WO2019178500A1 - Synthetic dna vectors and methods of use - Google Patents

Synthetic dna vectors and methods of use Download PDF

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Publication number
WO2019178500A1
WO2019178500A1 PCT/US2019/022511 US2019022511W WO2019178500A1 WO 2019178500 A1 WO2019178500 A1 WO 2019178500A1 US 2019022511 W US2019022511 W US 2019022511W WO 2019178500 A1 WO2019178500 A1 WO 2019178500A1
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Prior art keywords
dna vector
dna
vector
isolated
aav genome
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French (fr)
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Bruce C. SCHNEPP
Philip R. Johnson
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Limelight Bio Inc
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Limelight Bio Inc
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Priority to KR1020207028218A priority Critical patent/KR20200132893A/ko
Priority to EP19766752.0A priority patent/EP3765023A4/en
Priority to CA3092088A priority patent/CA3092088A1/en
Priority to JP2020545324A priority patent/JP2021516953A/ja
Priority to CN202311181118.4A priority patent/CN117757842A/zh
Priority to US16/980,914 priority patent/US20210002667A1/en
Priority to MX2020009579A priority patent/MX2020009579A/es
Priority to CN201980019118.XA priority patent/CN111954528A/zh
Application filed by Limelight Bio Inc filed Critical Limelight Bio Inc
Priority to AU2019233901A priority patent/AU2019233901A1/en
Publication of WO2019178500A1 publication Critical patent/WO2019178500A1/en
Priority to IL276923A priority patent/IL276923A/en
Anticipated expiration legal-status Critical
Priority to JP2023222352A priority patent/JP2024041819A/ja
Priority to US18/626,384 priority patent/US20240247284A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention features synthetic DNA vectors.
  • Gene therapy involves transduction of heterologous genes into target cells to correct a genetic defect underlying a disorder in a subject.
  • a variety of transduction approaches have been developed for use in gene therapy over the past several decades.
  • traditional bacterial plasmid DNA vectors represent a versatile tool in gene delivery but can present limitations owing to their bacterial origin.
  • Plasmid DNA vectors include bacterial genes, such as antibiotic resistance genes and origins of replication.
  • plasmid DNA vectors include bacterial signatures, such as CpG motifs.
  • bacterial expression systems for producing plasmid DNA vectors involves 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, e.g., by transcriptional silencing.
  • rAAV vectors Recombinant adeno-associated viral (rAAV) vectors have an established record of high-efficiency gene transfer in a variety of model systems and are now being tested as therapeutic modalities in a wide range of human diseases.
  • Genomes of rAAV vectors can persist in vivo (e.g., in post-mitotic cells) as circular episomes. After infection, single-stranded rAAV DNA is converted to double stranded circular DNA in the cell nucleus and persists in an episomal form for the life of the cell.
  • AAV vector systems can involve additional drawbacks, such as a limited packaging capacity of about 4.5 Kb, immunogenicity of viral proteins, and manufacturing difficulties.
  • non-viral isolated circular DNA vectors that replicate the in vivo persistence of rAAV vectors.
  • the DNA vectors provided herein are non-immunogenic and are not limited to the AAV packaging capacity of about 4.5 Kb.
  • the invention also features methods of producing the circular DNA vector (e.g., in vitro, in the absence of bacterial expression systems), pharmaceutical compositions including the circular DNA vector, and methods of using the vectors described herein, e.g., for inducing persistent episomal expression of a heterologous gene and for treating a disease associated with a defective gene.
  • the invention provides an isolated circular DNA vector including one or more heterologous genes, wherein the DNA vector lacks an origin of replication (e.g., a bacterial origin of replication) and/or a drug-resistance gene (e.g., as part of a bacterial plasmid).
  • an isolated circular DNA vector including one or more heterologous genes may lack an origin of replication (e.g., a bacterial origin of replication).
  • an isolated circular DNA vector including one or more heterologous genes may lack a drug-resistance gene (e.g., as part of a bacterial plasmid).
  • an isolated circular DNA vector including one or more heterologous genes may lack an origin of replication (e.g., a bacterial origin of replication) and a drug-resistance gene (e.g., as part of a bacterial plasmid).
  • the DNA molecule lacks bacterial plasmid DNA.
  • 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).
  • the DNA vector lacks an RNA polymerase arrest site (e.g., an RNA polymerase II (RNAPII) arrest site).
  • RNAPII RNA polymerase II
  • the isolated circular DNA vector includes one or more heterologous genes encoding a therapeutic protein configured to treat a Mendelian-heritable retinal dystrophy (e.g., Leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome).
  • a Mendelian-heritable retinal dystrophy e.g., Leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy,
  • the one or more heterologous genes can be ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT-172, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN,
  • the 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, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USH2A, and HMCN1 , wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • heterologous genes selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT-172, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USH2A, and HMCN1 , wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • the one or more heterologous genes encode a therapeutic protein configured to treat a retinal dystrophy (e.g., a Mendelian-heritable retinal dystrophy, e.g., a retinal dystrophy selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome).
  • a retinal dystrophy e.g., a Mendelian-heritable retinal dystrophy, e.g., a retinal dystrophy selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retin
  • an isolated circular DNA vector having one or more heterologous genes encoding a therapeutic protein (e.g., an antibody or portion thereof, a growth factor, an interleukin, an interferon, an anti-apoptosis factor, a cytokine, or an anti-diabetic factor), wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • a therapeutic protein e.g., an antibody or portion thereof, a growth factor, an interleukin, an interferon, an anti-apoptosis factor, a cytokine, or an anti-diabetic factor
  • the invention provides an isolated circular DNA vector having one or more heterologous genes including a trans-splicing molecule, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • the invention provides an isolated circular DNA vector comprising one or more heterologous genes encoding a liver-secreted therapeutic protein, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • the therapeutic protein is secreted into blood.
  • the invention provides an isolated circular DNA vector comprising one or more heterologous genes, wherein the DNA vector: (a) includes a terminal repeat sequence; and (b) lacks an origin of replication and/or a drug resistance gene.
  • the invention provides an isolated linear DNA molecule having a plurality of identical amplicons, wherein each of the plurality of identical amplicons comprises a heterologous gene encoding a therapeutic protein (e.g., a therapeutic protein configured to treat a retinal dystrophy, e.g., a Mendelian-heritable retinal dystrophy), wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a recombination site.
  • a therapeutic protein e.g., a therapeutic protein configured to treat a retinal dystrophy, e.g., a Mendelian-heritable retinal dystrophy
  • the retinal dystrophy is selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, age related macular degeneration (AMD), stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • the one or more heterologous genes are selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT-172, C3, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USH2A, and HMCN1 .
  • the invention provides an isolated linear DNA molecule having a plurality of identical amplicons, wherein each of the plurality of identical amplicons including a heterologous gene selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT- 172, C3, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USH2A, and HMCN1 , wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a heterologous gene selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT- 172, C3, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USH2A, and HMCN1
  • the a heterologous gene encodes a therapeutic protein configured to treat a retinal dystrophy (e.g., a Mendelian-heritable retinal dystrophy, e.g., LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert
  • a retinal dystrophy e.g., a Mendelian-heritable retinal dystrophy, e.g., LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert
  • CSNB-1 C retinitis pigmentosa
  • AMD stickler syndrome
  • microcephaly and choriorretinopathy retinitis pigmentosa
  • CSNB 2 Usher syndrome, or Wagner syndrome
  • an isolated linear DNA molecule having a plurality of identical amplicons, wherein each of the plurality of identical amplicons includes a heterologous gene encoding antibody or portion thereof, a coagulation factor, a growth factor, a hormone, an interleukin, an interferon, an anti-apoptosis factor, an anti-tumor factor, a cytokine, and an anti-diabetic factor, wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a
  • the invention features an isolated linear DNA molecule having a plurality of identical amplicons, wherein each of the plurality of identical amplicons includes a heterologous gene comprising a trans-splicing molecule, wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a recombination site.
  • the invention provides an isolated linear DNA molecule having a plurality of identical amplicons, wherein each of the plurality of identical amplicons includes a heterologous genes encoding a liver-secreted therapeutic protein (e.g., a therapeutic protein secreted into blood), wherein the DNA molecule lacks an origin of replication and/or a drug resistance gene.
  • a liver-secreted therapeutic protein e.g., a therapeutic protein secreted into blood
  • the DNA molecule lacks an origin of replication and/or a drug resistance gene.
  • the circular DNA vector or linear DNA molecule further includes one or more terminal repeat sequences (e.g., one or more inverted terminal repeat (ITR) sequences (e.g., two ITR sequences) or portion thereof (e.g., two A elements, B elements, C elements, or D elements), or long terminal repeat (LTR) sequences (e.g., two LTR sequences)).
  • ITR inverted terminal repeat
  • LTR long terminal repeat
  • the terminal repeat sequence is at least 10 base pairs (bp) in length (e.g., from 10 bp to 500 bp, from 12 bp to 400 bp, from 14 bp to 300 bp, from 16 bp to 250 bp, from 18 bp to 200 bp, from 20 bp to 180 bp, from 25 bp to 170 bp, from 30 bp to 1 60 bp, or from 50 bp to 150 bp, e.g., from 10 bp to 15 bp, from 15 bp to 20 bp, from 20 bp to 25 bp, from 25 bp to 30 bp, from 30 bp to 35 bp, from 35 bp to 40 bp, from 40 bp to 45 bp, from 45 bp to 50 bp, from 50 bp to 55 bp, from 55 bp to 60 bp, from 60 bp to 65 bp
  • the invention features an isolated linear DNA molecule including a plurality of identical amplicons, wherein each of the plurality of identical amplicons includes a heterologous gene, wherein the DNA molecule: (a) comprises a terminal repeat sequence (e.g., any of the aforementioned terminal repeat sequences) ; and (b) lacks an origin of replication and/or a drug resistance gene.
  • a terminal repeat sequence e.g., any of the aforementioned terminal repeat sequences
  • the circular DNA vector further includes a heterologous gene (e.g., one or more heterologous genes). In some embodiments, the one or more heterologous genes are greater than
  • 4.5 Kb in length e.g., the one or more heterologous genes, together or each alone, are from 4.5 Kb to 25 Kb, from 4.6 Kb to 24 Kb, from 4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from 5.0 Kb to 20 Kb, from 5.5 Kb to 18 Kb, from 6.0 Kb to 1 7 Kb, from 6.5 Kb to 16 Kb, from 7.0 Kb to 15 Kb, from
  • 1 1 .5 Kb or from 10.0 Kb to 1 1 .0 Kb in length, e.g., from 4.5 Kb to 8 Kb, from 8 Kb to 10 Kb, from 10 Kb to 15 Kb, from 15 Kb to 20 Kb in length, or greater, e.g., from 4.5 Kb to 5.0 Kb, from 5.0 Kb to 5.5 Kb, from
  • Kb to 6.0 Kb from 6.0 Kb to 6.5 Kb, from 6.5 Kb to 7.0 Kb, from 7.0 Kb to 7.5 Kb, from 7.5 Kb to 8.0 Kb, from 8.0 Kb to 8.5 Kb, from 8.5 Kb to 9.0 Kb, from 9.0 Kb to 9.5 Kb, from 9.5 Kb to 10 Kb, from 10 Kb to 10.5 Kb, from 10.5 Kb to 1 1 Kb, from 1 1 Kb to 1 1 .5 Kb, from 1 1 .5 Kb to 12 Kb, from 12 Kb to 12.5 Kb, from 12.5 Kb to 13 Kb, from 13 Kb to 13.5 Kb, from 13.5 Kb to 14 Kb, from 14 Kb to 14.5 Kb, from 14.5 Kb to 15 Kb, from 15 Kb to 15.5 Kb, from 15.5 Kb to 16 Kb, from 16 Kb to 16.5 Kb, from 16.5 Kb to 17 Kb, from 17 Kb to 17.5 Kb,
  • heterologous genes may be the same gene or different genes (e.g., they may encode peptides that interact functionally (e.g., as part of a signaling pathway) or structurally (e.g., through dimerization, e.g., a heavy and light chain of an antibody or fragment thereof)).
  • the heterologous gene of the circular DNA vector includes one or more trans-splicing molecules.
  • the circular DNA vector is a monomeric circular vector, a dimeric circular vector, a trimeric circular vector, 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).
  • the circular DNA vector includes a promoter sequence upstream of the one or more heterologous genes. Additionally or alternatively, the circular DNA vector can include a polyadenylation site downstream of the one or more heterologous genes.
  • the circular DNA vector includes the following elements, operatively linked from 5’ to 3’ or from 3’ to 5’: (i) a promoter sequence; (ii) one or more heterologous genes; (iii) a polyadenylation site; and (iv) a terminal repeat sequence (e.g., one or more terminal repeat sequences (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))).
  • ITR inverted terminal repeat
  • LTR long terminal repeat
  • the invention features methods of producing an isolated circular DNA vector (e.g., any of the circular DNA vectors described herein).
  • the method includes: (i) providing a sample including a circular DNA molecule including an AAV genome (e.g., a recombinant AAV (rAAV) genome, e.g., an AAV episome), wherein the AAV genome includes a heterologous gene and a terminal repeat sequence (e.g., one or more terminal repeat sequences (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
  • AAV genome e.g., a recombinant AAV (rAAV) genome, e.g., an AAV episome
  • rAAV recombinant AAV
  • AAV genome e.g., an AAV episome
  • the AAV genome includes a heterologous gene and a terminal repeat
  • the method further includes column purifying the isolated DNA vector to purify supercoiled DNA from the isolated DNA vector.
  • the supercoiled DNA can be monomeric supercoiled DNA.
