described herein (e.g., circular DNA vectors and / or DNA vectors comprising DD elements as described herein) or any of the pharmaceutically active ingredients thereof to the subject. In some embodiments, the disease is an ocular disease. In some embodiments, the subject is treated for Leber congenital amaurosis (LCA, e.g., LCA 10) using a DNA vector having the heterologous CEP290 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, the subject is treated for Stargardt disease using a DNA vector having the heterologous ABCA4 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, the subject is treated for pseudoxanthoma elasticum using a DNA vector having the heterologous ABCC6 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for rod-cone dystrophy (e.g., rod-cone dystrophy 7) using a DNA vector containing a heterologous RIMS1 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for exudative vitreoretinopathy using a DNA vector containing a heterologous LRP5 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for Joubert syndrome using a DNA vector containing a heterologous CC2D2A gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for CSNB-1C using a DNA vector containing a heterologous TRPM1 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for age-related macular degeneration using a DNA vector containing a heterologous C3 gene or a portion thereof (e.g., as part of a trans-splicing molecule).In some embodiments, subjects are treated for retinitis pigmentosa 71 using a DNA vector containing a heterologous IFT172 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for Stickler syndrome (e.g., Stickler syndrome 2) using a DNA vector containing a heterologous COL11A1 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for microcephaly and chorioretinopathy using a DNA vector containing a heterologous TUBGCP6 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for retinitis pigmentosa (e.g., recessive RP) using a DNA vector containing a heterologous KIAA1549 gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for CSNB 2 using a DNA vector containing a heterologous CACNA1F gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for Usher syndrome (e.g., Usher syndrome type 1B) using a DNA vector containing a heterologous MYO7A gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for Wagner syndrome using a DNA vector containing a heterologous VCAN gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for Usher syndrome type 2A using a DNA vector containing a heterologous USH2A gene or a portion thereof (e.g., as part of a trans-splicing molecule). In some embodiments, subjects are treated for AMD 1 using a DNA vector containing a heterologous HMCN1 gene or a portion thereof (e.g., as part of a trans-splicing molecule).

[0129] One of the vectors of the present invention (for example, the circular DNA vectors and / or DNA vectors containing DD elements described herein) is used in a mixture of 10 μg to 10 mg of DNA (for example, 25 μg to 5.0 mg, 50 μg to 2.0 mg, or 100 μg to 1.0 mg of DNA, for example, 10 μg to 20 μg, 20 μg to 30 μg, 30 μg to 40 μg, 40 μg to 50 μg, 50 μg to 75 μg, 75 μg to 100 μg, 100 μg to 200 μg, 200 μg to 300 μg, 300 μg to 400 μg, 400 μg to 500 μg, 500 μg to 1.0 mg, 1.0 mg to 5.0 mg, or 5.0 mg to 10 mg). The target can be administered a dose of DNA in mg (for example, approximately 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 600 μg, 700 μg, 750 μg, approximately 1.0 mg, 2.0 mg, 2.5 mg, 5.0 mg, 7.5 mg, or approximately 10 mg of DNA).

[0130] In some embodiments, administration of the DNA vectors of the present invention (e.g., circular DNA vectors and / or DNA vectors comprising DD elements as described herein) or compositions thereof is non-immunogenic or less likely to induce an immune response in a subject compared to administration of other gene therapy vectors (e.g., plasmid DNA vectors and viral vectors). Methods for evaluating the immunogenicity of the vectors are described above.

[0131] The synthetic DNA vectors provided herein (e.g., circular DNA vectors and / or DNA vectors containing DD elements) are considered suitable for repeated administration due to their ability to infect target cells without inducing an immune response or by inducing a reduced immune response compared to AAV vectors, as described above. Accordingly, the present invention provides a method for repeated administration of the vectors and pharmaceutical compositions described herein. Any of the aforementioned doses can be repeated at appropriate frequencies and durations. In some embodiments, the subject receives doses at frequencies of about twice a day, about once a day, about five times a week, about four times a week, about three times a week, about twice a week, about once a week, about twice a month, about once a month, about once every six weeks, about once every two months, about once every three months, about once every four months, twice a year, once a year, or less. In some embodiments, the number and frequency of doses correspond to the turnover rate of the target cells. It will be understood that in long-lived, postmitted target cells transfected with the vectors described herein, a single dose of the vector may be sufficient to maintain heterologous gene expression in the target cells for a substantial period. Therefore, in other embodiments, the DNA vectors provided herein may be administered to a subject in a single dose. The number of opportunities for heterologous nucleic acids to be delivered to the subject may be required to maintain clinical (e.g., therapeutic) benefits.

[0132] The method of the present invention comprises administering a DNA vector (e.g., a circular DNA vector as described herein, and / or a DNA vector comprising a DD element) or a pharmaceutically acceptable composition thereof via any suitable route. DNA vectors or their pharmaceutical compositions can be administered systemically or locally, for example, intravenously, intraocularly (e.g., intravitreously, subretinally, by eye drops, intraocularly, intraorbitally, intravitreously (e.g., by intravitreal injection), intradermally, intrahepatically, intracerebrally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intrapleurally, intratracheally, intraarachnoidally, intranasally, intravaginally, intrarectally, intratumorally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, orally, locally, transdermally, by inhalation, by aerosolization, by injection (e.g., by jet injection), by electroporation, by implantation, by infusion (e.g., by continuous infusion), by local perfusion directly immersing target cells, by catheter, by lavage, in a cream, or in a lipid composition.

[0133] Additionally or alternatively, the vector can be administered ex vivo to host cells, such as by explanting cells from an individual patient, and then, after selecting the cells incorporating the vector, the host cells can be re-implanted into the patient. Thus, in some aspects, this disclosure provides transfected host cells for treating diseases and a method of administering them.

[0134] The transfection efficiency of any of the vectors described herein can be evaluated using methods known in the art or any method described herein. Isolation of transfected cells can also be performed according to standard techniques. For example, cells containing heterogenes may express visible markers, such as fluorescent proteins (e.g., GFP) or other reporter proteins encoded by the heterogene sequence, which are useful for identifying and isolating one or more cells containing heterogenes. Cells containing heterogenes may also express selectable markers derived from the gene. For example, the survival of cells under certain conditions, such as exposure to cytotoxic substances or deficiency of nutrients or substrates normally required for survival, may depend on the expression or absence of expression of selectable markers. Therefore, the survival or absence of survival of cells under such conditions allows for the identification and isolation of cells or colonies of cells containing heterogenes. Cells containing heterogenes can also be characterized by assaying the presence of heterogenes contained in the vector by examining the host cell nucleic acid sequence (e.g., RNA sequence, e.g., mRNA sequence) by Southern blotting or PCR analysis, etc.

