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WO2025160245A1 - Viral and non-viral nanoplasmid vectors with improved production - Google Patents

Viral and non-viral nanoplasmid vectors with improved production

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Publication number
WO2025160245A1
WO2025160245A1 PCT/US2025/012704 US2025012704W WO2025160245A1 WO 2025160245 A1 WO2025160245 A1 WO 2025160245A1 US 2025012704 W US2025012704 W US 2025012704W WO 2025160245 A1 WO2025160245 A1 WO 2025160245A1
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Prior art keywords
seq
sequence
replication
origin
closed circular
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French (fr)
Inventor
James A. Williams
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Aldevron LLC
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Aldevron LLC
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Publication of WO2025160245A1 publication Critical patent/WO2025160245A1/en
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    • 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
    • C12N15/67—General methods for enhancing the expression
    • C12N15/69—Increasing the copy number of the vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
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    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/55—Vectors comprising a special origin of replication system from bacteria

Definitions

  • the present invention relates to recombinant DNA molecules, i.e., vectors, useful for viral and non-viral gene therapy, viral and non-viral cell therapy, and more particularly, for improving viral and non-viral vector manufacturing yield and quality, reducing transfection associated toxicity, improving transposition from non-viral transposon vectors, improving packaging titers from viral vectors, improving expression of viral and non-viral vector encoded genes, and for eliminating viral vector and non-viral vector mediated antibiotic selection marker gene transfer.
  • vectors useful for viral and non-viral gene therapy, viral and non-viral cell therapy, and more particularly, for improving viral and non-viral vector manufacturing yield and quality, reducing transfection associated toxicity, improving transposition from non-viral transposon vectors, improving packaging titers from viral vectors, improving expression of viral and non-viral vector encoded genes, and for eliminating viral vector and non-viral vector mediated antibiotic selection marker gene transfer.
  • Such recombinant DNA molecules are useful in biotechnology, ex vivo gene therapy, transgenic organisms, gene therapy, therapeutic vaccination, agriculture and DNA vaccines.
  • E. coli plasmids have long been an important source of recombinant DNA molecules used by researchers and by industry.
  • Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines.
  • Plasmids are also utilized in gene therapy or gene replacement applications, wherein the desired gene product is expressed from the plasmid after administration to a patient.
  • Plasmids are also utilized as in vitro transcription template vectors for mRNA vaccines and therapeutics. Plasmids are also utilized in non-viral transposon vectors for gene therapy or gene replacement applications, wherein the desired gene product is expressed from the genome after transposition from the plasmid and genome integration. Plasmids are also utilized in viral vectors for gene therapy or gene replacement applications, wherein the desired gene product is packaged in a transducing virus particle after transfection of a production cell line, and is then expressed from the virus in a target cell after viral transduction.
  • minicircle vectors are superior to plasmid vectors for production of AAV vectors (improved transducing unit titers, see Table 1) and transposon vectors (increased transposition, see Table 1).
  • improved performance due to improved expression duration with short backbone minicircle vectors should also be observed with short bacterial backbone plasmid vectors up to 1.1 kb.
  • viral vectors such as AAV, lentiviral and retroviral vectors, and transposon vectors contained structured DNA sequences at their termini.
  • AAV vectors contain flanking ITRs
  • Lentiviral and Retroviral vectors contain flanking LTRs.
  • MIP Mini-Intronic Plasmid
  • MIP vectors with longer spacer regions ⁇ Ikb can be made
  • a drawback of the MIP intron strategy is that it requires cloning of a replication and selection encoding intron into the eukaryotic region, which is not possible or desired with many vectors.
  • a drawback of the minicircle strategy to create short bacterial region AAV, Lentiviral, Retroviral or transposon vectors is that methods to manufacture minicircle vectors are expensive and not easily scalable.
  • E. coli-based manufacturing systems have been developed in which, after plasmid production, the bacterial region and the eukaryotic region are separated and circularized into a minicircle (eukaryotic region) and a bacterial region circle via the action of phage recombinases on recognition sequences in the plasmid.
  • a restriction enzyme is then utilized to digest the bacterial region circle at a unique site to eliminate this difficult to remove contaminant.