  • the heterologous gene is any of the heterologous genes described in any previous aspect, e.g., a heterologous gene that encodes a therapeutic protein configured to treat a retinal dystrophy (e.g., a Mendelian-heritable retinal dystrophy, a retinal dystrophy selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, age related macular degeneration (AMD), stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome; a hererologous gene that includes one or more of the following: ABCA4, CEP290, ABCC6, RIMS1 , LRP5,
  • COL1 1 A1 TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USH2A, and HMCN1 ; a heterologous gene that encodes antibody or portion thereof, a coagulation factor, a growth factor, a hormone, an interleukin, an interferon, an anti-apoptosis factor, an anti-tumor factor, a cytokine, and an anti-diabetic factor; and/or a heterologous gene that is a trans-splicing molecule.
  • the polymerase can be a thermophilic polymerase or a polymerase having high processivity through GC-rich residues (e.g., compared to a reference polymerase).
  • the polymerase is a phage polymerase.
  • the phage polymerase is Phi29 DNA polymerase.
  • the invention provides a method of producing an isolated circular DNA vector, the method including: (i) providing a sample including a circular DNA molecule including an AAV genome (e.g., an AAV episome), wherein the AAV genome includes a heterologous gene and a DD element; (ii) amplifying the AAV genome using a first polymerase-mediated rolling-circle amplification (e.g., an isothermal polymerase-mediated rolling circle amplification) to generate a first linear concatamer; (iii) digesting the first linear concatamer using a restriction enzyme to generate a first AAV genome; (iv) cloning the first AAV genome into a plasmid vector; (v) identifying a plasmid clone including a terminal repeat sequence (e.g., one or more terminal repeat sequences (e.g., one or more inverted terminal repeat (ITR) sequences (e.g., two ITR sequences) or long terminal repeat (LTR)
  • the invention features in vitro methods of producing a therapeutic circular DNA vector, the method including: (i) providing a sample including a circular DNA molecule including an AAV genome (e.g., a recombinant AAV (rAAV) genome, e.g., an AAV episome), wherein the AAV genome includes a heterologous gene and a terminal repeat sequence (e.g., one or more terminal repeat sequences (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))); (ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification (e.g., an isothermal polymerase-mediated rolling circle amplification) to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate an AAV genome; and (iv) allowing the AAV genome to
  • the polymerase is a phage polymerase (e.g., Phi29 DNA polymerase).
  • the method further includes column purifying the isolated DNA vector to purify supercoiled DNA from the isolated DNA vector.
  • the supercoiled DNA can be monomeric supercoiled DNA.
  • open relaxed circular DNA is separated from supercoiled DNA in the column purification and can be discarded.
  • a pharmaceutical composition including any one or more of the aforementioned circular DNA vectors and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is non-immunogenic (e.g., substantially devoid of bacterial components, such as bacterial signatures, e.g., CpG motifs). In some embodiments, the pharmaceutical composition is substantially devoid of viral particles.
  • the invention features a method of inducing expression (e.g., episomal expression) of a heterologous gene in a subject in need thereof, the method including administering to the subject a pharmaceutical composition including any of the aforementioned circular DNA vectors and a pharmaceutically acceptable carrier (e.g., a non-immunogenic pharmaceutical composition).
  • a pharmaceutical composition including any of the aforementioned circular DNA vectors and a pharmaceutically acceptable carrier (e.g., a non-immunogenic pharmaceutical composition).
  • the invention features methods of treatment using the circular DNA vectors and compositions described herein (e.g., any of the circular DNA vectors or compositions thereof of the preceding aspects).
  • the invention includes a method of treating a disorder in a subject (e.g., an ocular disorder, e.g., a retinal dystrophy, e.g., a Mendelian-heritable retinal dystrophy), the method including administering to the subject a pharmaceutical composition of any of the preceding aspects in a therapeutically effective amount.
  • a disorder in a subject e.g., an ocular disorder, e.g., a retinal dystrophy, e.g., a Mendelian-heritable retinal dystrophy
  • the pharmaceutical composition is administered repeatedly (e.g., about twice per day, about once per day, about five times per week, about four times per week, about three times per week, about twice per week, about once per week, about twice per month, about once per month, about once every six weeks, about once every two months, about once every three months, about once every four months, about twice per year, about once yearly, or less frequently).
  • the pharmaceutical composition is administered locally (e.g., ocularly,
  • the subject is being treated for leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.
  • LCA congenital amaurosis
  • Stargardt Disease pseudoxanthoma elasticum
  • rod cone dystrophy rod cone dystrophy
  • exudative vitreoretinopathy Joubert Syndrome
  • CSNB-1 C age-related macular degeneration
  • retinitis pigmentosa stickler syndrome
  • microcephaly and choriorretinopathy retinitis pigmentosa
  • CSNB 2 Usher syndrome, or Wagner syndrome.
  • the invention features non-viral isolated DNA vectors that replicate the in vivo persistence of rAAV vectors by including a double D (DD) element in a DNA molecule that is devoid of bacterial plasmid DNA.
  • DD double D
  • the DNA vectors provided herein are non-immunogenic and are not limited to the AAV packaging capacity of about 4.5 Kb.
  • the invention also features methods of producing the DD-containing DNA vector, pharmaceutical compositions including the DD-containing DNA vector, and methods of using the vectors described herein, e.g., for inducing episomal expression of a heterologous gene and for treating a disease associated with a defective gene.
  • the invention provides an isolated DNA vector including a DD element, wherein the DNA vector lacks an origin of replication (e.g., a bacterial origin of replication) and/or a drug-resistance gene (e.g., as part of a bacterial plasmid).
  • an isolated DNA vector including a DD element may lack an origin of replication (e.g., a bacterial origin of replication).
  • an isolated DNA vector including a DD element may lack a drug-resistance gene (e.g., as part of a bacterial plasmid).
  • an isolated DNA vector including a DD element may lack an origin of replication (e.g., a bacterial origin of replication) and a drug-resistance gene (e.g., as part of a bacterial plasmid).
  • the DNA molecule lacks bacterial plasmid DNA.
  • the DNA vector lacks an immunogenic bacterial signature (e.g., one or more bacterial-associated CpG motifs, e.g., unmethylated CpG motifs).
  • the DNA vector lacks an RNA polymerase arrest site (e.g., an RNA polymerase II (RNAPII) arrest site).
  • RNAPII RNA polymerase II
  • the invention features an isolated DNA vector including a DD element and a bacterial origin of replication and/or a drug resistance gene (e.g., as part of a bacterial plasmid).
  • the DNA vector further includes a heterologous gene (e.g., one or more heterologous genes).
  • the one or more heterologous genes are greater than 4.5 Kb in length (e.g., the one or more heterologous genes, together or each alone, are from 4.5 Kb to 25 Kb, from 4.6 Kb to 24 Kb, from 4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from 5.0 Kb to 20 Kb, from 5.5 Kb to 18 Kb, from 6.0 Kb to 17 Kb, from 6.5 Kb to 16 Kb, from 7.0 Kb to 15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kb to 13 Kb, from 8.5 Kb to 12.5 Kb, from 9.0 Kb to 12.0 Kb, from 9.5 Kb to 1 1 .5 Kb, or from 10.0 Kb to 1 1 .0 Kb in length, e.
  • the heterologous genes may be the same gene or different genes (e.g., they may encode peptides that interact functionally (e.g., as part of a signaling pathway) or structurally (e.g., through dimerization, e.g., a heavy and light chain of an antibody or fragment thereof)).
  • the heterologous gene includes one or more trans-splicing molecules.
  • the DNA vector is a circular vector (e.g., a monomeric circular vector, a dimeric circular vector, a trimeric circular vector, etc.). In some embodiments, the DNA vector is a monomeric circular vector.
  • the DNA vector includes a promoter sequence upstream of the one or more heterologous genes. Additionally or alternatively, the DNA vector can include a polyadenylation site downstream of the one or more heterologous genes. Thus, in some embodiments, the DNA vector includes the following elements, operatively linked from 5’ to 3’ or from 3’ to 5’: (i) a promoter sequence;
  • the invention features methods of producing an isolated DNA vector (e.g., any of the DNA vectors described herein), the method including: (i) providing a sample including a circular DNA molecule including an AAV genome (e.g., a recombinant AAV (rAAV) genome, e.g., an AAV episome), wherein the AAV genome includes a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase (e.g., phage-polymerase)-mediated rolling-circle amplification (e.g., an isothermal polymerase (e.g., phage polymerase)-mediated rolling circle amplification) to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate an AAV genome; and (iv) allowing the AAV genome to self-ligate to produce an isolated DNA vector including the heterologous gene and the DD element.
  • AAV genome e.g.
  • the polymerase can be a thermophilic polymerase or a polymerase having high processivity through GC-rich residues (e.g., compared to a reference polymerase).
  • the polymerase is a phage polymerase.
  • the phage polymerase is Phi29 DNA polymerase.
  • the invention provides a method of producing an isolated DNA vector, the method including: (i) providing a sample including a circular DNA molecule including an AAV genome (e.g., an AAV episome), wherein the AAV genome includes a heterologous gene and a DD element; (ii) amplifying the AAV genome using a first polymerase-mediated rolling-circle amplification (e.g., an isothermal polymerase-mediated rolling circle amplification) to generate a first linear concatamer; (iii) digesting the first linear concatamer using a restriction enzyme to generate a first AAV genome; (iv) cloning the first AAV genome into a plasmid vector; (v) identifying a plasmid clone including a DD element; (vi) digesting the plasmid clone including the DD element to generate a second AAV genome; (vii) allowing the second AAV genome to self-ligate to produce a circular DNA template; (i) providing
  • the invention features in vitro methods of producing a therapeutic DNA vector, the method including: (i) providing a sample including a circular DNA molecule including an AAV genome (e.g., a recombinant AAV (rAAV) genome, e.g., an AAV episome), wherein the AAV genome includes a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase-mediated rolling- circle amplification (e.g., an isothermal polymerase-mediated rolling circle amplification) to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate an AAV genome; and (iv) allowing the AAV genome to self-ligate to produce an isolated DNA vector including the heterologous gene and the DD element.
  • the polymerase is a phage polymerase (e.g., Phi29 DNA polymerase).
  • a pharmaceutical composition including the DNA vector of any of the preceding aspects and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is non-immunogenic (e.g., substantially devoid of immunogenic components, such as bacterial signatures, e.g., CpG motifs).
  • the pharmaceutical composition is substantially devoid of viral particles.
  • the invention features a method of inducing expression (e.g., episomal expression) of a heterologous gene in a subject in need thereof, the method including administering to the subject a pharmaceutical composition including the DNA vector of any of the preceding aspects and a pharmaceutically acceptable carrier (e.g., a non-immunogenic pharmaceutical composition).
  • a pharmaceutical composition including the DNA vector of any of the preceding aspects and a pharmaceutically acceptable carrier (e.g., a non-immunogenic pharmaceutical composition).
  • the expression is induced in the liver of the subject.
  • the liver can secrete a therapeutic protein encoded by the heterologous gene (e.g., into the blood).
  • the invention features methods of treatment using the DNA vectors and compositions described herein (e.g., any of the vectors or compositions of the preceding aspects).
  • the invention includes a method of treating a disorder in a subject (e.g., an ocular disorder, e.g., a retinal dystrophy, e.g., a Mendelian-heritable retinal dystrophy), the method including administering to the subject a pharmaceutical composition of any of the preceding aspects in a therapeutically effective amount.
  • a disorder in a subject e.g., an ocular disorder, e.g., a retinal dystrophy, e.g., a Mendelian-heritable retinal dystrophy
  • the pharmaceutical composition is administered repeatedly (e.g., about twice per day, about once per day, about five times per week, about four times per week, about three times per week, about twice per week, about once per week, about twice per month, about once per month, about once every six weeks, about once every two months, about once every three months, about once every four months, about twice per year, about once yearly, or less frequently).
  • the pharmaceutical composition is administered locally (e.g., ocularly, (e.g., intravitreally), intrahepatic, intracerebral, intramuscular, by aerosolization, intradermal, transdermal, or subcutaneous). In other embodiments, the pharmaceutical composition is administered systemically (e.g., intravenously).
  • the subject is being treated for leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.
  • LCA congenital amaurosis
  • Stargardt Disease pseudoxanthoma elasticum
  • rod cone dystrophy rod cone dystrophy
  • exudative vitreoretinopathy Joubert Syndrome
  • CSNB-1 C age-related macular degeneration
  • retinitis pigmentosa stickler syndrome
  • microcephaly and choriorretinopathy retinitis pigmentosa
  • CSNB 2 Usher syndrome
  • Wagner syndrome or Wagner syndrome.
  • FIG. 1 is a schematic diagram showing the formation of a terminal repeat sequence (in this case, a double D (DD) element) of AAV2.
  • AAV2 inverted terminal repeats are 145-bp in length and located at each end of the AAV genome.
  • the ITR contains inverted sequences (designated as A, B, C, and D) that can base-pair and form a hairpin-like structure.
  • a single ITR contains two "A", "B", and "C” regions, and a single "D” region.
  • Two ITRs can recombine to form a DD element that is 165 bp in length and is similar to a single ITR but now contains two "D” regions.
  • FIGS. 2A-2I are a series of illustrations showing exemplary ITR sequences for various AAV serotypes, showing locations and sequences of A, B, C, and D elements within an ITR.
  • FIG. 2A is an illustration of an AAV1 ITR.
  • FIG. 2B is an illustration of an AAV2 ITR.
  • FIG. 2C is an illustration of an AAV3 ITR.
  • FIG. 2D is an illustration of an AAV4 ITR.
  • FIG. 2E is an illustration of an AAV5 ITR.