[0135] The following embodiments do not limit the scope of the embodiments described herein. Those skilled in the art will recognize that modifications intended to be covered by the spirit and scope of the invention can be made in the following embodiments. [Examples]

[0136] Recombinant AAV (rAAV) vectors have a proven track record of highly efficient gene delivery in various model systems and are currently being tested as a therapeutic mode for a wide range of human diseases. Studies in animals and humans have shown that rAAV vector genomes persist in vivo primarily as circular episomes. This invention is based on the discovery that such persistence can be reproduced using synthetic techniques to generate circular DNA vectors. Molecular analysis of rAAV episomatic genomes isolated from both animals and humans reveals that these circular genomes contain terminal repeat sequences. In some of the following examples, the terminal repeat sequences identified within the rAAV episomatic genomes contain double D(DD) elements, which are the result of recombination of inverted terminal repeats (ITRs) located at each end of the linear AAV genome, as shown in Figure 1. Because DNA produced within bacteria contains unique bacterial signatures (CpG motifs) that can cause in vivo plasmid and gene expression loss, as well as impurities from the bacteria themselves (endotoxins, bacterial genomic DNA, and RNA), such synthetic DNA vectors can reduce immunogenicity and inflammation in the host compared to vectors produced within bacteria.

[0137] Example 1. Synthesis of a DNA vector having a DD element Stage 1 - Generation of rAAV2-eGFP virus and subsequent cell transduction We obtained the plasmid pAAV-BASIC-EGFP, which contains an AAV2 ITR adjacent to an expression cassette consisting of a CMV enhancer / promoter driving the eGFP protein and a BGHpA signaling pathway (Vector Biolabs, Malvern, PA). This plasmid was used in a triple transfection strategy in HEK293T cells to generate the rAAV2-eGFP viral vector. The other two plasmids used in the triple transfection were the AAV helper plasmids pRep-Cap2 (Part No. 0912; Applied Viromics, Fremont, CA) and pHELP (Part No. 0913; Applied Viromics, Fremont, CA). Cells were transfected using a calcium phosphate kit (Profection Mammalian Transfection System, Part No. TM012; Promega, Madison, WI). 48 hours after transfection, cells were lysed by freeze / thaw and treated with benzonase to produce crude viral lysates. The viral titer in the crude lysate was 5.3 × 10⁶ by qPCR. 12 The number of DNase-resistant particles (DRPs) / mL was determined to be 1 × 10⁶. To construct a circular rAAV genome, rAAV2-eGFP virus was introduced into HEK293T cells at a rate of 1 × 10⁶. 5 Infection was carried out at the multiplicity of infection (MOI). Figure 4 summarizes this process.

[0138] Cloning and characterization of rAAV genomes with step 2-DD elements A summary of the cloning and characterization of rAAV genomes containing DD elements is shown in Figure 5. Infected cells were collected 7 days after infection, and whole-cell DNA was extracted from the cells using the DNeasy blood and tissue kit (Qiagen; Germantown, MD). To remove residual linear rAAV genome, the DNA was treated with a plasmid-safe DNase (Lucigen, Middleton, WI) that specifically degrades the linear DNA while leaving the double-stranded circular rAAV genome intact. The residual circular rAAV genome was amplified using the TEMPLIPHI® kit (Part No. 25640010, GE Healthcare; Pittsburgh, PA). The TEMPLIPHI® kit contains Phi29 polymerase, which uses isothermal rolling circle amplification (RCA) for exponential amplification of circular DNA using bacteriophage Phi29 DNA polymerase. The result of Phi29 amplification is a long linear concatemer of DNA. This DNA is then digested with an enzyme (EcoRI) that performs a single cut within the rAAV genome to generate a unit-length genome, which is then cloned into the pBlueScript II KS+ plasmid (Part No. 212207, Agilent Technologies; Chicago, IL).

[0139] The resulting clone's DD element was sequenced, and clone "TG-18" was identified as having a 165 bp long intact DD element (without deletions or reconstructions). The sequence of clone TG-18 is shown in Figure 6A.

[0140] Step 3 - Preparation of a template for DD vector generation Since the rAAV genome containing the DD element (clone TG-18) was identified, the next step was to generate a circular template for downstream production of the DD vector. Plasmid TG-18 was digested with the restriction enzyme EcoRI to release a linear, unit-length rAAV genome from the plasmid backbone. The linear fragments were then autoligated (rather than ligated with heterologous DNA fragments) to reconstruct the circular rAAV genome. Any linear fragments that did not ligate to form a circular product were removed by plasmid-safe DNase treatment. This process is illustrated in Figure 7.

[0141] Step 4 - Generation of DD vectors in a test tube The circular rAAV genome generated in Step 3 contains a bacterial signature that is derived from bacteria and may have reduced persistence in the host and / or be immunogenic. In Step 4, this circular template is amplified in vitro to produce more rAAV genomes lacking the bacterial signature and contaminants. This is an advantage over conventional gene transfer vectors generated in bacteria. For in vitro generation, the circular template is amplified using the TEMPLIPHI® kit (Part# 25640010, GE Healthcare, Pittsburgh, PA). The TEMPLIPHI® kit contains Phi29 polymerase, which uses isothermal rolling circle amplification (RCA) for exponential amplification of circular DNA using bacteriophage Phi29 DNA polymerase. The result of Phi29 amplification is a long linear concatemer of DNA. The amplified DNA was examined to verify that the DD elements were faithfully replicated by the Phi29 DNA polymerase. The results are shown in Figure 8.

[0142] The amplified DNA was first digested with SwaI, which cleaves both sides of the DD element (Figure 9) and releases a 244 bp long fragment. The SwaI fragment from the amplified DNA was the same size as the SwaI fragment from the original TG-18 pBlueScript plasmid (Figure 10, arrow), demonstrating that Phi29 can amplify the DD element. The integrity of the amplified DD element was further analyzed by digestion with AhdI, which cleaves within the DD element. AhdI cleaves once within the DD vector and digests the concatemer DNA into a 2.1 kb unit-length genome, as demonstrated in Figure 11 (arrow).

[0143] Since it was demonstrated that the DD elements within the DD vector could be faithfully amplified, the next step was to construct the final circular DD vector product. An overview of the production strategy is shown in Figures 12–14. The circular rAAV genome generated in step 3 was amplified using Phi29 polymerase with isothermal RCA for exponential amplification of circular DNA using bacteriophage Phi29 DNA polymerase. The result of Phi29 amplification was a long linear concatemer of DNA (Figure 13A). This DNA was then digested with an enzyme (EcoRI) that cleaves once within the rAAV genome to produce an AAV genome (i.e., a unit-length AAV genome; Figure 13A). This AAV genome was then autoligated to reconstruct the circular rAAV genome (Figure 14A). Any linear fragments that were not ligated to form a circular product were removed by plasmaid-safe DNase treatment.

[0144] Step 5 - Confirmation of gene expression in the DD vector The final step in the in vitro generation process is to confirm that the DD vector is biologically active (i.e., expresses the transgene in cultured cells). A DD-containing DNA vector containing an eGFP expression cassette as a heterologous gene was transfected into HEK293T cells using Lipofectamine2000 (Life Technologies, Carlsbad, CA). After 48 hours, the cells were analyzed for GFP expression by immunofluorescence (Figures 15A and 15B) or Western blotting (Figure 16).

[0145] Example 2. Synthesis of circular DNA vectors A monomeric DNA vector was generated that does not contain bacterial plasmid DNA sequences and is completely synthesized in vitro (without requiring replication within bacteria). Therefore, the synthetic DNA vector can confer transgene DNA that behaves like AAV viral DNA to a given target cell without requiring the virus itself. This strategy offers several advantages over viral vectors. Firstly, it makes it possible to deliver genes that are too large to package in common viral vectors. Furthermore, repeated administration is possible because there are no viral proteins that would trigger an immune response and prevent repeated administration of another viral vector. In addition, the in vitro synthesis process has greater potential for more efficient production compared to other viral vectors.