  • a solution is needed to develop mRNA, AAV, lentiviral, retroviral or transposon vector containing short spacer regions preferably less than 1000 bp that can be efficiently manufactured without replication intermediates or poor production.
  • the vectors do not encode a protein-based selection marker.
  • the vectors are minimalized to eliminate all non-essential sequences.
  • vectors useful for viral and non-viral gene therapy [0014] in embodiments, disclosed are vectors useful for viral and non-viral gene therapy, [0015] hi embodiments, disclosed are vectors useful for viral and non-viral cell therapy, [0016] hi embodiments, disclosed are vectors for improving viral and non-viral vector manufacturing yield and quality,
  • vectors for reducing transfection associated toxicity are disclosed.
  • vectors for improving transposition from non-viral transposon vectors are disclosed.
  • vectors for improving packaging titers from viral vectors are disclosed.
  • vectors for improving expression of viral and non-viral vector encoded transgenes are disclosed.
  • vectors for eliminating antibiotic resistance marker gene transfer by viral and non-viral vectors are disclosed.
  • Retroviral vector, Retroviral envelope vector and Retroviral packaging vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
  • AAV vector and AAV helper vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
  • Retroviral vector methods and compositions with improved viral transducing unit production that utilize a Pol Ill-dependent origin of replication are disclosed.
  • AAV vector and AAV helper vector methods and compositions with improved viral transducing unit production that utilize a Pol Ill-dependent origin of replication are disclosed.
  • Retroviral vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
  • AAV vector and AAV helper vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
  • Retroviral vector, retroviral envelope vector and retroviral packing vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
  • AAV vector and AAV helper vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
  • AAV vector and AAV helper vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
  • Improved non-viral transposon and transposase vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
  • RNA selectable marker oriented to transcribe divergent from a structured DNA sequence including with a structured DNA sequence insert
  • each of the above improvements and the improvements described below is, by way of example but not limitation, relative to what is achieved in similar or identical circumstances, but with a different recombinant DNA molecule that does not include a Pol-III dependet origin of replication, and/or one or more primosomal assembly sites, and/or a marker, such as an RNA selectable marker, oriented to transcribe in a direction divergent from a structured DNA sequence.
  • the disclosed vectors provide improved viral and non-viral vector manufacturing yield.
  • the disclosed vectors provide improved viral and non-viral vector manufacturing quality.
  • the disclosed vectors provide viral vectors with improved packaging titers.
  • the disclosed vectors provide non-viral transposon vectors with improved transposition.
  • the disclosed vectors provide viral and non-viral vectors with improved expression of encoded transgenes. [0088] In embodiments, the disclosed vectors provide viral and non-viral vectors that eliminate antibiotic resistance marker gene transfer.
  • the disclosed vectors provide viral and non-viral vectors with reduced transfection associated toxicity.
  • a covalently closed circular recombinant DNA molecule can include a backbone and an insert, where the backbone can include a Pol Ill-dependent origin of replication, a selectable marker and a first primosomal assembly site, where the first primosomal assembly site is positioned downstream of the Pol Ill-dependent origin of replication in the direction of replication, and where the insert comprises a structured DNA sequence.
  • an antibiotic marker free covalently closed circular recombinant DNA molecule can include a backbone and an insert, where the backbone can include an origin of replication and an RNA selectable marker, where the insert can include a structured DNA sequence, and where the RNA selectable marker is oriented to transcribe in a direction divergent from the structured DNA sequence.
  • the RNA selectable marker can instead be substituted by an antibiotic selectable marker and the recombinant DNA molecule can be a covalently closed circular recombinant DNA molecule, where the antibiotic selectable marker is oriented to transcribe in a direction divergent from the structured DNA sequence.
  • divergent can refer to a direction away from a structured DNA sequence proximate to either end of the insert.
  • the structured DNA sequence can be within 1000 bp of the origin of replication.
  • the backbone can be less than 1000 bp.
  • the backbone and the insert can be operably linked such that there is no intervening sequence between the backbone and the insert.
  • the position of a feature can be located on either strand of a covalently closed circular recombinant DNA molecule.