  • FIG. 2F is an illustration of an AAV6 ITR.
  • FIG. 2G is an illustration of an AAV7 ITR.
  • FIG. 2H is an illustration of a partial AAV8 ITR.
  • FIG. 2I is an illustration of a partial AAV9 ITR.
  • FIG. 3A is a flow-chart showing exemplary steps of DD vector production and characterization process described in the Examples.
  • the first step is to generate or obtain a viral rAAV vector that contains an expression cassette (e.g., heterologous gene) needed for downstream function.
  • the virus infects cells in vitro and forms a circular, double-stranded episome with a DD element.
  • the circular rAAV genome is cloned from the cells and sequenced to confirm presence of a DD element. This can then be used to generate a plasmid-based template for in vitro DD vector production using rolling circle amplification (steps 3 and 4).
  • the final step is to confirm DD vector gene expression in vitro before proceeding with in vivo studies.
  • FIG. 3B is a flow-chart showing exemplary steps of synthetic circular vector production and characterization process described in the Examples.
  • the first step is to generate or obtain a viral rAAV vector that contains an expression cassette (e.g., heterologous gene) needed for downstream function.
  • the virus infects cells in vitro and forms a circular, double-stranded episome with a terminal repeat sequence (in this case, a DD element).
  • the circular rAAV genome is cloned from the cells and sequenced to confirm presence of a DD element. This can then be used to generate a plasmid-based template for in vitro DD vector production using rolling circle amplification (steps 3 and 4).
  • the final step is to confirm DD vector gene expression in vitro before proceeding with in vivo studies.
  • FIG. 4 is a schematic diagram showing a process for generating circular rAAV genomes in vitro.
  • a plasmid with a rAAV genome of interest is transfected with additional AAV production plasmids (triple transfection) to produce a rAAV viral vector (serotype 2) that contains the packaged genome.
  • the resulting virus infects HEK293T cells, in which circular rAAV genomes are produced.
  • FIG. 5 is a schematic diagram showing a rolling-circle amplification reaction for detection of rAAV circular genomes.
  • Total cellular DNA was digested with a restriction enzyme that does not cut within the AAV genome (in this case Avrll). The DNA was then treated with Plasmid-Safe DNase that degrades linear fragments but leaves circular, double-stranded DNA intact. The digestion reaction served as a template for linear rolling-circle amplification using random primers and Phi29 DNA polymerase. Large, linear concatameric arrays were produced following amplification of circular AAV episomes. The linear arrays were subsequently 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 genome was then cloned into the pBlueScript vector for further sequence analysis.
  • FIGS. 6A-6J is a series of illustrations showing exemplary sequences of various AAV2 terminal repeat sequences (in this case, DD elements).
  • FIG. 6A is an illustration of a standard DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5 ⁇ 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 (SEQ ID NO: 9).
  • FIG. 6A is an illustration of a standard DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5 ⁇ 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 (SEQ ID NO: 9).
  • FIG. 9 is an illustration of a standard DD element including, operatively linked in a 5’
  • FIG. 6B is an illustration of a standard DD element including, operatively linked in a 5’-to-3’ configuration, 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 (SEQ ID NO: 10).
  • FIG. 6C is an illustration of a DD element without B elements including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5 ⁇ element, a 5’ C element, a 3’ C element, a 3 ⁇ element, and a 3’ D element (SEQ ID NO: 1 1 ).
  • FIG. 1 1 is an illustration of a standard DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5’ A element, a 5’ B element, a 3’ B element, a 5’ C element, a 3’ C element
  • FIG. 6D is an illustration of a DD element without C elements including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5 ⁇ element, a 5’ B element, a 3’ B element, a 3’ A element, and a 3’ D element (SEQ ID NO: 12).
  • FIG. 6E is an illustration of a DD element without B and C elements including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5’ A element, a 3’ A element, and a 3’ D element (SEQ ID NO: 13).
  • FIG. 6F is an illustration of a DD element without A, B, and C elements including, operatively linked in a 5’-to-3’ configuration, a 5’ D element and a 3’ D element (SEQ ID NO: 14).
  • FIG. 6G is an illustration of a DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5 ⁇ element, a 5’ C element, a nucleic acid sequence in place of a 3 ⁇ element, and a 3’ D element (SEQ ID NO: 15).
  • FIG. 1 is an illustration of a DD element without A, B, and C elements including, operatively linked in a 5’-to-3’ configuration, a 5’ D element and a 3’ D element (SEQ ID NO: 14).
  • FIG. 6G is an illustration of a DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5 ⁇ element, a 5’ C element,
  • FIG. 6H is an illustration of a DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5’ A element, an overlapped 5’ C element with a 3 ⁇ element, and a 3’ D element (SEQ ID NO: 16).
  • FIG. 6I is an illustration of a DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a partial 5’ A element, a partial 3’ A element, and a 3’ D element (SEQ ID NO: 17).
  • 6J is an illustration of a DD element including, operatively linked in a 5’-to-3’ configuration, a 5’ D element, a 5 ⁇ element, a partial 3’ A element, and a 3’ D element (SEQ ID NO: 18).
  • FIG. 7 is a schematic illustration showing generation of plasmid-derived circular template.
  • Plasmid TG-18 is first digested with EcoRI to release a linear rAAV genome containing a terminal repeat sequence (DD element; represented as a bowtie). The ends of the linear fragment are ligated together to form a double-stranded circle.
  • DD element a terminal repeat sequence
  • FIG. 8 is a photograph of an agarose gel containing bands of DNA at different steps of the template formation process.
  • Lane 1 is the linear DNA fragment released from the pBlueScript vector.
  • Lane 2 is the result of self-ligation of the linear fragment from Lane 1 . Multiple DNA forms are present and include circular and linear DNA of various sizes resulting from the ligation of one or multiple DNA fragments. Lane 3 shows the DNA remaining after treatment with plasmid-safe DNase that degrades linear, but not circular, DNA.
  • FIG. 9 is a schematic diagram showing a process for analyzing Phi29 fidelity on amplifying the terminal repeat sequence (DD element).
  • a bacteria-derived circular DD vector serves as a template for linear rolling-circle amplification using random primers and Phi29 DNA polymerase.
  • Large, linear concatameric arrays are produced following amplification of circular AAV episomes.
  • the linear arrays are subsequently digested by restriction enzyme digestion to evaluate the presence of the DD element.
  • the Swal enzyme cuts on either side of the DD element to produce a 244-bp fragment.
  • the Ahdl enzyme cuts once within the DD element and digests the concatamers into unit-length fragments of 2.1 Kb.
  • FIG. 10 is a photograph of an agarose gel showing the results of a Swat digestion of amplified DNA.
  • DNA amplified from either 1 ng or 6 ng of the TG-18 plasmid template was digested with Swal to produce a 244-bp fragment (Lanes 2 and 3, arrow). This is the same size fragment released from the original TG-18 plasmid vector (Lane 1 ).
  • DNA amplified from a plasmid template lacking the DD element (TG-dDD) that was produced by removing the DD element from TG-18 using a Swal digest (Lanes 4 and 5).
  • FIG. 1 1 is a photograph of an agarose gel showing Ahdl digestion of amplified DNA. Ahdl cuts once with in the DD element. DNA amplified from either 1 ng or 6 ng of the TG-18 plasmid template was digested with Ahdl to produce a 2.1 -kb fragment (Lanes 1 and 2, arrow). Also included is DNA amplified from a plasmid template lacking the DD element (TG-dDD; lanes 3 and 4). This DNA should not be digested with Ahdl as it does not contain the DD element.
  • FIG. 12A is a schematic diagram showing self-ligation of a bacterial plasmid-derived template.
  • a plasmid having a terminal repeat sequence-containing vector (here, a DD element-containing vector) is first digested with EcoRI to release a linear rAAV genome containing a terminal repeat sequence (a DD element) within the gene sequence represented as a bowtie. The ends of the linear fragment are ligated together to form a double-stranded circle.
  • FIG. 12B is a photograph of an agarose gel showing DNA at different steps of the template formation process.
  • Lane 1 is the linear DNA fragment released from the pBlueScript vector. This fragment contains the CMV promoter, eGFP cDNA, BGHpA, and the DD element.
  • Lane 2 is the result of self-ligation of the linear fragment from Lane 1 . Multiple DNA forms are present and includes circular as well as linear DNA of various sizes resulting from the ligation of one or multiple DNA fragments.
  • Lane 3 shows the DNA remaining after treatment with plasmid-safe DNase that degrades linear, but not circular, DNA.
  • FIG. 13A is a schematic diagram showing the production of linear concatamers by Phi29 polymerase.
  • the bacteria-derived template shown in FIGS. 1 1 A and 1 1 B served as a template for linear RCA using random primers and Phi29 DNA polymerase.
  • Large, linear concatameric arrays were produced following amplification of circular AAV episomes.
  • the linear arrays were subsequently digested into unit-length monomeric AAV genomes by restriction enzyme digestion with EcoRI.
  • FIG. 13B is a photograph of an agarose gel showing size fractionated digested DNA.
  • FIG. 14A is a schematic drawing of an in vitro-derived rAAV genome that has been self-ligated from linear form into a circular product.
  • FIG. 14B is a photograph of an agarose gel showing the resulting monomeric circular DNA vector illustrated in FIG. 14A. The majority of the DNA is monomeric supercoiled circular DNA.
  • FIG. 15A is a photomicrograph showing GFP fluorescence of cells transfected with the synthetic vector characterized in FIG. 14B. Fluorescence was detected using a Spectramax MiniMax300 Imaging Cytometer.
  • FIG. 15B is a photomicrograph showing GFP fluorescence of cells transfected with the original plasmid containing the rAAV genome. Fluorescence was detected using a Spectramax MiniMax300 Imaging Cytometer.
  • FIG. 16 is a photograph of a Western blot showing GFP expression by cells transfected with pBS alone (lane 1 ), an in vitro-produced TG-18-derived DD vector (lane 2), an in vitro-produced TG-18-derived vector without the DD element (lane 3), a plasmid-derived TG-18-derived DD vector (lane 4), and a plasmid-derived TG-18-derived vector without the DD element (lane 5). Bands showing anti-tubulin staining are shown as a control.
  • FIG. 17 is a schematic diagram showing an exemplary process for producing synthetic DNA vectors using rolling circle amplification. This process includes column purification to separate open circle DNA molecules from supercoiled DNA monomers.
  • the present invention features non-viral DNA vectors that provide long-term transduction of quiescent cells (e.g., post-mitotic cells) in a manner similar to AAV vectors.
  • the invention is based, in part, on the development of an in vitro, cell-free system to synthetically produce circular AAV-like DNA vectors (e.g., DNA vectors containing a terminal repeat sequence, such as a DD element) by isothermal rolling-circle amplification and ligation-mediated circularization (as opposed to bacterial expression and site-specific recombination, for example).
  • the present methods allow for improved scalability and manufacturing efficiency in production of circular AAV-like DNA vectors.
  • the vectors produced by these methods are designed to overcome many of the problems associated with plasmid-DNA vectors, e.g., problems discussed in Lu et al., Mol. Ther. 2017, 25(5): 1 1 87-98, which is incorporated herein by reference in its entirety.
  • problems associated with plasmid-DNA vectors e.g., problems discussed in Lu et al., Mol. Ther. 2017, 25(5): 1 1 87-98, which is incorporated herein by reference in its entirety.
  • CpG islands and/or bacterial plasmid DNA sequences such as RNAPII arrest sites
  • transcriptional silencing can be reduced or eliminated, resulting in increased persistence of the heterologous gene.
  • immunogenic components e.g., bacterial endotoxin, DNA, or RNA, or bacterial signatures, such as CpG motifs
  • retinal dystrophies e.g., Mendelian-heritable retinal dystrophies.
  • the vectors of the present invention include synthetic DNA vectors that: (i) are substantially devoid of bacterial plasmid DNA sequences (e.g., RNAPII arrest sites, origins of replication, and/or resistance genes) and other bacterial signatures (e.g., immunogenic CpG motifs); and/or (ii) can be synthesized and amplified entirely in a test tube (e.g., replication in bacteria is unnecessary, e.g., bacterial origins of replication and bacterial resistance genes are unnecessary).
  • the vectors contain a DD element characteristics of AAV vectors.
  • the invention allows a target cell (e.g., a retinal cell) to be transduced with a DNA vector having a heterologous gene that behaves like AAV viral DNA (e.g., having low transcriptional silencing and enhanced persistence), without needing the virus itself.
  • a target cell e.g., a retinal cell
  • a DNA vector having a heterologous gene that behaves like AAV viral DNA (e.g., having low transcriptional silencing and enhanced persistence), without needing the virus itself.
  • the term“circular vector” or“circular DNA vector” refers to a nucleic acid molecule in a circular form. Such circular form is typically capable of being amplified into concatamers by rolling circle amplification.
  • a linear double-stranded nucleic acid having conjoined strands at its termini e.g., covalently conjugated backbones, e.g., by hairpin loops or other structures
  • a“Mendelian-heritable retinal dystrophy” refers to a disorder of the retina that follows a Mendelian inheritance pattern with variable penetrance (i.e., complete or reduced
  • a Mendelian-heritable retinal dystrophy may occur as a result of (a) single mutation in one allele (as in a dominant disorder) or (b) a single mutation in each allele (as in a recessive disorder).
  • the mutation can be a point mutation, an insertion, a deletion, or a splice variant mutation.
  • Mendelian-heritable retinal dystrophies include Leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB- 1 C, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • Mendelian-heritable retinal dystrophies do not include multifactorial disorders with multiple genetic associations that together the likelihood of developing the disease, such as age-related macular degeneration (AMD).