[0146] Figure 17 shows an exemplary process for constructing a synthetic circular DNA vector. A supercoiled monomeric DNA template was amplified using phi29 polymerase to produce linear concatemer DNA with restriction sites defining boundaries between repeating DNA fragments. The concatemer was digested using a restriction enzyme that cleaves DNA into unit-length fragments. Next, DNA ligase was added to induce self-ligation of the DNA fragments, producing a mixture of DNA structures containing relaxed open-circular DNA monomers and supercoiled DNA monomers. This mixture was column-purified using a thiophilic aromatic adsorption chromatography resin (Plasmidselect Xtra, GE Healthcare 28-4024-01) that has the selectivity to separate the supercoiled covalent closed-ring form of plasmid DNA from the open-ring form. The supercoiled DNA monomers obtained from this purification were recovered and can be used in the methods described herein, or alternatively, may serve as templates for further amplification.

[0147] Example 3. Characterization of in vivo persistence - GFP expression To characterize the degree of persistence of the synthetic circular DNA vector of the present invention, mice were administered three compositions, each containing a different DNA vector: (1) plasmid CAG-GFP (SEQ ID NO: 42) as a negative control for persistence; (2) ΔDD CAG-GFP (a synthetic circular DNA vector lacking a DD element); and (3) DD CAG-GFP (a synthetic circular DNA vector containing a DD element). Each group consisted of a total of 32 mice (8 mice at each time point), and each composition was administered by hydrostatic injection at a dose of 10 μg of DNA per mouse. Eight mice from each group were sacrificed at the following time points: 2 weeks, 4 weeks, 8 weeks, and 16 weeks, and liver tissue was collected and processed at each time point. GFP expression in hepatocytes was quantified according to a known method and compared between groups at each time point. If hepatocytes from mice administered with synthetic cyclic CAG-GFP express higher levels of GFP compared to hepatocytes from mice administered with plasmid CAG-GFP, then synthetic cyclic CAG-GFP is considered to have high persistence.

[0148] Example 4. Characterization of in vivo persistence - mSEAP expression Another study to characterize the degree of persistence of the synthetic circular DNA vector of the present invention involves heterologous expression of mouse secreted alkaline phosphatase (mSEAP), which is not endogenously expressed in mice. In this experiment, mice were administered four compositions, each containing a different DNA vector: (1) plasmid CAG-mSEAP as a negative control for persistence; (2) plasmid CAG-mSEAP-ΔCpG lacking the CpG motif; (3) ΔDD CAG-mSEAP-ΔCpG lacking the DD element and the CpG motif; and (4) DD CAG-mSEAP ΔCpG containing the DD element and lacking the CpG motif. Each group consisted of 12 mice, and each composition was administered by hydrostatic injection at a dose of 20 μg of DNA per mouse. Two mice from each group were sacrificed at the following time points: 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 24 weeks, and 200 μL of blood was collected. The serum concentration of mSEAP was quantified in each sample according to a known method and compared among the groups at each time point.

[0149] By comparing mSEAP concentrations between experimental groups, the effect of CpG motifs and / or DD elements on persistence is quantified. For example, serum mSEAP levels are nearly identical between experimental groups at the initial time point; however, mice administered with a vector having higher persistence show higher mSEAP concentrations at the later time point.

[0150] List of types Some aspects of the technology described herein may be defined according to any of the following numbered items: 1. An isolated DNA vector containing double D (DD) elements, the DNA molecule lacking replication origins and / or drug resistance genes. 2. A DNA vector of item 1 that lacks bacterial plasmid DNA. 3. A DNA vector lacking either an immunogenic bacterial signature and / or an RNA polymerase stop site, either item 1 or 2. 4. An isolated DNA vector containing DD elements and bacterial replication origins and / or drug resistance genes. 5. A DNA vector containing one or more heterologous genes, one of the DNA vectors listed in items 1-4. 6. A DNA vector of item 5 in which the heterologous gene exceeds 4.5 Kb in length. 7. A circular DNA vector, one of the DNA vectors listed in items 1-6. 8. The DNA vector described in item 7, where the circular vector is a monomeric circular vector. 9. A DNA vector containing a promoter sequence upstream of one or more heterologous genes, one of items 6-8. 10. A DNA vector containing one or more polyadenylation sites downstream of one or more heterologous genes, one of the DNA vectors listed in items 6-9. 11. A DNA vector of item 10, in which one or more heterologous genes contain a trans-splicing molecule. 12. A DNA vector of item 10 or 11 in which the following elements: (i) promoter sequence; (ii) one or more heterologous genes; (iii) polyadenylation site; and (iv) DD element, are functionally linked in the 5' to 3' direction. 13. A method for generating an isolated DNA vector, comprising the following steps: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome comprising heterogeneous genes and DD elements; (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) digesting the concatemers using restriction enzymes to produce unit-length linear DNA molecules; and (iv) autoligating the unit-length linear DNA molecules to generate an isolated DNA vector comprising heterogeneous genes and DD elements. 14. A method for generating an isolated DNA vector, comprising the following steps: (i) providing a sample containing a circular DNA molecule containing a heterologous gene and an AAV genome containing a DD element; (ii) amplifying the AAV genome using a first polymerase-mediated rolling circle amplification to produce a first linear concatemer; (iii) digesting the first linear concatemer using restriction enzymes to produce a first unit-length linear DNA molecule; (iv) cloning the first unit-length linear DNA molecule into a plasmid vector; (v) identifying a plasmid clone containing a DD element; (vi) digesting the plasmid clone containing a DD element to produce a second unit-length linear DNA molecule; (vii) autoligating the second unit-length linear DNA molecule to generate a circular DNA template; (viii) amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification to produce a second linear concatemer; (ix) (x) a step of digesting the second linear concatemer using restriction enzymes to produce a third unit-length linear DNA molecule; and (x) a step of autoligating the third unit-length linear DNA molecule to produce an isolated DNA vector containing heterogenes and DD elements. 15. The polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, as per the method of item 13 or 14. 16. The polymerase is Phi29 DNA polymerase, according to one of the methods in items 13-15. 17. An in vitro method for generating a therapeutic DNA vector, comprising the following steps: (i) providing a sample containing a circular DNA molecule containing an AAV genome containing heterogeneous genes and DD elements; (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) digesting the concatemers using restriction enzymes to produce unit-length linear DNA molecules; and (iv) autoligating the unit-length linear DNA molecules to generate a therapeutic DNA vector containing heterogeneous genes and DD elements. 18. Polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, as described in item 17. 19. The method of item 17 or 18, wherein the polymerase is Phi29 DNA polymerase. 20. A pharmaceutical composition comprising one DNA vector from items 1 to 12 and a pharmaceutically acceptable carrier. 21. A non-immunogenic pharmaceutical composition of item 20. 22. A method for inducing the episomal expression of a heterologous gene in a subject requiring such expression, comprising the step of administering to the subject one of items 1 to 11 isolated DNA vectors or a pharmaceutical composition of item 20 or 21. 23. A method for treating a disorder in a subject, comprising the step of administering to the subject a therapeutically effective dose of one isolated DNA vector from item 1 to 12 or a pharmaceutical composition of item 20 or 21. 24. The method of item 22 or 23, wherein an isolated DNA vector or pharmaceutical composition is repeatedly administered. 25. One of the methods described in items 22-24, wherein an isolated DNA vector or pharmaceutical composition is administered topically. 26. The method of item 25, wherein an isolated DNA vector or pharmaceutical composition is administered into the vitreous humor. 27. The disability is an eye disability, one of the methods described in items 22-26. 28. The eye disorder is one of the following, items 22-27: Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome.