  • a first primosomal assembly site can be on one strand while a second primosomal assembly site can be on the opposite strand.
  • SEQ ID NO: 29 encodes two primosomal assembly sites, but the sequence provided has one in the sense orientation and one in the antisense orientation.
  • a backbone can, in some embodiments, be a bacterial replication-selection region of the present disclosure.
  • the origin of replication can be a Pol Ill-dependent origin of replication such as, by way of example but not limitation, an R6K origin, ColE2 origin or ColE2 -related origin.
  • the Pol Ill-dependent origin of replication does not require Pol 1.
  • the Pol Ill-dependent origin of replication can be an R6K origin of replication such as, by way of example but not limitation, an R6K gamma origin.
  • the R6K origin of replication can have a sequence that has at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24.
  • the Pol Ill-dependent origin can be an R6K origin that is a 6 iteron R6K gamma origin or a 7 iteron R6K gamma origin (such as, by way of example, but not limitation, SEQ ID NO: 18) or having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity thereto.
  • the iteron repeat can be selected from any one of SEQ ID NOs: 19-23.
  • the selectable marker can be an RNA selectable marker.
  • the selectable marker can be an antibiotic selectable marker.
  • the RNA selectable marker can be an RNA-OUT RNA selectable marker.
  • the RNA-OUT RNA selectable marker can be an RNA-IN regulating RNA-OUT functional variant with at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7.
  • the RNA selectable marker can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6.
  • the first primosomal assembly site can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
  • the backbone can further include a second primosomal assembly site positioned downstream of the origin of replication in the direction of replication.
  • the second primosomal assembly site can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
  • the backbone can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29 such that first primosomal assembly site and the second primosomal assembly site are downstream of the origin of replication in the direction of replication.
  • the recombinant DNA molecule can be antibiotic free.
  • the backbone can include a sequence with at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the structured DNA sequence can be any structured DNA sequence described in embodiments of this disclosure.
  • the structured DNA sequence can be selected from an inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and a eukaryotic promoter-enhancer sequence.
  • the insert can be any type of vector described in the present disclosure.
  • the insert can be a transposon vector, a transposase vector, a mRNA vector, an AAV vector, a Lentiviral vector.
  • the structured DNA sequence can be an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promoter-enhancer sequence.
  • the structured DNA sequence can be an inverted repeat sequence.
  • the AAV vector can encode AAV ITRs.
  • the AAV vector can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 35 (5’ inverted terminal repeat sequence) and a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36 (3’ inverted terminal repeat sequence).
  • the insert can include one or more inverted repeats of an AAV ITR.
  • said recombinant DNA molecule is an AAV ITR containing non- viral vector
  • the structured DNA sequence can be a direct repeat sequence or an eukaryotic origin of replication.
  • the structured DNA sequence can be selected from an homopolymeric repeat such as a polyA repeat, a SV40 origin of replication, a viral LTR, a lentiviral LTR, a retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
  • an homopolymeric repeat such as a polyA repeat, a SV40 origin of replication, a viral LTR, a lentiviral LTR, a retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
  • the homopolymeric sequence can be a polyA repeat.
  • the homopolymeric sequence such as, by way of example, but not limitation a polyA repeat, can include from about 3 to about 500 residues or more.
  • the polyA repeat can include the sequence of any one of SEQ ID NOs: 37-39.
  • the recombinant DNA molecule can be a mRNA vector that includes one or more homopolymeric sequences.
  • the selectable marker can be an RNA selectable marker oriented to transcribe in a direction divergent from the structured DNA sequence.
  • said recombinant DNA molecule is selected from viral vector, lentiviral vector, retroviral vector, AAV vector, Ad vector, non-viral transposon vector, Sleeping Beauty transposon vector, PiggyBac transposon vector, Tol2 transposon vector, and polyA containing mRNA vector.
  • the recombinant DNA molecules of the foregoing embodiments, or of any embodiment of the present disclosure can be replicated by providing a cell containing the recombinant DNA molecule and subjecting the cell to a fermentation cell, where the cell and the fermentation process are of any embodiments described herein.
  • an existing recombinant DNA molecule can be prepared by substituting the origin of replication and/or selectable marker to arrive at the recombinant DNA molecule of any of the embodiments of the disclosure and to have the features thereof.