  • AMD age-related macular degeneration
  • terminal repeat sequence refers to a portion of a nucleic acid molecule having a sequence of nucleotides, wherein the sequence is repeated in adjacent portions of a nucleic acid molecule.
  • the sequences may be repeated in the same or reverse direction (e.g., ABCDABCD or ABCDDCBA, respectively).
  • terminal repeat sequences can be, or be derived from (e.g., products of ligation of), inverted terminal repeat sequences (ITRs) or long terminal repeat sequences (LTRs).
  • ITR-derived terminal repeat sequences may have repeated A elements, B, elements, C elements, and/or D elements (wherein A, B, C, and D elements are defined by SEQ ID NOs: 31 -37 and depicted in FIGS. 2A-2H).
  • each of FIGS. 6A-6J are terminal repeat sequences, and all DD elements (e.g., SEQ ID NOs: 9 or 10) are examples of a terminal repeat sequence.
  • a terminal repeat sequence can have a structure that results from homologous recombination (e.g., intermolecular homologous recombination or intramolecular homologous recombination).
  • inverted terminal repeat refers to the stretch of nucleic acid that exists in AAV and/or recombinant AAV (rAAV) that can form a T-shaped palindromic structure, that is required for completing AAV lytic and latent life cycles, as described in Muzyczka and Berns, Fields Virology 2001 , 2: 2327-2359.
  • rAAV recombinant AAV
  • DD element a type of terminal repeat sequence which is a DNA structure having a 5’ D element (i.e.
  • a nucleic acid sequence with at least 80% homology e.g., 80%, 85%, 90%, 95%, or 100% homology
  • a nucleic acid sequence with at least 80% homology e.g., 80%, 85%, 90%, 95%, or 100% homology
  • a nucleic acid sequence with at least 80% homology e.g., 80%, 85%, 90%, 95%, or 100% homology
  • a 5’ D element is 100% homologous to the nucleic acid sequence of SEQ ID NO: 1 and/or a 3’ D element is 1 00% homologous to the nucleic acid sequence of SEQ ID NO: 8.
  • DD element can be generated by joining two AAV inverted terminal repeats (ITRs) from the same molecule (intramolecular recombination) or different molecules (intermolecular recombination) by ligation, as shown in FIG. 1 . Such ligation can occur between ITRs of any AAV serotype, exemplary structures of which are shown in FIGS. 2A-2I.
  • a DD element contains 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 portion(s) thereof, operatively linking the 3’ end of the 5’ D element with the 5’ end of the 3’ D element. In some embodiments, no heterologous genes are present between the 3’ end of the 5’ D element and the 5’ end of the 3’ element.
  • the sequences of exemplary DD elements derived from AAV2 are shown by each of FIGS. 6A-6J.
  • DD elements from other AAV serotypes e.g., AAV1 , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 may be used. Representative 5’ and 3’ D elements from AAV serotypes 1 -7 are provided below.
  • heterologous gene refers to a gene that does not naturally occur as part of a viral genome.
  • a heterologous gene can be a mammalian gene, e.g., a therapeutic gene, e.g., a mammalian gene that encodes a therapeutic protein.
  • a heterologous gene encodes a protein or portion thereof that is defective or absent in the target cell and/or subject.
  • the heterologous gene contains one or more exons encoding a protein that is defective or absent in the target cell and/or subject.
  • the heterologous gene includes one or more trans-splicing molecules, e.g., as described in WO 2017/087900, which is incorporated herein by reference in its entirety.
  • a heterologous gene includes a therapeutic nucleic acid, such as a therapeutic RNA (e.g., microRNA).
  • a“trans-splicing molecule” has three main elements: (a) a binding domain that confers specificity by tethering the trans-splicing molecule 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 the target gene, which can replace one or more exons in the target gene (e.g., one or more mutated exons).
  • target gene e.g., pre-mRNA
  • a splicing domain e.g., a splicing domain having a 3’ or 5’ splice site
  • a coding sequence configured to be trans-spliced onto the target gene, which can replace one or more exons in the target gene (e.g., one or more mutated exons).
  • promoter refers to a sequence that regulates transcription of a heterologous gene operably linked to the promoter. Promoters provide the sequence sufficient to direct transcription and/or recognition sites for RNA polymerase and other transcription factors required for efficient transcription and can direct cell-specific expression. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, kozak sequences, and introns). Examples of promoters known in the art and useful in the viral vectors described herein include the CMV promoter, CBA promoter, smCBA promoter, and those promoters derived from an immunoglobulin gene, SV40, or other tissue specific genes.
  • a vector or composition e.g., a pharmaceutical composition containing a DNA vector of the invention
  • an immunogenic component such as an immunogenic bacterial signature
  • Methods for screening compositions for presence of immunogenic components include in vitro and in vivo animal assays according to methods known in the art.
  • a vector or composition that is substantially devoid of an immunogenic component is non-immunogenic.
  • non-immunogenic means that a vector or composition does not elicit a measurable inflammatory response (e.g., a phenotype associated with toll-like receptor signaling) in a therapeutically relevant dose.
  • Methods for screening compositions for presence of immunogenic components include in vitro and in vivo animal assays according to methods known in the art.
  • a suitable in vitro assay for determining whether a vector or composition is non-immunogenic involves culturing human peripheral blood mononuclear cells (PBMC) or human PBMC-derived myeloid cells (e.g., monocytes) in the presence of the vector or composition and measuring the amount of IL-1 b, IL-6, and/or IL-12 in the culture after eight hours. If the concentration of IL-1 b, IL-6, and/or IL-12 is not increased in the sample containing the vector or composition, relative to a negative control, the vector or composition is non-immunogenic.
  • PBMC peripheral blood mononuclear cells
  • PBMC-derived myeloid cells e.g., monocytes
  • nucleic acid molecule refers to a nucleic acid molecule comprising multiple copies of the same or substantially the same nucleic acid sequences (e.g., subunits) that are typically linked in a series.
  • the term “isolated” means artificially produced.
  • the term “isolated” refers to a DNA vector that is: (i) amplified in vitro, for example, by rolling-circle amplification or polymerase chain reaction (PCR); (ii) recombinantly produced by molecular cloning; (iii) purified, as by restriction endonuclease cleavage and gel electrophoretic fractionation, or column chromatography; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated DNA vector is one which is readily manipulable by recombinant DNA techniques well-known in the art.
  • nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated, but a nucleic acid sequence existing in its native state in its natural host is not.
  • An isolated DNA vector may be substantially purified, but need not be.
  • a“vector” refers to a nucleic acid molecule capable of carrying a heterologous gene into a target cell in which the heterologous gene can then be replicated, processed, and/or expressed in the target cell. After a target cell or host cell processes the genome of the vector (e.g., by generating a DD element), the genome is not considered a vector.
  • a“target cell” refers to any cell that expresses a target gene and which the vector infects or is intended to infect.
  • Vectors can infect target cells that reside in a subject (in situ) or target cells in culture.
  • target cells of the invention are post-mitotic cells.
  • Target cells include both vertebrate and invertebrate animal cells (and cell lines of animal origin). Representative examples of vertebrate cells include mammalian cells, such as humans, rodents (e.g., rats and mice), and ungulates (e.g., cows, goats, sheep and swine).
  • Target cells include ocular cells, such as retinal cells.
  • target cells can be stem cells (e.g., pluripotent cells (i.e. , a cell whose descendants can differentiate into several restricted cell types, such as hematopoietic stem cells or other stem cells) or totipotent cells (i.e. , a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells, and somatic stem cells e.g., hematopoietic cells)).
  • stem cells e.g., pluripotent cells (i.e. , a cell whose descendants can differentiate into several restricted cell types, such as hematopoietic stem cells or other stem cells) or totipotent cells (i.e. , a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells, and somatic stem cells e.g., hematopoietic cells)).
  • pluripotent cells i.e. , a cell whose descendants can differentiate into several restricted cell types, such as
  • target cells include oocytes, eggs, cells of an embryo, zygotes, sperm cells, and somatic (non-stem) mature cells from a variety of organs or tissues, such as hepatocytes, neural cells, muscle cells and blood cells (e.g., lymphocytes).
  • a “host cell” refers to any cell that harbors a DNA vector of interest.
  • a host cell may be used as a recipient of a DNA vector as described by the disclosure.
  • the term includes the progeny of the original cell which has been transfected.
  • a "host cell” as used herein may refer to a cell which has been transfected with a heterologous gene (e.g., by a DNA vector described herein). It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • the term“subject” includes any mammal in need of the methods of treatment or prophylaxis described herein.
  • the subject is a human.
  • Other mammals in need of such treatment or prophylaxis include dogs, cats, or other domesticated animals, horses, livestock, laboratory animals, including non-human primates, etc.
  • the subject may be male or female.
  • the subject has a disease or disorder caused by a mutation in the target gene.
  • the subject is at risk of developing a disease or disorder caused by a mutation in the target gene.
  • the subject has shown clinical signs of a disease or disorder caused by a mutation in the target gene.
  • the subject may be any age during which treatment or prophylactic therapy may be beneficial.
  • the subject is 0-5 years of age, 5-10 years of age, 10-20 years of age, 20-30 years of age, 30-50 years of age, 50-70 years of age, or more than 70 years of age.
  • an“effective amount” or“effective dose” of a vector or composition thereof refers to an amount sufficient to achieve a desired biological and/or pharmacological effect, e.g., when delivered to a cell or organism according to a selected administration form, route, and/or schedule.
  • the absolute amount of a particular vector or composition that is effective can vary depending on such factors as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc.
  • an“effective amount” can be contacted with cells or administered to a subject in a single dose or through use of multiple doses.
  • Persistence refers to the duration of time during which a gene is expressible in a cell. Persistence of a DNA vector, or persistence of a heterologous gene within a DNA vector, can be quantified relative to 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 invention), using any gene expression characterization method known in the art. In some embodiments, a control vector lacks a DD element. Additionally or alternatively, persistence can be quantified at any given time point following administration of the vector.
  • a heterologous gene of a DNA vector of the invention persists for at least six months after administration if its expression is detected in situ six months after administration of the vector.
  • a gene“persists” in a target cell if its transcription is detectable at three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, two years, or longer after administration.
  • a gene is said to persist if any detectable fraction of the original expression level remains (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) after a given period of time after administration (e.g., three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, two years, or longer after administration).
  • any detectable fraction of the original expression level remains (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) after a given period of time after administration (e.g., three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, two years, or longer after administration).
  • a“mutation” refers to any aberrant nucleic acid sequence that causes a defective (e.g., non-functional, reduced function, aberrant function, less than normal amounts produced) protein product. Mutations include base pair mutations (e.g., single nucleotide polymorphisms), missense mutations, frameshift mutations, deletions, insertions, and splice mutations.
  • the terms“disorder associated with a mutation” or“mutation associated with a disorder” refer to a correlation between a disorder and a mutation.
  • a disorder associated with a mutation is known or suspected to be wholly or partially, or directly or indirectly, caused by the mutation.
  • a subject having the mutation may be at risk of developing the disorder, and the risk may additionally depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or a different gene), or environmental factors.
  • treatment is defined as reducing the progression of a disease, reducing the severity of a disease symptom, retarding progression of a disease symptom, removing a disease symptom, or delaying onset of a disease.
  • the term“prevention” of a disorder is defined as reducing the risk of onset of a disease, e.g., as a prophylactic therapy for a subject who is at risk of developing a disorder associated with a mutation.
  • a subject can be characterized as“at risk” of developing a disorder by identifying a mutation associated with the disorder, according to any suitable method known in the art or described herein.
  • a subject who is at risk of developing a disorder has one or more mutations associated with the disorder.
  • a subject can be characterized as“at risk” of developing a disorder if the subject has a family history of the disorder.
  • administering refers to delivering the composition, or an ex vivo-treated cell, to the subject in need thereof, e.g., having a mutation or defect in the targeted gene.
  • the method involves delivering the composition by subretinal injection to the photoreceptor cells or other ocular cells.
  • intravitreal injection to ocular cells or injection via the palpebral vein to ocular cells may be employed.
  • the composition is administered intravenously. Still other methods of administration may be selected by one of skill in the art, in view of this disclosure.
  • a pharmaceutically acceptable composition is approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which a vector or composition of the invention is administered. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA., 2 nd edition, 2005.
  • a and“an” mean“one or more of.”
  • “a gene” is understood to represent one or more such genes.
  • the terms“a” and“an,”“one or more of a (or an),” and“at least one of a (or an)” are used interchangeably herein.
  • the term“about” refers to a value within ⁇ 10% variability from the reference value, unless otherwise specified.
  • Synthetic DNA vectors having DD elements can persist intracellularly (e.g., in quiescent cells, such as post-mitotic cells) as episomes, e.g., in a manner similar to AAV vectors.
  • Vectors provided herein can be naked DNA vectors, devoid of components inherent 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).
  • circular DNA vectors featuring a heterologous gene without an origin of replication and/or a drug resistance gene, herein referred to as circular DNA vectors.
  • the present invention provides circular DNA vectors that are produced synthetically.
  • Synthetic circular DNA vectors can persist intracellularly (e.g., in quiescent cells, such as post mitotic cells) as episomes, e.g., in a manner similar to AAV vectors.
  • Vectors provided herein can be naked DNA vectors, devoid of components inherent 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).
  • the DNA vector is persistent in vivo (e.g., the circularity and non-bacterial nature (i.e. , by in vitro synthesis) are associated with long-term transcription or expression of a heterologous gene of the DNA vector).
  • the persistence of the circular DNA vector is from 5% to 50% greater, 50% to 100% greater, one-fold to five-fold, or five-fold to ten-fold (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, one-fold, two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, or more) greater than a reference vector (e.g., a circular vector produced in bacteria or having one or more bacterial signatures not present in the vector of the invention).