[0151] The following numbered additional items further define some aspects of the invention described herein: 1. An isolated circular DNA vector containing one or more heterologous genes, which lacks replication origins and / or drug resistance genes. 2. A DNA vector of item 1 that lacks bacterial plasmid DNA. 3. A DNA vector lacking either an immunogenic bacterial signature and / or an RNA polymerase stop site, either item 1 or 2. 4. A DNA vector from any of items 1-3 that substantially lacks a CpG island. 5. A DNA vector from any of items 1-4, further containing terminal repeat sequences. 6. A DNA vector according to item 5, with terminal repeat sequences of at least 10 bp in length. 7. A DNA vector containing a heterologous gene longer than 4.5 kb, one of the DNA vectors listed in items 1-6. 8. A double-stranded DNA vector, one of items 1-7. 9. The double-stranded DNA vector described in item 8 is a monomer. 10. A DNA vector containing a promoter sequence upstream of one or more heterologous genes, one of the DNA vectors listed in items 1-9. 11. A DNA vector containing one or more polyadenylation sites downstream of one or more heterologous genes, one of items 1-10. 12. A DNA vector containing one or more heterologous genes and a trans-splicing molecule, one of the DNA vectors listed in items 1-11. 13. A DNA vector of item 11 or 12, in which the following elements: (i) promoter sequence; (ii) one or more heterologous genes; (iii) polyadenylation site; and (iv) terminal repeat sequence are functionally linked in the 5' to 3' direction. 14. A method for generating an isolated DNA vector, comprising the following steps: (i) providing a sample containing a circular DNA vector containing an AAV genome containing heterologous genes; (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) digesting the concatemers using restriction enzymes to produce multiple AAV genomes; and (iv) autoligating each of the multiple AAV genomes to generate an isolated DNA vector containing heterologous genes. 15. Method 14 for AAV genomes containing terminal repeat sequences. 16. The method of item 14 or 15, further comprising the step of purifying supercoiled DNA from the isolated DNA vector by column purification of an isolated DNA vector containing heterologous genes. 17. A method for generating an isolated DNA vector, comprising the following steps: (i) providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and terminal repeat sequences; (ii) amplifying the AAV genome using a first polymerase-mediated rolling circle amplification to produce a first linear concatemer; (iii) digesting the first linear concatemer using restriction enzymes to produce a first AAV genome; (iv) cloning the first AAV genome into a plasmid vector; (v) identifying a plasmid clone containing terminal repeat sequences; (vi) digesting the plasmid clone containing terminal repeat sequences to produce a second AAV genome; (vii) autoligating the second AAV genome to generate a circular DNA template; (viii) amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification to produce a second linear concatemer; (ix) (x) a step of digesting the second linear concatemer with restriction enzymes to construct a third AAV genome; and a step of autoligating the third AAV genome to generate an isolated DNA vector containing heterogeneous genes and terminal repeat sequences. 18. Polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, using one of the methods described in items 14-17. 19. The polymerase is Phi29 DNA polymerase, according to one of the methods in items 14-18. 20. An in vitro method for generating a therapeutic DNA vector, comprising the following steps: (i) providing a sample containing a circular DNA vector containing an AAV genome containing heterologous genes; (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) digesting the concatemers using restriction enzymes to produce an AAV genome; and (iv) autoligating the AAV genome to generate a therapeutic DNA vector containing heterologous genes. 21. The method of item 20, further comprising the step of purifying supercoiled DNA from an isolated DNA vector containing heterologous genes by column purification. 22. The polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, according to the method of item 20 or 21. 23. The polymerase is Phi29 DNA polymerase, according to one of the methods in items 20-22. 24. A pharmaceutical composition comprising one DNA vector from items 1 to 13 and a pharmaceutically acceptable carrier. 25. A non-immunogenic pharmaceutical composition of item 24. 26. A method for inducing the episomal expression of a heterologous gene in a subject requiring such expression, comprising the step of administering to the subject one of the isolated DNA vectors from items 1 to 13 or a pharmaceutical composition of item 24 or 25. 27. A method for treating a disorder in a subject, comprising the step of administering to the subject a therapeutically effective dose of one isolated DNA vector from item 1 to 13 or a pharmaceutical composition of item 24 or 25. 28. The method of item 26 or 27, wherein an isolated DNA vector or pharmaceutical composition is repeatedly administered. 29. One of the methods described in items 26-28, wherein an isolated DNA vector or pharmaceutical composition is administered topically. 30. The method of item 29, wherein an isolated DNA vector or pharmaceutical composition is administered into the vitreous humor. 31. The disability is an eye disability, one of the methods from items 26-30. 32. The method of item 31, in which the eye disorder is LCA, Stargardt disease, pseudoxanthoma elasticum, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome. 33. An isolated DNA vector containing a double D (DD) element, which lacks a replication origin and / or a drug resistance gene. 34. A DNA vector of item 33 that lacks bacterial plasmid DNA. 35. A DNA vector lacking an immunogenic bacterial signature and / or an RNA polymerase stop site, either one of items 33 or 34. 36. An isolated DNA vector containing a DD element and bacterial replication origins and / or drug resistance genes. 37. A DNA vector from any one of items 33-36, further containing one or more heterologous genes. 38. DNA vectors for item 36 in which heterologous genes exceed 4.5 Kb in length. 39. A circular DNA vector, one of the DNA vectors listed in items 33-38. 40. The DNA vector in item 39, where the circular vector is a monomeric circular vector. 41. A DNA vector containing a promoter sequence upstream of one or more heterologous genes, one of items 38-40. 42. A DNA vector containing one or more polyadenylation sites downstream of one or more heterologous genes, as specified in items 38-41. 43. A DNA vector of item 42 in which one or more heterologous genes contain a trans-splicing molecule. 44. A DNA vector of item 42 or 43, in which the following elements: (i) promoter sequence; (ii) one or more heterologous genes; (iii) polyadenylation site; and (iv) DD element, are functionally linked in the 5' to 3' direction. 45. A method for generating an isolated DNA vector, comprising the following steps: (i) providing a sample comprising a circular DNA vector comprising an AAV genome comprising heterogeneous genes and DD elements; (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) digesting the concatemers using restriction enzymes to produce multiple AAV genomes; and (iv) autoligating each of the multiple AAV genomes to generate an isolated DNA vector comprising heterogeneous genes and DD elements. 46. ​​A method for generating an isolated DNA vector, comprising the following steps: (i) providing a sample comprising a circular DNA vector comprising an AAV genome containing heterologous genes and DD elements; (ii) amplifying the AAV genome using a first polymerase-mediated rolling circle amplification to produce a first linear concatemer; (iii) digesting the first linear concatemer using restriction enzymes to produce a first AAV genome; (iv) cloning the first AAV genome into a plasmid vector; (v) identifying a plasmid clone containing DD elements; (vi) digesting the plasmid clone containing DD elements to produce a second AAV genome; (vii) autoligating the second AAV genome to generate a circular DNA template; (viii) amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification to produce a second linear concatemer; (ix) (x) a step of digesting the second linear concatemer with restriction enzymes to produce a third AAV genome; and (x) a step of autoligating the third AAV genome to generate an isolated DNA vector containing heterologous genes and DD elements. 47. The polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, as described in item 45 or 46. 48. The polymerase is Phi29 DNA polymerase, according to one of the methods in items 45-47. 49. An in vitro method for generating a therapeutic DNA vector, comprising the following steps: (i) providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and DD elements; (ii) amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce a linear concatemer; (iii) digesting the concatemer using restriction enzymes to produce an AAV genome; and (iv) autoligating the AAV genome to generate a therapeutic DNA vector containing heterogeneous genes and DD elements. 50. Polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, as described in item 49. 51. The method of item 49 or 50, wherein the polymerase is Phi29 DNA polymerase. 52. A pharmaceutical composition comprising one DNA vector from items 33 to 44 and a pharmaceutically acceptable carrier. 53. A non-immunogenic pharmaceutical composition of item 52. 54. A method for inducing the episomal expression of a heterologous gene in a subject requiring such expression, comprising the step of administering to the subject one of the DNA vectors from items 33 to 45 or a pharmaceutical composition of item 52 or 53. 55. A method for treating a disorder in a subject, comprising the step of administering to the subject a therapeutically effective dose of one DNA vector from item 33 to 44 or a pharmaceutical composition of item 52 or 53. 56. The method of item 54 or 55, wherein an isolated DNA vector or pharmaceutical composition is repeatedly administered. 57. One of the methods described in items 54-56, wherein an isolated DNA vector or pharmaceutical composition is administered topically. 58. The method of item 57, wherein an isolated DNA vector or pharmaceutical composition is administered into the vitreous humor. 59. The disability is an eye disability, one of the methods described in items 54-58. 60. The eye disorder is one of the following, items 54-59: Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome.