  • the origin of replication is a Pol I-dependent origin of replication
  • it can be a pUC origin, pMBl origin, and ColEl origin.
  • the primosomal assembly site(s), selectable marker and origin or replication can be in any order relative to a structured DNA sequence and within the backbone.
  • the primosomal assembly site(s) can be between the origin of replication and the selectable marker or at either end of the origin of replication and selectable marker.
  • the origin of replication and the selectable marker can be in either order with respect to the structured DNA sequence or insert.
  • the following configurations represent certain embodiments where the RNA selectable marker transcribes in a direction diverent from the structured DNA sequence:
  • a R6K origin can include multiple iterons such as, by way of example, but not limitation, 6 or 7 iterons.
  • the present technology provides a method for improving the replication of a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a Pol I-dependent origin of replication, and ii) an insert comprising a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication and eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication; b) modifying the covalently closed circular recombinant molecule of a) such that the Pol I-dependent origin of replication is replaced with a Pol Ill-dependent origin of replication whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has improved replication.
  • said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin.
  • said Pol Ill-dependent origin of replication is an R6K gamma replication origin.
  • said Pol Ill-dependent origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said structured DNA sequence is selected from polyA repeat, SV40 origin of replication, viral LTR, Lentiviral LTR, Retroviral LTR, transposon IR/DR repeat, Sleeping Beauty transposon IR/DR repeat, AAV ITR, CMV enhancer, and SV40 enhancer.
  • said improved replication is selected from reduced production of replication intermediates and increased plasmid copy number.
  • the present technology provides a method for improving the replication of a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication and eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication; b) modifying the covalently closed circular recombinant molecule of a) such that the antibiotic selectable marker is replaced with an RNA selectable marker and the Pol I-dependent origin of replication is replaced with a Pol Ill-dependent origin of replication, whereby the resultant Pol Ill-dependent origin of replication covalently closed
  • said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin.
  • said Pol Ill- dependent origin of replication is an R6K gamma replication origin.
  • said Pol Ill-dependent origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • said RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with a Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region with at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • said structured DNA sequence is selected from polyA repeat, SV40 origin of replication, viral LTR, Lentiviral LTR, Retroviral LTR, transposon IR/DR repeat, Sleeping Beauty transposon IRT)R repeat, AAV ITR, CMV enhancer, and SV40 enhancer.
  • said improved replication is selected from reduced production of replication intermediates, and increased plasmid copy number.
  • the current technology provides an antibiotic marker free covalently closed circular recombinant DNA molecule comprising: a) an antibiotic marker free insert comprising a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence; b) a Pol Ill-dependent origin of replication comprising an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24; and c) an RNA-OUT RNA selectable marker comprising an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • said R6K gamma replication origin and said RNA- OUT RNA selectable marker comprise a R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region with at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the present technology provides a method for improving AAV vector viral transducing unit production from a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from AAV vector, AAV rep cap vector, Ad helper vector, and Ad helper rep cap vector; b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill- dependent origin of replication covalently closed
  • said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin.
  • said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said RNA-OUT RNA selectable marker is an RNA- IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the current technology provides a method for improving Retroviral or Lentiviral vector viral transducing unit production from a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from Retroviral vector, Lentiviral vector, Retroviral envelope vector, Lentiviral envelope vector, Retroviral packaging vector and Lentiviral packaging vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-se
  • said Pol I- dependent origin of replication is selected from the group consisting of: pUC origin, pMB 1 origin, and ColEl origin.
  • said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replicationselection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • said Pol I- dependent origin of replication is selected from the group consisting of: pUC origin, pMB 1 origin, or ColEl origin.
  • said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, SEQ ID NO: 7.
  • RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replicationselection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • current technology provides a method for improving expression from a covalently closed circular viral vector or non-viral transposon plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from Lentiviral vector, Retroviral vector, and AAV vector or non-viral transposon vector; and b) modifying the covalently closed circular recombinant molecule of (a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA- OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol III- dependent origin of replication
  • said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin.