  • a reference vector e.g., a circular vector produced in bacteria or having one or more bacterial signatures not present in the vector of the invention.
  • the circular DNA vector of the invention persists for one week to four weeks, from one month to four months, from four months to one year, from one year to five years, from five years to twenty years, or from twenty years to fifty years (e.g., at least one week, at least two weeks, at least one month, at least four months, at least one year, at least two years, at least five years, at least ten years, at least twenty years, at least thirty years, at least forty years, or at least fifty years).
  • the DNA vector includes a DD element, which may be associated with increased persistence.
  • a DNA vector may be a circular DNA vector.
  • the circular DNA vector may be monomeric, dimeric, trimeric, tetrameric, pentameric, hexameric, etc.
  • the circular DNA vector is monomeric. In other preferred embodiments, the circular DNA vector is a monomeric, supercoiled circular DNA molecule. In some embodiments, the DNA vector is nicked. In some embodiments, the DNA vector is open circular. In some embodiments, the DNA vector is double-stranded circular.
  • the DNA vector may include a DD element.
  • the DNA vector e.g., the circular DNA vector, e.g., the monomeric circular DNA vector
  • the DNA vector includes, operatively linked in the 5’ to 3’ direction: (i) a 5’ D element, (ii) a heterologous gene, and (iii) a 3’ D element.
  • the DNA vector comprises, operatively linked in the 5’ to 3’ direction: (i) a 5’ D element, (ii) a promoter, (iii) a heterologous gene, and (iv) a 3’ D element.
  • the DNA vector comprises, operatively linked in the 5’ to 3’ direction: (i) a 5’ D element, (ii) a promoter, (iii) a heterologous gene, (iv) a polyadenylation site, and (v) a 3’ D element.
  • a DNA vector may include, operatively linked in a 5’ to 3’ direction: (i) a 5 ⁇ element, (ii) 5’ D element, (iii) a heterologous gene, (iv) a 3’ D element, and (v) a 5 ⁇ element.
  • the DNA vector includes, in a 5’ to 3’ direction: (i) a 5’ A element, (ii) 5’ D element, (iii) a promoter, (iv) a heterologous gene, (v) a 3’ D element, and (vi) a 5 ⁇ element.
  • the DNA vector includes, in a 5’ to 3’ direction: (i) a 5 ⁇ element, (ii) 5’ D element, (iii) a promoter, (iv) a heterologous gene, (v) a polyadenylation site, (vi) a 3’ D element, and (vii) a 5 ⁇ element.
  • the DNA vector includes, in a 5’ to 3’ direction: (i) a 5’ C element, (ii) a 5’ A element, (iii) 5’ D element, (iv) a heterologous gene, (v) a 3’ D element, (vi) a 3 ⁇ element, and (vii) a 3’ B element.
  • the DNA vector includes, in a 5’ to 3’ direction: (i) a 5’ C element, (ii) a 5 ⁇ element, (iii) 5’ D element, (iv) a promoter, (v) a heterologous gene, (vi) a 3’ D element, (vii) a 3 ⁇ element, and (viii) a 3’ B element.
  • the DNA vector includes, in a 5’ to 3’ direction: (i) a 5’ C element, (ii) a 5 ⁇ element, (iii) 5’ D element, (iv) a promoter, (v) a heterologous gene, (vi) a
  • polyadenylation site (vii) a 3’ D element, (viii) a 3 ⁇ element, and (ix) a 3’ B element.
  • 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.
  • the DNA vector includes, operatively linked in a 5’ to 3’ direction: (i) a heterologous gene and (ii) a DD element.
  • the DNA vector includes, in a 5’ to 3’ direction: (i) a promoter, (ii) a heterologous gene, and (iii) DD element.
  • the DNA vector includes, in a 5’ to 3’ direction: (i) a heterologous gene, (ii) a polyadenylation site, and (iii) a DD element. In some embodiments, the DNA vector includes, in a 5’ to 3’ direction: (i) a promoter, (ii) a heterologous gene, (iii) a polyadenylation site, and (iv) a DD element.
  • vectors and compositions provided herein include terminal repeat sequences, which may be derived, e.g., from ITRs, LTRs, or other terminal structures, e.g., as a result of circularization.
  • the terminal repeat sequence can be at least 1 0 base pairs (bp) in length (e.g., from 10 bp to 500 bp, from 12 bp to 400 bp, from 14 bp to 300 bp, from 16 bp to 250 bp, from 18 bp to 200 bp, from 20 bp to 180 bp, from 25 bp to 170 bp, from 30 bp to 160 bp, or from 50 bp to 150 bp, e.g., from 10 bp to 15 bp, from 15 bp to 20 bp, from 20 bp to 25 bp, from 25 bp to 30 bp, from 30 bp to 35 bp, from 35 bp, from 35 bp
  • a terminal repeat sequence of a synthetic vector can be a DD element (e.g., a DD element derived from, and/or containing one or more portions of an ITR).
  • a 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.
  • DD elements can be derived from an AAV of any serotype, e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.
  • the DD element comprises two D elements directly joined to one another, for example, in the configuration shown in FIG. 6F.
  • the DD element has the nucleic acid sequence of SEQ ID NO: 14.
  • 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.
  • a DD element of the present invention has at least one additional element separating the 5’ D element from the 3’ D element, such as one or more A elements; one or more B elements; and/or one or more C elements, which may be arranged in any suitable order.
  • the DD element comprises, operatively linked in a 5’-to-3’ configuration: (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
  • a nucleic acid sequence having at least 80% homology e.g., 80%, 85%, 90%, 95%, or 100% homology
  • one or more internal nucleic acids e.g., non-heterologous nucleic acids
  • 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 the nucleic acid sequence of any one of SEQ ID NOs: 8, 20, 22, 24, 26, 28, 30, 39, or 41 .
  • the one or more nucleic acids of (ii) is from 1 -125 nucleic acids, 2-100 nucleic acids, 5-80 nucleic acids, or 10-50 nucleic acids, e.g., 1 -20 nucleic acids, 20-40 nucleic acids, 40-60 nucleic acids, 60-80 nucleic acids, 80-100 nucleic acids, or 100-125 nucleic acids, e.g., 1 -5 nucleic acids, 5-10 nucleic acids, 10-1 5 nucleic acids, 15-20 nucleic acids, 20-25 nucleic acids, 25-30 nucleic acids, 30- 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-
  • the DD element comprises two D elements (e.g., a 5’ D element (e.g.,
  • SEQ ID NO: 1 19, 21 , 23, 25, 27, 29, 38, or 40
  • a 3’ D element e.g., SEQ ID NO: 8, 20, 22, 24, 26, 28, 30, 39, or 41
  • a elements e.g., a 5 ⁇ element (e.g., SEQ ID NO: 2) and a 3 ⁇ element (e.g., SEQ ID NO: 7)
  • 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)
  • C elements e.g., SEQ ID NOs: 1 -8.
  • the nucleic acid sequences of SEQ ID NOs: 1 -8 may be operatively linked in order in a 5’ to 3’ direction, for example, as shown in FIG. 6A.
  • the DD element comprises the nucleic acid sequence of SEQ ID NO: 9.
  • SEQ ID NOs: 1 -8 can be operatively linked in any suitable order.
  • the DD element comprises the nucleic acid sequence of SEQ ID NO: 10.
  • SEQ ID NOs: 1 and 8 flank the remaining elements and/or nucleic acids within the D element.
  • SEQ ID NOs: 1 -8 can each be directly linked or indirectly linked (e.g., operatively linked) to one another, e.g., SEQ ID NOs: 1 -8 can be operatively linked in a 5’ to 3’ direction.
  • the DD element comprises 1 -100 additional nucleic acids (e.g., 3-50 nucleic acids, e.g., 3-10 nucleic acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or more additional nucleic acids) positioned between the 5’ D element and the 3’ D element (e.g., between one, two, three, four, five, or more of the following pairs of elements: a 5’ D element and a 5’ A element, a 5’ D element and a 5’ B element, a 5’ D element and a 3’ B element, a 5’ D element and a 5’ C element, a 5’ D element and a 3’ C element, a 5’ D element and a 3 ⁇ element, a 5’ D element and a 3’ D element, a 5’ A element
  • Additional nucleic acids may serve, for example, as restriction sites, as shown by the Ahdl sites in FIGS. 6A and 6B.
  • one or more of elements A, B, or C are absent.
  • FIG. 6C shows a AAV2-derived DD element without B elements.
  • the DD element of the invention may have a nucleic acid sequence having 80%, 85%,
  • FIG. 6D shows a DD element without C elements.
  • the DD element of the invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 12.
  • the DD element does not comprise B or C elements, such as shown in FIG. 6E.
  • the DD element of the invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 13.
  • one or more of elements A, B, or C may be replaced by a dissimilar nucleic acid sequence, such as in FIG. 6G, which shows a suitable DD element having a different nucleic acid sequence in place of its 3’ A element.
  • the DD element comprises SEQ ID NOs: 1 -3 and 8.
  • the DD element of the invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 1 5.
  • one or more nucleic acids overlap between two adjacent elements.
  • the overlapping nucleic acids need not be repeated.
  • An example of such a DD element is shown in FIG. 6H, where the 3’ end of the 5’ C element overlaps with the 5’ end of the 3’ A element.
  • the DD element of the invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 16.
  • Nucleic acid sequences between the 5’ and 3’ D elements may be portions of any one or more of the 5’ or 3’ A elements, 5’ or 3’ B elements, or 5’ or 3’ C elements.
  • the DD element comprises one or more partial A elements, such as shown in FIGS. 6I and 6J.
  • a partial A element may comprise a nucleic acid sequence having six or more consecutive matching nucleic acids as SEQ ID NOs: 2 or 7 (e.g., 6-40, 8-35, 1 0-30, or 15-25, e.g., 6, 7, 8, 9, 1 0, 1 1 , 12, 13, 14, 15, 16, 17, 18,
  • the DD element of the 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 invention may have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 18.
  • nucleic acid sequences of AAV2-derived DD elements and sub-elements thereof are provided in Table 2, below.
  • any of the vectors of the present invention can be used to insert a heterologous gene into a target cell.
  • a broad range of heterologous genes may be delivered to target cells by way of the present vectors.
  • the heterologous gene is configured to transfect a target cell having a mutation associated with a disease which can be treated by expression of the heterologous gene, e.g., a gene encoding a therapeutic protein, e.g., a protein that is defective or absent in the target cell and/or subject.
  • the heterologous gene may encode all or a portion of (e.g., as part of a trans splicing molecule) an ocular protein, such as CEP290, ABCA4, ABCC6, RIMS1 , LRP5, CC2D2A,
  • an ocular protein such as CEP290, ABCA4, ABCC6, RIMS1 , LRP5, CC2D2A,
  • exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosis agents, coagulation factors, anti-tumor factors.
  • Therapeutic proteins may include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropin, 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- 1 1 , IL- 12, IL- 13, IL- 14, IL-15, IL-16, IL- 17, and/or IL-18.
  • heterologous genes encoding polypeptides of interest can be included as part of the vectors of the invention, including for example, growth hormones to promote growth in a transgenic animal, or insulin-like growth factors (IGFs), a-anti-trypsin, erythropoietin (EPO), factors VIII, IX, X, and XI of the blood clotting system, LDL-receptor, GATA-1 , etc.
  • IGFs insulin-like growth factors
  • EPO erythropoietin
  • factors VIII IX, X, and XI of the blood clotting system
  • LDL-receptor erythropoietin
  • GATA-1 GATA-1
  • the nucleic acid sequence may include a suicide gene encoding, e.g., apoptotic or apoptosis-related enzymes and genes including AIF, Apaf, (e.g., Apaf-1 , Apaf-2, or Apaf-3) APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x.sub.L, Bcl-x.sub.S, bik, CAD, Calpain, Caspases e.g.
  • AIF apoptotic or apoptosis-related enzymes and genes including AIF, Apaf, (e.g., Apaf-1 , Apaf-2, or Apaf-3) APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x.sub.L, Bcl-x.sub.S, bik, CAD, Calpain, Caspases
  • Caspase-1 Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase- 6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-1 1 , or Granzyme B, ced-3, ced-9,
  • the heterologous gene encodes an antibody, or a portion, fragment, or variant thereof.
  • Antibodies include fragments that are capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab’, di-scFv, sdAb (single domain antibody) and (Fab’)2 (including a chemically linked F(ab’)2).
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to crystallize readily.
  • Antibodies also include chimeric antibodies and humanized antibodies. Furthermore, for all antibody constructs provided herein, variants having the sequences from other organisms are also contemplated. Thus, if a human version of an antibody is disclosed, one of skill in the art will appreciate how to transform the human sequence based antibody into a mouse, rat, cat, dog, horse, etc. sequence. Antibody fragments also include either orientation of single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc.
  • a single polynucleotide of a heterologous gene encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
  • Antibody fragments also include nanobodies (e.g., sdAb, an antibody having a single, monomeric domain, such as a pair of variable domains of heavy chains, without a light chain).
  • Multispecific antibodies e.g., bispecific antibodies, trispecific antibodies, etc.
  • the heterologous gene includes a reporter sequence, which can be useful in verifying heterologous gene expression, for example, in specific cells and tissues.
  • Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding b-lactamase, b -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • the reporter sequences When associated with regulatory elements which drive their expression, the reporter sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunohistochemistry for example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for b-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.
  • the heterologous gene does not include a coding sequence.