[0152] The following numbered additional items further define some aspects of the invention described herein: 1. An isolated circular DNA vector comprising one or more heterologous genes encoding a therapeutic protein configured to treat Mendelian retinal dystrophy, wherein the DNA vector lacks replication origins and / or drug resistance genes. 2. A DNA vector for item 1, wherein Mendelian hereditary retinal dystrophy is selected from the group consisting of Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome. 3. A DNA vector of item 1 or 2, wherein one or more heterologous genes are selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1. 4. An isolated circular DNA vector comprising one or more heterologous genes selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1, wherein the DNA vector lacks replication origins and / or drug resistance genes. 5. A DNA vector of item 4, wherein one or more heterologous genes encode a therapeutic protein configured to treat a Mendelian hereditary retinal dystrophy selected from the group consisting of LCA, Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome. 6. An isolated circular DNA vector comprising one or more heterologous genes encoding a therapeutic protein selected from the group consisting of antibodies or a portion thereof, growth factors, interleukins, interferons, anti-apoptotic factors, cytokines, and anti-diabetic factors, wherein the DNA vector lacks replication origins and / or drug resistance genes. 7. An isolated circular DNA vector comprising one or more heterologous genes including a trans-splicing molecule, wherein the DNA vector lacks replication origins and / or drug resistance genes. 8. An isolated circular DNA vector comprising one or more heterologous genes encoding therapeutic proteins secreted from the liver, wherein the DNA vector lacks replication origins and / or drug resistance genes. 9. A DNA vector from item 8 that secretes therapeutic proteins into the bloodstream. 10. A DNA vector containing terminal repeat sequences, one of the DNA vectors listed in items 1-9. 11. A DNA vector of item 10, having a terminal repeat sequence of at least 10 bp in length. 12. An isolated circular DNA vector containing one or more heterologous genes, (a) Contains terminal repeat sequences; and (b) Lacking replication origins and / or drug resistance genes, The DNA vector. 13. A DNA vector lacking bacterial plasmid DNA, one of items 1-12. 14. (a) Immunogenic bacterial signature; and / or (b) RNA polymerase arrest site A DNA vector lacking any of the following: items 1-13. 15. A DNA vector from any of items 1-14 that substantially lacks a CpG island. 16. A DNA vector containing a heterologous gene longer than 4.5 kb, one of the DNA vectors listed in items 1-15. 17. A double-stranded DNA vector, one of the options listed in items 1-15. 18. The DNA vector described in item 17, in which the double-stranded vector is a monomer. 19. A DNA vector containing a promoter sequence upstream of one or more heterologous genes, one of items 1-18. 20. A DNA vector containing one or more polyadenylation sites downstream of one or more heterologous genes, one of the DNA vectors listed in items 1-19. 21. The following elements: (i) Promoter sequence; (ii) One or more heterogeneous genes; (iii) Polyadenylated sites; and (iv) Terminal repeat sequences A DNA vector of item 20, in which the 5' to 3' directions are functionally linked. 22. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises a heterogene encoding a therapeutic protein configured to treat retinal dystrophy, wherein the DNA molecule lacks (a) replication origins and / or drug resistance genes; and (b) recombination sites. 23. Item 22 DNA molecules selected from the group consisting of LCA, Stargardt disease, pseudoxanthoma elasticum, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, age-related macular degeneration (AMD), Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome. 24. A DNA molecule of item 22 or 23 in which one or more heterogenes are selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1. 25. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises a heterogene selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1, wherein the DNA molecule lacks (a) replication start sites and / or drug resistance genes; and (b) recombination sites. 26. A DNA molecule of item 25, wherein heterologous genes encode a therapeutic protein configured to treat Mendelian hereditary retinal dystrophy selected from the group consisting of LCA, Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, AMD, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome. 27. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises heterologous genes encoding antibodies or parts thereof, coagulation factors, growth factors, hormones, interleukins, interferons, anti-apoptotic factors, antitumor factors, cytokines, and antidiabetic factors, wherein the DNA molecule lacks (a) replication origins and / or drug resistance genes; and (b) recombination sites. 28. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises heterogeneous genes including trans-splicing molecules, wherein the DNA molecule lacks (a) replication origins and / or drug resistance genes; and (b) recombination sites. 29. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises a heterogene encoding a therapeutic protein secreted from the liver, and the DNA molecule lacking replication origins and / or drug resistance genes. 30. The therapeutic protein is secreted into the bloodstream from the DNA molecule of item 29. 31. Any one of the DNA molecules from items 22-30, in which each identical amplicon contains a terminal repeat sequence. 32. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises heterogeneous genes, wherein the DNA molecule (a) comprises terminal repeat sequences; and (b) lacks replication origins and / or drug resistance genes. 33. A DNA molecule of item 31 or 32, with a terminal repeat sequence of at least 10 bp in length. 34. One DNA molecule from items 31-33 whose terminal repeat sequence is a DD element. 35. A method for generating an isolated DNA vector, (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterologous genes; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) The step of digesting concatemers using restriction enzymes to produce multiple AAV genomes; and (iv) The step of generating an isolated DNA vector containing heterologous genes by autoligating each of the multiple AAV genomes. Includes, The heterologous gene, (a) Encoding a therapeutic protein configured to treat Mendelian hereditary retinal dystrophy; (b) Selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1; (c) Encoding antibodies or parts thereof, coagulation factors, growth factors, hormones, interleukins, interferons, anti-apoptotic factors, antitumor factors, cytokines, and antidiabetic factors; (d) a trans-splicing molecule; and / or (e) Encoding a therapeutic protein secreted from the liver, The method. 36. Method 35 for AAV genomes containing terminal repeat sequences. 37. The method of item 35 or 36, further comprising the step of purifying supercoiled DNA from an isolated DNA vector containing heterologous genes by column purification. 38. A method for generating an isolated DNA vector, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and terminal repeat sequences; (ii) The step of amplifying the AAV genome using the first polymerase-mediated rolling circle amplification to produce the first linear concatemer; (iii) The first step of digesting the first linear concatemer using restriction enzymes to construct the first AAV genome; (iv) The first AAV genome is cloned into a plasmid vector; (v) The step of identifying plasmid clones containing terminal repeat sequences; (vi) The step of digesting the plasmid clone containing terminal repeat sequences to construct a second AAV genome; (vii) The second AAV genome is autoligated to generate a circular DNA template; (viii) A step of amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification to produce a second linear concatemer; (ix) The step of digesting the second linear concatemer using restriction enzymes to construct a third AAV genome; and (x) A third AAV genome is autoligated to generate an isolated DNA vector containing heterogeneous genes and terminal repeat sequences. 39. Polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, one of the methods described in items 35-38. 40. The polymerase is Phi29 DNA polymerase, according to one of the methods in items 35-39. 41. An in vitro method for generating therapeutic DNA vectors, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterologous genes; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) The step of digesting concatemers using restriction enzymes to construct the AAV genome; and (iv) The step of generating a therapeutic DNA vector containing heterologous genes by autoligating the AAV genome. 42. The method of item 41, further comprising the step of purifying supercoiled DNA from an isolated DNA vector containing heterologous genes by column purification. 43. The polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, as described in item 41 or 42. 44. The polymerase is Phi29 DNA polymerase, according to one of the methods in items 41-43. 45. A pharmaceutical composition comprising one DNA vector from items 1 to 21 and a pharmaceutically acceptable carrier. 46. ​​A non-immunogenic pharmaceutical composition of item 45. 47. A method for inducing the episomal expression of a heterologous gene in a subject requiring such expression, comprising the step of administering to the subject one of the isolated DNA vectors from items 1 to 21 or a pharmaceutical composition of item 45 or 46. 48. A method for treating a disorder in a subject, comprising the step of administering to the subject a therapeutically effective dose of one isolated DNA vector from item 1 to 21 or a pharmaceutical composition of item 43 or 44. 49. The method of item 47 or 48, wherein an isolated DNA vector or pharmaceutical composition is repeatedly administered. 50. One of the methods described in items 47-49, wherein an isolated DNA vector or pharmaceutical composition is administered topically. 51. The method of item 50, wherein an isolated DNA vector or pharmaceutical composition is administered into the vitreous humor. 52. The disability is an eye disability, one of the methods described in items 47-51. 53. If the eye disorder is Mendelian hereditary retinal dystrophy, the method of item 52. 54. The method of item 53, in which the eye disorder is LCA, Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome. 55. An isolated DNA vector comprising a double D (DD) element, wherein the DNA vector lacks a replication origin and / or a drug resistance gene. 56. A DNA vector of item 55 that lacks bacterial plasmid DNA. 57. A DNA vector lacking an immunogenic bacterial signature and / or an RNA polymerase stop site, either one of items 55 or 56. 58. An isolated DNA vector containing a DD element and bacterial replication origins and / or drug resistance genes. 59. A DNA vector containing one or more heterologous genes, one of any of items 55-57. 60. DNA vectors for item 59 in which heterologous genes exceed 4.5 Kb in length. 61. A circular DNA vector, one of items 55-60. 62. The DNA vector in item 61, where the circular vector is a monomeric circular vector. 63. A DNA vector containing a promoter sequence upstream of one or more heterologous genes, as specified in items 60-62. 64. A DNA vector containing one or more polyadenylation sites downstream of one or more heterologous genes, as specified in items 60-63. 65. A DNA vector of item 64 in which one or more heterologous genes contain a trans-splicing molecule. 66. The following elements: (i) Promoter sequence; (ii) One or more heterogeneous genes; (iii) Polyadenylated sites; and (iv) DD element A DNA vector of item 64 or 65, functionally linked in the 5' to 3' direction. 67. A method for generating an isolated DNA vector, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and DD elements; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) The step of digesting concatemers using restriction enzymes to produce multiple AAV genomes; and (iv) The step of generating an isolated DNA vector containing heterogeneous genes and DD elements by autoligating multiple AAV genomes. 68. A method for generating an isolated DNA vector, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and DD elements; (ii) The step of amplifying the AAV genome using the first polymerase-mediated rolling circle amplification to produce the first linear concatemer; (iii) The first step of digesting the first linear concatemer using restriction enzymes to construct the first AAV genome; (iv) The first AAV genome is cloned into a plasmid vector; (v) The step of identifying plasmid clones containing the DD element; (vi) Digesting the plasmid clone containing the DD element to construct a second AAV genome; (vii) The second AAV genome is autoligated to generate a circular DNA template; (viii) A step of amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification to produce a second linear concatemer; (ix) The step of digesting the second linear concatemer using restriction enzymes to construct a third AAV genome; and (x) A third AAV genome is autoligated to generate an isolated DNA vector containing heterologous genes and DD elements. 69. The polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, as described in item 67 or 68. 70. The polymerase is Phi29 DNA polymerase, according to one of the methods in items 67-69. 71. An in vitro method for generating therapeutic DNA vectors, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and DD elements; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) The step of digesting concatemers using restriction enzymes to construct the AAV genome; and (iv) The step of generating a therapeutic DNA vector containing heterogeneous genes and DD elements by autoligating the AAV genome. 72. Polymerase-mediated rolling circle amplification is isothermal rolling circle amplification, as described in item 71. 73. The method of item 71 or 72, wherein the polymerase is Phi29 DNA polymerase. 74. A pharmaceutical composition comprising one DNA vector from items 55-66 and a pharmaceutically acceptable carrier. 75. A non-immunogenic pharmaceutical composition of item 74. 76. A method for inducing the episomal expression of a heterologous gene in a subject requiring such expression, comprising the step of administering to the subject one of the isolated DNA vectors from items 55 to 66 or a pharmaceutical composition of item 74 or 75. 77. A method for treating a disorder in a subject, comprising the step of administering to the subject a therapeutically effective dose of one isolated DNA vector from item 55 to 66 or a pharmaceutical composition of item 74 or 75. 78. The method of item 76 or 77, wherein an isolated DNA vector or pharmaceutical composition is repeatedly administered. 79. Any one of the methods described in items 76-78, wherein an isolated DNA vector or pharmaceutical composition is administered topically. 80. The method of item 79, wherein an isolated DNA vector or pharmaceutical composition is administered into the vitreous humor. 81. The disability is an eye disability, one of the methods from items 76-80. 82. If the eye disorder is Mendelian hereditary retinal dystrophy, the method of item 81. 83. The eye disorder is LCA, Stargardt disease, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome, in any way described in items 76-82.