  • said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the present technology provides a method for eliminating antibiotic resistant marker gene transfer from a covalently closed circular viral vector plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker, and ii) an insert comprising an antibiotic resistance marker free eukaryotic region selected from viral vector, Lentiviral vector, Lentiviral packaging vector, Lentiviral envelope vector Retroviral vector, Retroviral envelope vector, Retroviral packaging vector, AAV vector, AAV rep cap vector, Ad helper vector, and Ad helper rep cap vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent
  • said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin.
  • said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the present technology provides a method for eliminating antibiotic resistant marker gene transfer from a covalently closed circular non-viral transposon plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker, and ii) an insert comprising an antibiotic resistance marker free eukaryotic region selected from non-viral transposon vector, non-viral transposase vector, Sleeping Beauty transposon vector, Sleeping Beauty transposase vector, PiggyBac transposon vector, PiggyBac transposase vector, Tol2 transposon vector, Tol2 transposase vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and
  • said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin.
  • said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the present technology provides an antibiotic marker free covalently closed circular recombinant DNA molecule comprising: a) an antibiotic marker free insert comprising a eukaryotic region selected from Lentiviral vector, Lentiviral envelope vector, Lentiviral packaging vector, Retroviral vector, Retroviral envelope vector, Retroviral packaging vector, AAV vector, AAV rep cap vector, Ad helper vector, Ad helper rep cap vector, non- viral transposon vector, and non-viral transposase vector; b) a Pol Ill-dependent origin of replication comprising an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24; and c) an RNA-OUT RNA selectable marker comprising an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO:
  • said R6K gamma replication origin and said RNA-OUT RNA selectable marker comprise a R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region with at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the present technology provides a method for reducing transfection associated toxicity from a covalently closed circular viral vector or non-viral transposon plasmid comprising: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from lentiviral vector, retroviral vector, AAV vector and non-viral transposon vector; modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill- dependent origin of replication covalent
  • said Pol I- dependent origin of replication is selected from the group consisting of: pUC origin, pMB 1 origin, and ColEl origin.
  • said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24.
  • said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7.
  • RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6.
  • said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replicationselection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the resultant Pol Ill-dependent replication origin plasmids have surprisingly improved manufacturing quality and yield than the parent pMBl, ColEl or pBR322 derived replication origin expression plasmid vector.
  • FIGS. 1A-1F depict the R6K origin (FIGS. 1A, IE, and IF), RNA-OUT selectable marker (FIG. IB), and 14 and 3 CpG R6K-RNA-OUT bacterial backbones (FIGS. 1C and ID);
  • FIGS. 2A-2B depict a Pol I-dependent pUC origin Sleeping Beauty transposon vector (FIG. 2A) and a Pol Ill-dependent R6K origin Sleeping Beauty transposon vector (FIG. 2B);
  • FIGS. 3A-3C depict Pol I-dependent pUC origin AAV vectors (FIGS. 3A and 3B) and a Pol Ill-dependent R6K origin AAV vectors (FIG. 3C); and
  • FIGS. 4A-4F depict Pol I-dependent pUC origin A60 polyA repeat encoding mRNA vectors (FIGS. 4A ⁇ 4B), a Pol Ill-dependent R6K origin A60 polyA repeat encoding mRNA vector (FIG. 4C), Pol I-dependent pUC origin A99 polyA repeat encoding mRNA vectors (FIGS. 4D-4E), and a Pol Ill-dependent R6K origin A99 polyA repeat encoding mRNA vector (FIG. 4F).
  • FIGS. 5A-5C depict the Pol Ill-dependent R6K origin R6K-RNA-OUT bacterial backbones from SEQ ID NO: 25 (A) SEQ ID NO: 27 (B) SEQ ID NO: 28 (C) SEQ ID NO: 34.
  • FIGS. 6A-6B depict the Pol Ill-dependent R6K origin AAV ITR encoding AAV vector (FIG. 6A) A 100 polyA repeat encoding mRNA vector (FIG. 6B)
  • FIGS. 7A-7B are BspQl linearizations of purified plasmid DNA from fermentation harvests of mRNA vector -NP (polyA 100 ⁇ ROUT R6K origin>) and mRNA vector -NP 7 iteron PAS R6K> ROUT> (polyA 100 R6K origin> PAS ROUT>).