  • Non-coding sequences such as shRNA, promoters, enhancers, sequences to mark DNA (e.g., for antibody recognition), PCR amplification sites, sequences that define restriction enzyme sites, site-specific recombinase recognition sites, sequences that are recognized by a protein that binds to and/or modifies nucleic acids, and linkers, may be included in the vector.
  • non-coding sequences include binding domains that bind a target intron.
  • the heterologous gene is from 0.1 Kb to 100 Kb in length (e.g., the heterologous gene is from 0.2 Kb to 90 Kb, from 0.5 Kb to 80 Kb, from 1 .0 Kb to 70 Kb, from 1 .5 Kb to 60 Kb, from 2.0 Kb to 50 Kb, from 2.5 Kb to 45 Kb, from 3.0 Kb to 40 Kb, from 3.5 Kb to 35 Kb, from 4.0 Kb to 30 Kb, from 4.5 Kb to 25 Kb, from 4.6 Kb to 24 Kb, from 4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from 5.0 Kb to 20 Kb, from 5.5 Kb to 1 8 Kb, from 6.0 Kb to 17 Kb, from 6.5 Kb to 16 Kb, from 7.0 Kb to 15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kb to 13 Kb, from 8.5 Kb to 12.5
  • Kb 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 1 1 Kb, about 12 Kb, about 13 Kb, about 14 Kb, about 15 Kb, about 16 Kb, about 17 Kb, about 18 Kb, about 19 Kb, about 20 Kb in length, or greater).
  • DNA vectors of the invention may include conventional control elements necessary which are operably linked to the heterologous gene in a manner which permits transcription, translation, and/or expression in a target cell.
  • Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e. , Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product.
  • RNA processing signals such as splicing and polyadenylation (polyA) signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (i.e. , Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product.
  • RNA processing signals such as splicing and polyadenylation (polyA) signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (i.e. , Kozak consensus sequence)
  • sequences that enhance protein stability i.e. , Kozak consensus sequence
  • Promoters useful as part of the DNA vectors described herein include constitutive and inducible promoters.
  • constitutive promoters include, a cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), an SV40 promoter, a dihydrofolate reductase promoter, a b- actin promoter, a phosphoglycerol kinase (PGK) promoter, and a EF1 a promoter.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • SV40 promoter a dihydrofolate reductase promoter
  • b- actin promoter a phosphoglycerol kinase (PGK) promoter
  • PGK phosphoglycerol kinase
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available from a variety of commercial sources.
  • inducible promoters regulated by exogenously supplied promoters include zinc-inducible sheep metallothionine (MT) promoters, dexamethasone-inducible mouse mammary tumor virus promoters, T7 polymerase promoter systems, ecdysone insect promoters, tetracycline-repressible systems, tetracycline- inducible systems, RU486-inducible systems, and rapamycin-inducible systems.
  • MT sheep metallothionine
  • dexamethasone-inducible mouse mammary tumor virus promoters T7 polymerase promoter systems
  • ecdysone insect promoters tetracycline-repressible systems
  • tetracycline- inducible systems RU486-inducible systems
  • rapamycin-inducible systems Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g.
  • the native promoter for the heterologous gene is used.
  • the native promoter may be preferred when it is desired that expression of the heterologous gene should mimic the native expression.
  • the native promoter may be used when expression of the heterologous gene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli.
  • other native expression control elements such as enhancer elements, polyadenylation sites, or Kozak consensus sequences may also be used to mimic native expression.
  • a polyadenylation (pA) sequence can be inserted following the heterologous gene and before the terminal repeat sequence.
  • a heterologous gene useful in the present disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the heterologous gene. Selection of introns and other common vector elements are conventional and many such sequences are available.
  • the precise nature of the regulatory sequences needed for gene expression in host cells may vary between species, tissues or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the disclosure may optionally include 5' leader or signal sequences.
  • a synthetic DNA vector e.g., a circular DNA vector as described herein and/or a DNA vector having a DD element.
  • the methods provided herein involve in vitro synthesis (e.g., in the absence of cells) rather by bacterial cell synthesis.
  • In vitro synthesis of DNA vectors e.g., circular DNA vectors as described herein and/or DNA vectors containing a DD element
  • a polymerase such as a phage polymerase (e.g., Phi29 polymerase).
  • Phi29 polymerase is particular useful to process replication of terminal repeat sequences, such as DD elements.
  • the polymerase used herein can be a thermophilic polymerase that has high processivity through GC-rich residues.
  • the polymerase used to replicate (e.g., amplify) the DD element is Phi29 polymerase. Particular methods of producing the DNA vectors of the invention are described in detail in the Examples, below.
  • a DNA vector e.g., a circular DNA vector as described herein
  • production of a DNA vector can begin with providing a sample having a circular DNA molecule including an AAV genome (e.g., a rAAV genome) having heterologous gene and a terminal repeat sequence (e.g., a DD element).
  • the sample can be a lysate or other preparation from a cell (e.g., a mammalian cell) that was infected with the AAV vector (e.g., rAAV vector).
  • Double stranded circular DNA can be obtained from the cell using standard DNA extraction/isolation techniques.
  • linear DNA is specifically degraded, e.g., using plasmid-safe DNase, to purify the circular DNA.
  • the double stranded circular DNA having the AAV genome can be amplified in vitro, in a cell-free preparation, by incubating the DNA with a polymerase (e.g., a phage polymerase, e.g., Phi29 DNA polymerase; TempliPhi kit, GE Healthcare), primers (e.g., random primers), and a nucleotide mixture (e.g., dNTP, e.g., dATP, dCTP, dGTP, and dTTP).
  • a polymerase e.g., a phage polymerase, e.g., Phi29 DNA polymerase; TempliPhi kit, GE Healthcare
  • primers e.g., random primers
  • a nucleotide mixture e.g., dNTP, e.g., dATP, dCTP, dGTP, and dTTP.
  • the polymerase e.g., phage polymerase, e.g., Phi29 polymerase
  • AAV genome e.g., an AAV genome including an intact terminal repeat sequence, e.g., a DD element
  • rolling-circle amplification e.g., isothermal rolling-circle amplification
  • Suitable polymerases include thermophilic polymerases and polymerases that feature high processivity through GC-rich residues.
  • the resulting concatamers can be digested using a restriction enzyme to cut once within the genome to generate unit-length linear AAV genomes including the heterologous gene and the terminal repeat sequence (e.g., a DD element)).
  • Self-ligation of this linear DNA molecule results in a circular, synthetic DNA vector of the invention, complete with the heterologous gene and the intact terminal repeat sequence (e.g., a DD element).
  • the linear DNA molecule can be cloned into a plasmid vector according to known techniques and characterized, as is illustrated in the Examples below, 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).
  • the synthetic DNA vector can be isolated from the bacterial components of a plasmid in which it was cloned, and bacterial signatures, such as bacterial CpG motifs, are absent from the isolated vector.
  • compositions including any of the DNA vectors (e.g., synthetic DNA vectors) described herein (e.g., DNA vectors containing a DD element and/or circular DNA vectors described above) in a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions described herein are substantially devoid of contaminates, such viral particles, viral capsid proteins, or peptide fragments thereof.
  • the pharmaceutical compositions provided herein are non-immunogenic.
  • non-immunogenic pharmaceutical compositions may be substantially devoid of 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), flagellin, lipoteichoic acid, peptidoglycan, and viral nucleic acids molecules, such as double-stranded RNA.
  • CpG motifs e.g., unmethylated CpG motifs or hypomethylated CpG motifs
  • endotoxins e.g., lipopolysaccharides (LPS), e.g., bacterial LPS
  • flagellin e.g., lipoteichoic acid
  • peptidoglycan e.g., peptidoglycan
  • viral nucleic acids molecules such as double-stranded RNA.
  • compositions described herein may be assessed for contamination by conventional methods and formulated into a pharmaceutical composition intended for a suitable route of administration.
  • Still other compositions containing the DNA vector may be formulated similarly with a suitable carrier.
  • Such formulation involves the use of a pharmaceutically and/or physiologically acceptable vehicle or carrier, particularly directed for administration to the target cell.
  • carriers suitable for administration to the target cells include buffered saline, an isotonic sodium chloride solution, or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, or diluents.
  • the carrier is a liquid for injection.
  • physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. A variety of such known carriers are provided in U.S. Patent No. 7,629,322, incorporated herein by reference.
  • the carrier is an isotonic sodium chloride solution.
  • the carrier is balanced salt solution.
  • the carrier includes tween. If the vector is to be stored long term, it may be frozen in the presence of glycerol or Tween20.
  • compositions containing vectors described herein include a surfactant.
  • useful surfactants such as Pluronic F68 (Poloxamer 188, also known as LUTROL® F68) may be included as they prevent AAV from sticking to inert surfaces and thus ensure delivery of the desired dose.
  • the carrier is isotonic sodium chloride solution and includes a surfactant Pluronic F68.
  • Delivery vehicles such as liposomes, nanoparticles, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the DNA vectors may be formulated for delivery by encapsulation in a lipid particle, a liposome, a vesicle, or a nanoparticle.
  • the DNA vector is complexed with a delivery vehicle such as a poloxamer and/or polycationic material.
  • compositions having any of the DNA vectors of the invention may contain a unit dose containing a quantity of DNA from 10 pg to 10 mg (e.g., from 25 pg to 5.0 mg, from 50 pg to 2.0 mg, or from 100 pg to 1 .0 mg of DNA, e.g., from 10 pg to 20 pg, from 20 pg to 30 pg, from 30 pg to 40 pg, from 40 pg to 50 pg, from 50 pg to 75 pg, from 75 pg to 100 pg, from 100 pg to 200 pg, from 200 pg to 300 pg, from 300 pg to 400 pg, from 400 pg to 500 pg, from 500 pg to 1 .0 mg, from 1 .0 mg to 5.0 mg, or from 5.0 mg to 10 mg of DNA, e.g., from 10 pg to 10 mg (e.g., from 25 pg to 5.0 mg, from
  • compositions contain at least about 0.01 % DNA vector by weight.
  • the pharmaceutical compositions may contain 0.01 % to 80% DNA vector by weight (e.g., from 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 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 DNA vector).
  • compositions of the invention can contain any of the synthetic circular DNA vectors described herein in monomeric form (e.g., greater than 50% monomeric, greater than 60% monomeric, greater than 70% monomeric, greater than 80% monomeric, greater than 90% monomeric, greater than 95% monomeric, greater than 97% monomeric, greater than 98% monomeric, or greater than 99% monomeric).
  • from 70% to 99.99% of the synthetic circular DNA vector molecules in the pharmaceutical composition are monomeric (e.g., from 70% to 99.9%, from 70% to 99.5%, from 70% to 99%, from 75% to 99.9%, from 75% to 99.5%, from 75% to 99%, from 80% to 99.9%, from 80% to 99.5%, from 80% to 99%, from 85% to 99.9%, from 85% to 99.5%, from 85% to 99%, from 90% to 99.9%, from 90% to 99.5%, from 90% to 99%, from 95% to 99.9%, from 95% to 99.5%, or from 95% to 99% of the synthetic circular DNA vector molecules in the pharmaceutical composition are monomeric, e.g., about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the synthetic circular DNA vector molecules in the pharmaceutical composition are monomeric).
  • heterologous gene in a subject in need thereof by administering to the subject any of the DNA vectors (e.g., circular DNA vectors as described herein and/or DNA vectors including a DD element) or pharmaceutical compositions thereof described herein.
  • Cells of a subject that contain a heterologous gene can be characterized by examining the nucleic acid sequence (e.g., an RNA sequence, e.g., an mRNA sequence) of the host cell, such as by Southern Blotting or PCR analysis, to assay for the presence of the heterologous gene contained in the vector.
  • the expression of the heterologous gene in the subject can be characterized (e.g., quantitatively or qualitatively) by monitoring the progress of a disease associated with a defect or mutation in the target gene
  • the expression (e.g., episomal expression) of the heterologous gene is confirmed by observing a decline in one or more symptoms associated with the disease.
  • the invention provides methods of treating a disease in a subject associated with a defect in a target gene (e.g., a gene corresponding to the heterologous gene) by administering to the subject any of the DNA vectors (e.g., circular DNA vectors as described herein and/or DNA vectors including a DD element) or pharmaceutical compositions thereof described herein.
  • a target gene e.g., a gene corresponding to the heterologous gene
  • the disease is an ocular disease.
  • the subject is being treated for Leber’s congenital amaurosis (LCA, e.g., LCA 10) using a DNA vector having a heterologous CEP290 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for Stargardt Disease using a DNA vector having a heterologous ABCA4 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for pseudoxanthoma elasticum using a DNA vector having a heterologous ABCC6 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for rod cone dystrophy (e.g., rod cone dystrophy 7) using a DNA vector having a heterologous RIMS1 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for exudative vitreoretinopathy using a DNA vector having a heterologous LRP5 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for Joubert Syndrome using a DNA vector having a heterologous CC2D2A gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for CSNB-1 C using a DNA vector having a heterologous TRPM1 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for age-related macular degeneration using a DNA vector having a heterologous C3 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for retinitis pigmentosa 71 using a DNA vector having a heterologous IFT172 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for stickler syndrome (e.g., stickler syndrome 2) using a DNA vector having a heterologous COL1 1 A1 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for microcephaly and choriorretinopathy using a DNA vector having a heterologous TUBGCP6 gene or portion thereof (e.g., as part of a trans splicing molecule).
  • the subject is being treated for retinitis pigmentosa (e.g., RP recessive) using a DNA vector having a heterologous KIAA1549 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for CSNB 2 using a DNA vector having a heterologous CACNA1 F gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for Usher syndrome (e.g., Usher syndrome type 1 B) using a DNA vector having a heterologous MY07A gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for Wagner syndrome using a DNA vector having a heterologous VCAN gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for Usher syndrome type 2A using a DNA vector having a heterologous USH2A gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • the subject is being treated for AMD 1 using a DNA vector having a heterologous HMCN1 gene or portion thereof (e.g., as part of a trans-splicing molecule).