[0153] Other embodiments All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication or patent application is specifically and individually incorporated by reference.

[0154] While the present invention has been described in relation to its particular aspects, it will be understood that the invention is subject to further modification, and that this application is intended to encompass any changes, uses, or adaptations of the invention, including deviations from the disclosure in accordance with the generally accepted principles of the invention, and that fall within known or customary practices in the art to which the invention pertains, and that can be applied to the essential features described herein, and that can be applied to the essential features described herein.

[0155] Other embodiments are within the scope of the attached claims.

[0156]

Claims

1. An isolated circular DNA vector comprising one or more heterologous genes encoding a therapeutic protein configured to treat Mendelian retinal dystrophy, wherein the DNA vector lacks replication origins and / or drug resistance genes.

2. The DNA vector according to claim 1, wherein the Mendelian hereditary retinal dystrophy is selected from the group consisting of Stargardt disease, Leber congenital amaurosis (LCA), pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome.

3. The DNA vector according to claim 1, wherein one or more heterologous genes are selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1.

4. An isolated circular DNA vector comprising one or more heterologous genes selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1, wherein the DNA vector lacks replication origins and / or drug resistance genes.

5. The DNA vector according to claim 4, wherein one or more heterologous genes encode a therapeutic protein configured to treat a Mendelian hereditary retinal dystrophy selected from the group consisting of Stargardt disease, LCA, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome.

6. An isolated circular DNA vector comprising one or more heterologous genes encoding a therapeutic protein selected from the group consisting of antibodies or a portion thereof, growth factors, interleukins, interferons, anti-apoptotic factors, cytokines, and anti-diabetic factors, wherein the DNA vector lacks replication origins and / or drug resistance genes.

7. An isolated circular DNA vector comprising one or more heterologous genes including a trans-splicing molecule, wherein the DNA vector lacks replication origins and / or drug resistance genes.

8. An isolated circular DNA vector comprising one or more heterologous genes encoding therapeutic proteins secreted from the liver, wherein the DNA vector lacks replication origins and / or drug resistance genes.

9. The DNA vector according to claim 8, wherein a therapeutic protein is secreted into the bloodstream.

10. A DNA vector according to claim 1, comprising a terminal repeat sequence.

11. The DNA vector according to claim 10, wherein the terminal repeat sequence is at least 10 bp long.

12. An isolated circular DNA vector containing one or more heterologous genes, (a) containing terminal repeat sequences; and (b) Lacking replication origins and / or drug resistance genes, The DNA vector.

13. The DNA vector according to claim 1, lacking bacterial plasmid DNA.

14. (a) immunogenic bacterial signature; and / or (b) RNA polymerase arrest site The DNA vector according to claim 1, lacking [a specific element].

15. The DNA vector according to claim 1, substantially lacking CpG islands.

16. The DNA vector according to claim 1, wherein the heterologous gene is longer than 4.5 kb.

17. The DNA vector according to claim 1, which is double-stranded.

18. The DNA vector according to claim 17, wherein the double-stranded vector is a monomer.

19. A DNA vector according to any one of claims 1 to 18, comprising a promoter sequence upstream of one or more heterologous genes.

20. The DNA vector according to claim 1, comprising a polyadenylation site downstream of one or more heterogenes.

21. The following elements: (i) Promoter sequence; (ii) One or more heterogeneous genes; (iii) Polyadenylated sites; and (iv) Terminal repeat sequences The DNA vector according to claim 20, wherein the elements are functionally linked in the 5' to 3' direction.

22. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises a heterogene encoding a therapeutic protein configured to treat retinal dystrophy, wherein the DNA molecule lacks (a) replication origins and / or drug resistance genes; and (b) recombination sites.

23. The DNA molecule according to claim 22, wherein the Mendelian hereditary retinal dystrophy is selected from the group consisting of Stargardt disease, LCA, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, age-related macular degeneration (AMD), Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome.

24. The DNA molecule according to claim 22 or 23, wherein one or more heterologous genes are selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1.

25. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises a heterogene selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1, wherein the DNA molecule lacks (a) replication start sites and / or drug resistance genes; and (b) recombination sites.

26. The DNA molecule according to claim 25, wherein heterologous genes encode a therapeutic protein configured to treat a Mendelian hereditary retinal dystrophy selected from the group consisting of Stargardt disease, LCA, pseudoxanthoma elastica, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, retinitis pigmentosa, AMD, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, and Wagner syndrome.

27. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises heterologous genes encoding antibodies or parts thereof, coagulation factors, growth factors, hormones, interleukins, interferons, anti-apoptotic factors, antitumor factors, cytokines, and antidiabetic factors, wherein the DNA molecule lacks (a) replication origins and / or drug resistance genes; and (b) recombination sites.

28. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises heterogeneous genes including trans-splicing molecules, wherein the DNA molecule lacks (a) replication origins and / or drug resistance genes; and (b) recombination sites.

29. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises a heterogene encoding a therapeutic protein secreted from the liver, and the DNA molecule lacking replication origins and / or drug resistance genes.

30. The DNA molecule according to claim 29, wherein a therapeutic protein is secreted into the bloodstream.

31. A DNA molecule according to any one of claims 22 to 30, wherein each identical amplicon contains a terminal repeat sequence.

32. An isolated linear DNA molecule comprising multiple identical amplicons, each of which comprises heterogeneous genes, wherein the DNA molecule (a) comprises terminal repeat sequences; and (b) lacks replication origins and / or drug resistance genes.

33. The DNA molecule according to claim 31 or 32, wherein the terminal repeat sequence is at least 10 bp long.

34. A DNA molecule according to any one of claims 31 to 33, wherein the terminal repeat sequence is a DD element.

35. A method for generating an isolated DNA vector, (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterologous genes; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) The step of digesting concatemers using restriction enzymes to produce multiple AAV genomes; and (iv) The step of generating an isolated DNA vector containing heterologous genes by autoligating each of the multiple AAV genomes. Includes, The heterologous gene, (a) Encoding a therapeutic protein configured to treat Mendelian hereditary retinal dystrophy; (b) Selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1; (c) Encoding antibodies or parts thereof, coagulation factors, growth factors, hormones, interleukins, interferons, anti-apoptotic factors, antitumor factors, cytokines, and antidiabetic factors; (d) a trans-splicing molecule; and / or (e) Encoding a therapeutic protein secreted from the liver, The method.

36. The method according to claim 35, wherein the AAV genome includes terminal repeat sequences.

37. The method according to claim 35 or 36, further comprising the step of purifying supercoiled DNA from an isolated DNA vector containing heterologous genes by column purification.

38. A method for generating an isolated DNA vector, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and terminal repeat sequences; (ii) The step of amplifying the AAV genome using the first polymerase-mediated rolling circle amplification to produce the first linear concatemer; (iii) The first step of digesting the first linear concatemer using restriction enzymes to construct the first AAV genome; (iv) The first step of cloning the AAV genome into a plasmid vector; (v) The step of identifying plasmid clones containing terminal repeat sequences; (vi) Digesting the plasmid clone containing terminal repeat sequences to construct a second AAV genome; (vii) The second AAV genome is autoligated to generate a circular DNA template; (viii) A step of amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification to produce a second linear concatemer; (ix) the step of digesting the second linear concatemer with restriction enzymes to construct a third AAV genome; and (x) A third AAV genome is autoligated to generate an isolated DNA vector containing heterogeneous genes and terminal repeat sequences.