  • FIG 8 depicts Table 1 : Minicircle applications with various viral and non- viral vector platforms.
  • FIG 9 depicts Table 2: pNTC multiple cloning site flanked R6K Origin-RNA-OUT selection marker vectors.
  • FIG 10 depicts Table 3: SV40 origin Lentiviral vectors: pUC versus R6K origin shake flask production yields/quality.
  • FIG 11 depicts Table 4: Sleeping Beauty Transposon vectors: pUC versus R6K origin shake flask production yields/quality.
  • FIG 12 depicts Table 5: AAV vectors: pUC versus R6K origin shake flask production yields/quality.
  • FIG 13 depicts Table 6: mRNA vectors: pUC versus R6K origin DH5a HyperGRO fermentation yields/quality.
  • FIG 14 depicts Table 7 : AAV helper vectors: pUC versus R6K origin plasmid production yields/quality.
  • FIG 15 depicts Table 8: AAV vectors: R6K origin versus R6K origin + primosomal assembly site shake flask production yields/quality.
  • FIG 16 depicts Table 9: AAV vectors: R6K origin versus R6K origin + primosomal assembly site shake flask production yields/quality.
  • FIG 17 depicts Table 10: AAV ITR vector: R6K origin with and without PAS HyperGRO Fermentation Yields/quality
  • FIG 18 depicts Table 11: mRNA polyA vector: R6K origin with and without PAS HyperGRO Fermentation Yields/quality
  • SEQ ID NO:1 R6K gamma origin
  • SEQ ID NO:2 1 CpG R6K gamma origin
  • SEQ ID NO:3 CpG free R6K gamma origin
  • SEQ ID NO:4 Extended R6K gamma origin
  • SEQ ID NO:5 RNA-OUT Selectable Marker
  • SEQ ID NO:6 RNA-OUT antisense repressor RNA
  • SEQ ID NO:7 2 CpG RNA-OUT Selectable Marker
  • SEQ ID NO:8 R6K gamma origin-RNA-OUT bacterial region flanked by Nhel and Kpnl restriction sites
  • SEQ ID NO:9 1 CpG R6K gamma origin-2 CpG RNA-OUT bacterial region flanked by Nhel and Kpnl restriction sites
  • SEQ ID NO:10 pNTC-NPl polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoR I Hind 111
  • SEQ ID NO:11 pNTC-NP2 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoR I Hind 111
  • SEQ ID NO: 12 pNTC-NP3 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoR I Hind 111
  • SEQ ID NO: 13 pNTC-NP4 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI/Hindin
  • SEQ ID NO:14 pNTC-NP5 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: KasEHindlll
  • SEQ ID NO:15 pNTC-NP6 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRESacI
  • SEQ ID NO:16 pNTC-NP7 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: BssHII-BssHII
  • SEQ ID NO:17 pNTC-3xCpG NP1 polylinker R6K-RNA-OUT polylinker cloning cassette: Hindlll-EcoRI
  • SEQ ID NO:20 R6K gamma origin 22 bp iteron repeat
  • SEQ ID NO:21 R6K gamma origin 22 bp iteron repeat
  • SEQ ID NO:22 R6K gamma origin 22 bp iteron repeat
  • SEQ ID NO:23 R6K gamma origin 22 bp iteron repeat
  • SEQ ID NO:24 1 CpG R6K gamma origin (7 iteron)
  • SEQ ID NO:25 R6K gamma origin (7 iteron)-RNA-OUT bacterial region
  • SEQ ID NO:26 1 CpG R6K gamma origin (7 iteron)-2 CpG RNA-OUT bacterial region
  • SEQ ID NO:31 PAS-BL
  • SEQ ID NO :33 R6K plasmid CpG free ssiA primosomal assembly site
  • SEQ ID NO:34 NP 7 iteron + R6K PAS RNA-OUT bacterial region
  • SEQ ID NO:35 AAV2 ITR
  • SEQ ID NO:36 AAV2 ITR
  • AAV vector Adeno-associated virus vector, an episomal viral vector. Includes self- complementary (sc) Adeno-associated virus vectors (scAAV) and single-stranded (ss) Adeno-associated virus vectors (ssAAV)
  • ampR Ampicillin Resistance gene
  • Antibiotic selectable marker A gene that confirs resistance to an antibiotic, e.g. ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene
  • Bacterial region Region of a plasmid vector required for propagation and selection in the bacterial host
  • ccc Covalently Closed Circular
  • cl Lambda repressor
  • cITs857 Lambda repressor further incorporating a C to T (Ala to Thr) mutation that confers temperature sensitivity.