  • any of the vectors of the present invention can be administered to a subject in a dosage from 10 pg to 10 mg of DNA (e.g., from 25 pg to 5.0 mg, from 50 pg to 2.0 mg, or from 100 pg to 1 .0 mg of DNA, e.g., from 10 pg to 20 pg, from 20 pg to 30 pg, from 30 pg to 40 pg, from 40 pg to 50 pg, from 50 pg to 75 pg, from 75 pg to 100 pg, from 100 pg to 200 pg, from 200 pg to 300 pg, from 300 pg to 400 pg, from 400 pg to 500 pg, from 500 pg to 1 .0 mg, from 1 .0 mg to 5.0 mg, or from 5.0 mg to 10 mg of DNA, e.g., about
  • a DNA vector of the invention e.g., a circular DNA vector as described herein and/or a DNA vector containing a DD element
  • administration of a DNA vector of the invention is non-immunogenic or less likely to induce an immune response in a subject compared with administration of other gene therapy vectors (e.g., plasmid DNA vectors and viral vectors).
  • gene therapy vectors e.g., plasmid DNA vectors and viral vectors.
  • the synthetic DNA vectors provided herein can be amenable to repeat dosing due to their ability to infect target cells without triggering an immune response, or inducing a reduced immune response relative to an AAV vector, as discussed above.
  • the invention provides methods of repeatedly administering the vectors and pharmaceutical compositions described herein. Any of the aforementioned dosing quantities may be repeated at a suitable frequency and duration.
  • the subject receives a dose about twice per day, about once per day, about five times per week, about four times per week, about three times per week, about twice per week, about once per week, about twice per month, about once per month, about once every six weeks, about once every two months, about once every three months, about once every four months, twice per year, once yearly, or less frequently.
  • the number and frequency of doses corresponds with the rate of turnover of the target cell. It will be understood that in long-lived post-mitotic target cells transfected using the vectors described herein, a single dose of vector may be sufficient to maintain expression of the heterologous gene within the target cell for a substantial period of time.
  • a DNA vector provided herein may be administered to a subject in a single dose.
  • the number of occasions in which heterologous nucleic acid is delivered to the subject can be that which is required to maintain a clinical (e.g., therapeutic) benefit.
  • Methods of the invention include administration of a DNA vector (e.g., a circular DNA vector as described herein and/or a DNA vector containing a DD element) or pharmaceutical composition thereof through any suitable route.
  • the DNA vector or pharmaceutical composition thereof can be administered systemically or locally, e.g., intravenously, ocularly (e.g., intravitreally, subretinally, by eye drop, intraocularly, intraorbitally), intramuscularly, intravitreally (e.g., by intravitreal injection), intradermally, intrahepatically, intracerebrally, intramuscularly, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, intratumorally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically
  • aerosolization by injection (e.g., by jet injection), by electroporation, by implantation, by infusion (e.g., by continuous infusion), by localized perfusion bathing target cells directly, by catheter, by lavage, in creams, or in lipid compositions.
  • vectors can be administered to host cells ex vivo, such as by cells explanted from an individual patient, followed by reimplantation of the host cells into a patient, e.g., after selection for cells which have incorporated the vector.
  • the disclosure provides transfected host cells and methods of administration thereof for treating a disease.
  • a cell comprising a heterologous gene can express a visible marker, such as a fluorescent protein (e.g., GFP) or other reporter protein, encoded by the sequence of the heterologous gene that aids in the identification and isolation of a cell or cells comprising the heterologous gene.
  • a cell containing a heterologous gene can also express a selectable marker from the gene.
  • survival of the cell under certain conditions may be dependent on expression or lack of expression of a selectable marker.
  • survival or lack of survival of cells under such conditions allows for identification and isolation cells or colonies of cells that contain a heterologous gene.
  • Cells containing a heterologous gene can also be characterized by examining the nucleic acid sequence (e.g., an RNA sequence, e.g., an mRNA sequence) of the host cell, such as by Southern Blotting or PCR analysis, to assay for the presence of the heterologous gene contained in the vector.
  • rAAV vectors have an established record of high-efficiency gene transfer in a variety of model systems and are now being tested as therapeutic modalities in a wide range of human diseases.
  • Studies in animals and humans have shown that rAAV vector genomes persist in vivo predominantly as circular episomes.
  • the present invention is based on the discovery that such persistence can be replicated using synthetic techniques to produce circular DNA vectors.
  • Molecular analysis of rAAV episomal genomes isolated from both animals and humans reveals that these circular genomes contain terminal repeat sequences.
  • terminal repeat sequences identified within rAAV episomal genomes include a Double D (DD) element, which is a result of recombination of the inverted terminal repeats (ITRs) located at each end of the linear AAV genome, shown in FIG. 1 .
  • DD Double D
  • ITRs inverted terminal repeats
  • Such synthetic DNA vectors can reduce immunogenicity and inflammation in the host relative to vectors generated in bacteria, since DNA produced in bacteria contains inherent bacterial signatures (CpG motifs) as well as impurities from the bacteria themselves (endotoxin, bacterial genomic DNA and RNA) that can lead to loss of the plasmid and gene expression in vivo.
  • Step 1 Production of rAA V2-eGFP virus, followed by cell transduction.
  • Plasmid pAAV-BASIC-EGFP was obtained (Vector Biolabs, Malvern, PA), which contained AAV2 ITRs flanking an expression cassette consisting of a CMV enhancer/promoter driving eGFP protein with a BGHpA signal.
  • the plasmid was used in a triple transfection strategy in HEK293T cells to produce rAAV2-eGFP viral vectors.
  • Two other plasmids used in the triple transfection were AAV helper plasmids pRep-Cap2 (Part No. 0912; Applied Viromics, Fremont, CA) and pHELP (Part No. 0913;
  • the cells were transfected using a calcium phosphate kit (Protection Mammalian Transfection System, Part No. TM012; Promega, Madison, Wl). At 48 hours post transfection, the cells were lysed by freeze/thaw and treated with benzonase to generate a crude viral lysate. The virus titer in the crude lysate was determined to be 5.3 c 10 12 DNase-resistant particles (DRP)/mL by qPCR. To generate circular rAAV genomes, HEK293T cells were infected with the rAAV2- eGFP virus with a multiplicity of infection (MOI) of 1 c 1 0 5 .
  • MOI multiplicity of infection
  • Step 2 Cloning and characterization of rAA V genome with DD element.
  • FIG. 5 A summary of the cloning and characterization of rAAV genome having a DD element is shown in FIG. 5.
  • Infected cells were harvested seven days post-infection and total cellular DNA was extracted from cells using a DNeasy Blood and Tissue kit (Qiagen; Germantown, MD).
  • plasmid-safe DNase Luciferase
  • Wl Middleton, Wl
  • Residual circular rAAV genomes were amplified using a TEMPLI PHITM kit (Part No. 25640010, GE Healthcare; Pittsburgh, PA).
  • the TEMPLIPHITM kit contains Phi29 polymerase that uses isothermal rolling circle amplification (RCA) for the exponential amplification of circular DNA using bacteriophage Phi29 DNA polymerase.
  • the result of Phi29 amplification is long linear concatamers of DNA.
  • This DNA is then digested with an enzyme (EcoRI) that cuts once within the rAAV genome to produce a unit-length genome that is cloned into pBlueScript II KS+ plasmid (Part No. 212207, Agilent Technologies; Chicago, IL).
  • DD elements within the resulting clones were sequenced, and clone "TG-18," was identified as having an intact DD element (no deletions or rearrangements) of 165 bp in length.
  • the sequence of clone TG-18 is shown in FIG. 6A.
  • Step 3 Generation of template for DD vector production
  • Step 4 Production of DD vector in a test tube
  • the circular rAAV genome produced in Step 3 originated in bacteria and contains bacterial signatures that have the potential to reduce persistence and/or to be immunogenic in the host.
  • Step 4 amplifies this circular template in a test tube to generate more rAAV genomes that are devoid of bacterial signatures and contaminants. This is an advantage over traditional gene transfer vectors produced in bacteria.
  • the circular template is amplified using a TEMPLIPHITM kit (Part# 25640010, GE Healthcare, Pittsburgh, PA).
  • the TEMPLIPHITM kit contains Phi29 polymerase that uses isothermal rolling circle amplification (RCA) for the exponential amplification of circular DNA using bacteriophage Phi29 DNA polymerase.
  • RCA isothermal rolling circle amplification
  • the result of Phi29 amplification is long linear concatamers of DNA.
  • the amplified DNA was first digested with Swal, which cuts on either side of the DD element (FIG. 9) to release a fragment of 244 bp in length.
  • the Swal fragment from the amplified DNA was the same size as the Swal fragment from the original TG-18 pBlueScript plasmid (FIG. 10, arrow), indicating that Phi29 can amplify the DD element.
  • the integrity of the amplified DD element was further analyzed by digestion with Ahdl that cuts within the DD element. Ahdl cuts once within the DD vector and digests the concatameric DNA into 2.1 kb unit-length genomes, as demonstrated in FIG. 1 1 (arrow).
  • the circular rAAV genome produced in Step 3 is amplified using Phi29 polymerase that uses isothermal RCA for the exponential amplification of circular DNA using bacteriophage Phi29 DNA polymerase.
  • the result of Phi29 amplification is long linear concatamers of DNA (FIG. 13A).
  • This DNA is then digested with an enzyme (EcoRI) that cuts once within the rAAV genome to produce an AAV genome (i.e., a unit-length AAV genome; FIG. 13A).
  • This AAV genome is then self-ligated to re-create a circular rAAV genome (FIG. 14A). Any linear fragments that were not ligated to form a circular product was eliminated by plasmid-safe DNase treatment.
  • Step 5 Confirmation of gene expression of DD vector
  • the last step in the in vitro production process is to confirm that the DD vector is biologically active (i.e., expresses the transgene in cultured cells).
  • DD-containing DNA vector containing the eGFP expression cassette as a heterologous gene was transfected into HEK293T cells using Lipofectamine 2000 (Life Technologies, Carlsbad, CA). Cells were analyzed 48 hours later for GFP expression by immunofluorescence (FIGS. 15A and 1 5B) or western blotting (FIG. 1 6).
  • Monomeric DNA vectors were produced in which the vectors contain no bacterial plasmid DNA sequences and are synthesized entirely in a test tube (no replication in bacteria required). Therefore, the synthetic DNA vectors can endow a given target cell with transgene DNA that behaves like AAV viral DNA— without needing the virus itself.
  • This strategy offers several advantages over viral vectors. First, it allows delivery of genes that are too large for packaging into common viral vectors. Furthermore, it enables repeat dosing, since there are no viral proteins that would trigger an immune response to prevent repeat dosing of another viral vector. In addition, the in vitro synthesis process has a greater potential for more efficient manufacturing relative to other viral vectors.
  • FIG. 1 An exemplary process for generating synthetic circular DNA vectors is shown in FIG.
  • Amplification of a supercoiled monomeric DNA template was performed using phi29 polymerase to generate linear concatameric DNA having a restriction site that defines the boundaries between repeated DNA fragments.
  • the concatamers were digested using a restriction enzyme that cleaves the DNA into unit-length fragments.
  • DNA ligase was added to induce self-ligation of the DNA fragments, generating a mixture of DNA structures including open relaxed circles and supercoiled DNA
  • This mixture was column purified using a thiophilic aromatic adsorption chromatography resin (Plasmidselect Xtra, GE Healthcare 28-4024-01 ), which has a selectivity that allows supercoiled covalently closed circular forms of plasmid DNA to be separated from open circular forms.
  • Supercoiled DNA monomer obtained from this purification was recovered and can be used in the methods described herein or, alternatively, may serve as a template for additional amplification.
  • mice are administered with three compositions, each including a different DNA vector: (1 ) plasmid CAG- GFP (SEQ ID NO: 42) as a negative control of persistence; (2) ADD CAG-GFP (a synthetic circular DNA vector lacking a DD element); and (3) DD CAG-GFP (a synthetic circular DNA vector having a DD element).
  • Each group contains 32 mice total (eight mice per time point), and each composition is administered at 10 pg DNA per mouse by hydrodynamic injection. Eight mice from each group are sacrificed at each of the following time points: two weeks, four weeks, eight weeks, and sixteen weeks, and liver tissue is harvested and processed at each time point.
  • Synthetic circular CAG-GFP is determined to be highly persistent if liver cells from mice administered with synthetic circular CAG-GFP express higher levels of GFP in comparison to liver cells from mice administered with plasmid CAG-GFP.
  • mice are administered with four compositions, each including a different DNA vector: (1 ) plasmid CAG-mSEAP as a negative control of persistence; (2) plasmid CAG-mSEAP-ACpG, which lacks CpG motifs; (3) ADD CAG-mSEAP-ACpG, which lacks a DD element and CpG motifs; and (4) DD CAG-mSEAP ACpG, which includes a DD element and lacks CpG motifs.
  • mSEAP mouse secreted alkaline phosphatase
  • Each group contains 12 mice, and each composition is administered at 20 pg DNA per mouse by hydrodynamic injection. Two mice from each group are sacrificed at each of the following time points: two weeks, four weeks, eight weeks, twelve weeks, sixteen weeks, and twenty-four weeks, and 200 mI_ blood is collected. Serum concentration of mSEAP is quantified in each sample according to known methods and compared across groups at each time point.