39. The method according to any one of claims 35 to 38, wherein the polymerase-mediated rolling circle amplification is isothermal rolling circle amplification.

40. The method according to any one of claims 35 to 39, wherein the polymerase is Phi29 DNA polymerase.

41. An in vitro method for generating therapeutic DNA vectors, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterologous genes; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) The step of digesting concatemers using restriction enzymes to construct the AAV genome; and (iv) The step of generating a therapeutic DNA vector containing heterologous genes by autoligating the AAV genome.

42. The method according to claim 41, further comprising the step of purifying supercoiled DNA from an isolated DNA vector containing heterologous genes by column purification.

43. The method according to claim 41 or 42, wherein the polymerase-mediated rolling circle amplification is isothermal rolling circle amplification.

44. The method according to any one of claims 41 to 43, wherein the polymerase is Phi29 DNA polymerase.

45. A pharmaceutical composition comprising a DNA vector according to any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

46. The pharmaceutical composition according to claim 45, which is non-immunogenic.

47. A method for inducing the episomal expression of a heterologous gene in a subject requiring such expression, comprising the step of administering to the subject an isolated DNA vector according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 45 or 46.

48. A method for treating a disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of an isolated DNA vector according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 43 or 44.

49. The method according to claim 47 or 48, wherein an isolated DNA vector or pharmaceutical composition is repeatedly administered.

50. The method according to any one of claims 47 to 49, wherein an isolated DNA vector or pharmaceutical composition is administered topically.

51. The method according to claim 50, wherein an isolated DNA vector or pharmaceutical composition is administered into the vitreous body.

52. The method according to any one of claims 47 to 51, wherein the impairment is an eye impairment.

53. The method according to claim 52, wherein the eye disorder is Mendelian hereditary retinal dystrophy.

54. The method according to claim 53, wherein the eye disorder is LCA, Stargardt disease, pseudoxanthoma elasticum, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome.

55. An isolated DNA vector comprising a double D (DD) element, wherein the DNA vector lacks replication origins and / or drug resistance genes.

56. The DNA vector according to claim 55, lacking bacterial plasmid DNA.

57. A DNA vector according to any one of claims 55 or 56, lacking an immunogenic bacterial signature and / or an RNA polymerase stop site.

58. An isolated DNA vector containing DD elements and bacterial replication origins and / or drug resistance genes.

59. A DNA vector according to any one of claims 55 to 57, further comprising one or more heterogenes.

60. The DNA vector according to claim 59, wherein the heterologous gene is longer than 4.5 kb.

61. A DNA vector according to any one of claims 55 to 60, which is a circular vector.

62. The DNA vector according to claim 61, wherein the circular vector is a monomeric circular vector.

63. A DNA vector according to any one of claims 60 to 62, comprising a promoter sequence upstream of one or more heterologous genes.

64. A DNA vector according to any one of claims 60 to 63, comprising a polyadenylation site downstream of one or more heterologous genes.

65. The DNA vector according to claim 64, wherein one or more heterologous genes include a trans-splicing molecule.

66. The following elements: (i) Promoter sequence; (ii) One or more heterogeneous genes; (iii) Polyadenylated sites; and (iv) DD element The DNA vector according to claim 64 or 65, wherein the elements are functionally linked in the 5' to 3' direction.

67. A method for generating an isolated DNA vector, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and DD elements; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) the step of digesting concatemers using restriction enzymes to produce multiple AAV genomes; and (iv) The step of generating an isolated DNA vector containing heterogeneous genes and DD elements by autoligating multiple AAV genomes.

68. A method for generating an isolated DNA vector, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and DD elements; (ii) The step of amplifying the AAV genome using the first polymerase-mediated rolling circle amplification to produce the first linear concatemer; (iii) The first step of digesting the first linear concatemer using restriction enzymes to construct the first AAV genome; (iv) The first step of cloning the AAV genome into a plasmid vector; (v) The step of identifying plasmid clones containing the DD element; (vi) Digesting the plasmid clone containing the DD element to construct a second AAV genome; (vii) The second AAV genome is autoligated to generate a circular DNA template; (viii) A step of amplifying the circular DNA template using a second polymerase-mediated rolling circle amplification to produce a second linear concatemer; (ix) the step of digesting the second linear concatemer with restriction enzymes to construct a third AAV genome; and (x) A third AAV genome is autoligated to generate an isolated DNA vector containing heterologous genes and DD elements.

69. The method according to claim 67 or 68, wherein the polymerase-mediated rolling circle amplification is isothermal rolling circle amplification.

70. The method according to any one of claims 67 to 69, wherein the polymerase is Phi29 DNA polymerase.

71. An in vitro method for generating therapeutic DNA vectors, including the following steps: (i) A step of providing a sample containing a circular DNA vector containing an AAV genome containing heterogeneous genes and DD elements; (ii) The step of amplifying the AAV genome using polymerase-mediated rolling circle amplification to produce linear concatemers; (iii) the step of digesting concatemers using restriction enzymes to construct the AAV genome; and (iv) The step of generating a therapeutic DNA vector containing heterogeneous genes and DD elements by autoligating the AAV genome.

72. The method according to claim 71, wherein the polymerase-mediated rolling circle amplification is isothermal rolling circle amplification.

73. The method according to claim 71 or 72, wherein the polymerase is Phi29 DNA polymerase.

74. A pharmaceutical composition comprising a DNA vector according to any one of claims 55 to 66 and a pharmaceutically acceptable carrier.

75. The pharmaceutical composition according to claim 74, which is non-immunogenic.

76. A method for inducing the episomal expression of a heterologous gene in a subject requiring such expression, comprising the step of administering to the subject an isolated DNA vector according to any one of claims 55 to 66 or a pharmaceutical composition according to claim 74 or 75.

77. A method for treating a disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of an isolated DNA vector according to any one of claims 55 to 66 or a pharmaceutical composition according to claim 74 or 75.

78. The method according to claim 76 or 77, wherein an isolated DNA vector or pharmaceutical composition is repeatedly administered.

79. The method according to any one of claims 76 to 78, wherein an isolated DNA vector or pharmaceutical composition is administered topically.

80. The method according to claim 79, wherein an isolated DNA vector or pharmaceutical composition is administered into the vitreous humor.

81. The method according to any one of claims 76 to 80, wherein the impairment is an eye impairment.

82. The method according to claim 81, wherein the eye disorder is Mendelian hereditary retinal dystrophy.

83. The method according to any one of claims 76 to 82, wherein the ocular disorder is LCA, Stargardt disease, pseudoxanthoma elasticum, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB-2, Usher syndrome, or Wagner syndrome.

84. The method according to any one of claims 76 to 80, wherein episomal expression is induced in the target liver.

85. The method according to claim 84, wherein the liver secretes a therapeutic protein encoded by a heterologous gene.

86. The method according to claim 85, wherein the liver secretes a therapeutic protein into the bloodstream.

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