  • CpG motifs and/or a DD element on persistence is quantified by comparing mSEAP concentration across the experimental groups. For example, serum mSEAP levels are approximately equivalent across experimental groups at early time points; however, mice administered with vectors having higher persistence exhibit greater concentrations of mSEAP at later time points.
  • An isolated DNA vector comprising a double D (DD) element, wherein the DNA molecule lacks an origin of replication and/or a drug resistance gene.
  • DD double D
  • An isolated DNA vector comprising a DD element and a bacterial origin of replication and/or a drug resistance gene.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase- mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate a unit-length linear DNA molecule; and (iv) allowing the unit-length linear DNA molecule to self-ligate to produce an isolated DNA vector comprising the heterologous gene and the DD element.
  • a method of producing an isolated DNA vector comprising:(i) providing a sample comprising a circular DNA molecule comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using a first polymerase-mediated rolling-circle amplification to generate a first linear concatamer; (iii) digesting the first linear concatamer using a restriction enzyme to generate 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 comprising a DD element; (vi) digesting the plasmid clone comprising the DD element to generate a second unit-length linear DNA molecule; (vii) allowing the second unit-length linear DNA molecule to self-ligate to produce a circular DNA template; (viii) amplifying the circular DNA template using second polymerase-mediated rolling
  • An in vitro method of producing a therapeutic DNA vector comprising: (i) providing a sample comprising a circular DNA molecule comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate a unit-length linear DNA molecule; and (iv) allowing the unit-length linear DNA molecule to self-ligate to produce a therapeutic DNA vector comprising the heterologous gene and the DD element.
  • a pharmaceutical composition comprising the DNA vector of any one of paragraphs 1 -12 and a pharmaceutically acceptable carrier.
  • a method of inducing episomal expression of a heterologous gene in a subject in need thereof comprising administering to the subject the isolated DNA vector of any one of paragraphs 1 -1 1 or the pharmaceutical composition of paragraph 20 or 21 .
  • a method of treating a disorder in a subject comprising administering to the subject the isolated DNA vector of any one of paragraphs 1 -12 or the pharmaceutical composition of paragraph 20 or 21 in a therapeutically effective amount.
  • ocular disorder is leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.
  • LCA congenital amaurosis
  • Stargardt Disease pseudoxanthoma elasticum
  • rod cone dystrophy rod cone dystrophy
  • exudative vitreoretinopathy Joubert Syndrome
  • CSNB-1 C age-related macular degeneration
  • retinitis pigmentosa stickler syndrome
  • microcephaly and choriorretinopathy retinitis pigmentosa
  • CSNB 2 Usher syndrome, or Wagner syndrome.
  • An isolated circular DNA vector comprising one or more heterologous genes, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene; (ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate multiple AAV genomes; and (iv) allowing each of the multiple AAV genomes to self-ligate to produce an isolated DNA vector comprising the heterologous gene.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a terminal repeat sequence; (ii) amplifying the AAV genome using a first polymerase-mediated rolling-circle amplification to generate a first linear concatamer; (iii) digesting the first linear concatamer using 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 a terminal repeat sequence; (vi) digesting the plasmid clone comprising the terminal repeat sequence to generate a second AAV genome; (vii) allowing the second AAV genome to self-ligate to produce a circular DNA template; (viii) amplifying the circular DNA template using second polymerase-mediated rolling-circle amplification to generate a second linear concatamer;
  • An in vitro method of producing a therapeutic DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene; (ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate an AAV genome; and (iv) allowing the AAV genome to self-ligate to produce a therapeutic DNA vector comprising the heterologous gene.
  • a pharmaceutical composition comprising the DNA vector of any one of paragraphs 1 -13 and a pharmaceutically acceptable carrier.
  • a method of inducing episomal expression of a heterologous gene in a subject in need thereof comprising administering to the subject the isolated DNA vector of any one of paragraphs 1 -13 or the pharmaceutical composition of paragraph 24 or 25.
  • 27. A method of treating a disorder in a subject, the method comprising administering to the subject the isolated DNA vector of any one of paragraphs 1 -13 or the pharmaceutical composition of paragraph 24 or 25 in a therapeutically effective amount.
  • the ocular disorder is LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB- 1 C, age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.
  • An isolated DNA vector comprising a double D (DD) element, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • DD double D
  • An isolated DNA vector comprising a DD element and a bacterial origin of replication and/or a drug resistance gene.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase- mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate multiple AAV genomes; and (iv) allowing each of the multiple AAV genomes to self-ligate to produce an isolated DNA vector comprising the heterologous gene and the DD element.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using a first polymerase-mediated rolling-circle amplification to generate a first linear concatamer; (iii) digesting the first linear concatamer using 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 a DD element; (vi) digesting the plasmid clone comprising the DD element to generate a second AAV genome; (vii) allowing the second AAV genome to self-ligate to produce a circular DNA template; (viii) amplifying the circular DNA template using second polymerase-mediated rolling-circle amplification to generate a second linear concatamer
  • An in vitro method of producing a therapeutic DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate an AAV genome; and (iv) allowing the AAV genome to self-ligate to produce a therapeutic DNA vector comprising the heterologous gene and the DD element.
  • a pharmaceutical composition comprising the DNA vector of any one of paragraphs 33-44 and a pharmaceutically acceptable carrier.
  • a method of inducing episomal expression of a heterologous gene in a subject in need thereof comprising administering to the subject the isolated DNA vector of any one of paragraphs 33-45 or the pharmaceutical composition of paragraph 52 or 53.
  • a method of treating a disorder in a subject comprising administering to the subject the isolated DNA vector of any one of paragraphs 33-44 or the pharmaceutical composition of paragraph 52 or 53 in a therapeutically effective amount.
  • ocular disorder is Leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.
  • LCA congenital amaurosis
  • Stargardt Disease pseudoxanthoma elasticum
  • rod cone dystrophy rod cone dystrophy
  • exudative vitreoretinopathy Joubert Syndrome
  • CSNB-1 C age-related macular degeneration
  • retinitis pigmentosa stickler syndrome
  • microcephaly and choriorretinopathy retinitis pigmentosa
  • CSNB 2 Usher syndrome, or Wagner syndrome.
  • An isolated circular DNA vector comprising one or more heterologous genes encoding a therapeutic protein configured to treat a Mendelian-heritable retinal dystrophy, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • Mendelian-heritable retinal dystrophy is selected from the group consisting of Leber’s congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • LCA congenital amaurosis
  • CSNB-1 C retinitis pigmentosa
  • stickler syndrome microcephaly and choriorretinopathy
  • retinitis pigmentosa CSNB 2
  • Usher syndrome and Wagner syndrome.
  • 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, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USH2A, and HMCN1 , wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • the one or more heterologous genes encode a therapeutic protein configured to treat a Mendelian-heritable retinal dystrophy selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • a Mendelian-heritable retinal dystrophy selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • An isolated circular DNA vector comprising one or more heterologous genes encoding a therapeutic protein selected from the group consisting of an antibody or portion thereof, a growth factor, an interleukin, an interferon, an anti-apoptosis factor, a cytokine, and an anti-diabetic factor, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • An isolated circular DNA vector comprising one or more heterologous genes comprising a trans-splicing molecule, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • An isolated circular DNA vector comprising one or more heterologous genes encoding a liver- secreted therapeutic protein, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • the DNA vector of paragraph 8 wherein the therapeutic protein is secreted into blood.
  • An isolated circular DNA vector comprising one or more heterologous genes, wherein the DNA vector: (a) comprises a terminal repeat sequence; and (b) lacks an origin of replication and/or a drug resistance gene.
  • An isolated linear DNA molecule comprising a plurality of identical amplicons, wherein each of the plurality of identical amplicons comprises a heterologous gene encoding a therapeutic protein configured to treat a retinal dystrophy, wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a recombination site.
  • Mendelian-heritable retinal dystrophy is selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, age related macular degeneration (AMD), stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • An isolated linear DNA molecule comprising a plurality of identical amplicons, wherein each of the plurality of identical amplicons comprises a heterologous gene selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT-172, C3, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USFI2A, and FIMCN1 , wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a recombination site.
  • a heterologous gene selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT-172, C3, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY07A, VCAN, USFI2
  • a heterologous gene encodes a therapeutic protein configured to treat a Mendelian-heritable retinal dystrophy selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, AMD, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • a Mendelian-heritable retinal dystrophy selected from the group consisting of LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1 C, retinitis pigmentosa, AMD, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.
  • An isolated linear DNA molecule comprising a plurality of identical amplicons, wherein each of the plurality of identical amplicons comprises a heterologous gene encoding antibody or portion thereof, a coagulation factor, a growth factor, a hormone, an interleukin, an interferon, an anti-apoptosis factor, an anti-tumor factor, a cytokine, and an anti-diabetic factor, wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a recombination site.
  • An isolated linear DNA molecule comprising a plurality of identical amplicons, wherein each of the plurality of identical amplicons comprises a heterologous gene comprising a trans-splicing molecule, wherein the DNA molecule lacks: (a) an origin of replication and/or a drug resistance gene; and (b) a recombination site.
  • An isolated linear DNA molecule comprising a plurality of identical amplicons, wherein each of the plurality of identical amplicons comprises a heterologous gene encoding a liver-secreted therapeutic protein, wherein the DNA molecule lacks an origin of replication and/or a drug resistance gene.
  • An isolated linear DNA molecule comprising a plurality of identical amplicons, wherein each of the plurality of identical amplicons comprises a heterologous gene, wherein the DNA molecule: (a) comprises a terminal repeat sequence; and (b) lacks an origin of replication and/or a drug resistance gene.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene; (ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate multiple AAV genomes; and (iv) allowing each of the multiple AAV genomes to self-ligate to produce an isolated DNA vector comprising the heterologous gene; wherein the heterologous gene: (a) encodes a therapeutic protein configured to treat a Mendelian-heritable retinal dystrophy; (b) is selected from the group consisting of ABCA4, CEP290, ABCC6, RIMS1 , LRP5, CC2D2A, TRPM1 , IFT-172, C3, COL1 1 A1 , TUBGCP6, KIAA1549, CACNA1 F, MY
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a terminal repeat sequence; (ii) amplifying the AAV genome using a first polymerase-mediated rolling-circle amplification to generate a first linear concatamer; (iii) digesting the first linear concatamer using 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 a terminal repeat sequence; (vi) digesting the plasmid clone comprising the terminal repeat sequence to generate a second AAV genome; (vii) allowing the second AAV genome to self-ligate to produce a circular DNA template; (viii) amplifying the circular DNA template using second polymerase-mediated rolling-circle amplification to generate a second linear concatamer
  • An in vitro method of producing a therapeutic DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene;(ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate an AAV genome; and (iv) allowing the AAV genome to self-ligate to produce a therapeutic DNA vector comprising the heterologous gene.
  • a pharmaceutical composition comprising the DNA vector of any one of paragraphs 1 -21 and a pharmaceutically acceptable carrier.
  • a method of inducing episomal expression of a heterologous gene in a subject in need thereof comprising administering to the subject the isolated DNA vector of any one of paragraphs 1 -21 or the pharmaceutical composition of paragraph 45 or 46.
  • 48. A method of treating a disorder in a subject, the method comprising administering to the subject the isolated DNA vector of any one of paragraphs 1 -21 or the pharmaceutical composition of paragraph 43 or 44 in a therapeutically effective amount.
  • the ocular disorder is LCA, Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB- 1 C, age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.
  • An isolated DNA vector comprising a double D (DD) element, wherein the DNA vector lacks an origin of replication and/or a drug resistance gene.
  • DD double D
  • An isolated DNA vector comprising a DD element and a bacterial origin of replication and/or a drug resistance gene.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase- mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate multiple AAV genomes; and (iv) allowing each of the multiple AAV genomes to self-ligate to produce an isolated DNA vector comprising the heterologous gene and the DD element.
  • a method of producing an isolated DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using a first polymerase-mediated rolling-circle amplification to generate a first linear concatamer; (iii) digesting the first linear concatamer using 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 a DD element; (vi) digesting the plasmid clone comprising the DD element to generate a second AAV genome; (vii) allowing the second AAV genome to self-ligate to produce a circular DNA template; (viii) amplifying the circular DNA template using second polymerase-mediated rolling-circle amplification to generate a second linear concata
  • An in vitro method of producing a therapeutic DNA vector comprising: (i) providing a sample comprising a circular DNA vector comprising an AAV genome, wherein the AAV genome comprises a heterologous gene and a DD element; (ii) amplifying the AAV genome using polymerase-mediated rolling-circle amplification to generate a linear concatamer; (iii) digesting the concatamer using a restriction enzyme to generate an AAV genome; and (iv) allowing the AAV genome to self-ligate to produce a therapeutic DNA vector comprising the heterologous gene and the DD element.
  • a pharmaceutical composition comprising the DNA vector of any one of paragraphs 55-66 and a pharmaceutically acceptable carrier.
  • a method of inducing episomal expression of a heterologous gene in a subject in need thereof comprising administering to the subject the isolated DNA vector of any one of paragraphs 55-66 or the pharmaceutical composition of paragraph 74 or 75.
  • a method of treating a disorder in a subject comprising administering to the subject the isolated DNA vector of any one of paragraphs 55-66 or the pharmaceutical composition of paragraph 74 or 75 in a therapeutically effective amount.
  • CSNB-1 C age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.

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CN111954528A (zh) 2020-11-17
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EP3765023A1 (en) 2021-01-20
EP3765023A4 (en) 2022-01-19
IL276923A (en) 2020-10-29
US20210002667A1 (en) 2021-01-07
KR20200132893A (ko) 2020-11-25
US20240247284A1 (en) 2024-07-25
MX2020009579A (es) 2020-11-09

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