  • cITs857 is a functional repressor at 28-30°C but is mostly inactive at 37-42°C. Also called cI857
  • cmv Cytomegalovirus
  • DNA replicon A genetic element that can replicate under its own control; examples include plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof
  • EGFP Enhanced green fluorescent protein
  • Eukaryotic expression vector A vector for expression of mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target eukaryotic organism using RNA Polymerase I, II or III promoters
  • Eukaryotic region The region of a plasmid that encodes eukaryotic sequences and/or sequences required for plasmid function in the target organism. This includes the region of a plasmid vector required for expression of one or more transgenes in the target organism including RNA Pol II enhancers, promoters, transgenes and polyA sequences. This also includes the region of a plasmid vector required for expression of one or more transgenes in the target organism using RNA Pol I or RNA Pol III promoters, RNA Pol I or RNA Pol III expressed transgenes or RNAs.
  • the eukaryotic region may optionally include other functional sequences, such as eukaryotic transcriptional terminators, supercoiling-induced DNA duplex destabilized (SIDD) structures, S/MARs, boundary elements, etc.
  • eukaryotic transcriptional terminators such as supercoiling-induced DNA duplex destabilized (SIDD) structures, S/MARs, boundary elements, etc.
  • SIDD supercoiling-induced DNA duplex destabilized
  • S/MARs supercoiling-induced DNA duplex destabilized
  • boundary elements etc.
  • the eukaryotic region contains flanking direct repeat LTRs
  • AAV vector the eukaryotic region contains flanking inverted terminal repeats
  • IR/DR termini e.g. Sleeping Beauty
  • the eukaryotic region may encode homology arms to direct targeted integration
  • Exon A nucleotide sequence encoded by a gene that is transcribed and present within a mature mRNA product after RNA splicing to remove introns has been completed
  • Expression vector A vector for expression of mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target organism.
  • gene of interest gene to be expressed in the target organism. Includes mRNA genes that encode protein or peptide antigens, protein or peptide therapeutics, and mRNA, shRNA, RNA or microRNA that encode RNA therapeutics, and mRNA, shRNA, RNA or microRNA that encode RNA vaccines, etc.
  • Homopolymeric repeat simple sequence DNA repeat of polyA, polyG, polyC or polyT. Can be from a few base pairs to several hundred consecutive base pairs
  • IM Intramuscular
  • immune response Antigen reactive cellular (e.g. antigen reactive T cells) or antibody (e.g. antigen reactive IgG) responses
  • Intron A nucleotide sequence encoded by a gene that is transcribed and subsequently removed from a mature mRNA product by RNA splicing
  • IR/DR Inverted Repeats which are each Directly Repeated twice.
  • Sleeping Beauty transposon IR/DR repeats are each Directly Repeated twice.
  • Iteron Directly repeated DNA sequences in a origin of replication that are required for replication initiation.
  • R6K origin iteron repeats are 22 bp
  • kan Kanamycin
  • kanR Kanamycin Resistance gene
  • a Sall site (GTCGAC) immediately upstream of the ATG start codon (GTCGACATG) is an effective kozak sequence
  • Lentiviral vector Integrative viral vector that can infect dividing and non-dividing cells. Also call Lentiviral transfer plasmid. Plasmid encodes Lentiviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with Lentiviral envelope and packaging plasmids required to make viral particles
  • Lentiviral envelope vector Plasmid encoding envelope glycoprotein
  • Lentiviral packaging vector One or two plasmids that express gag, pol and Rev functions required to package the Lentiviral transfer vector
  • minicircle Covalently closed circular plasmid derivatives in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site-specific recombination or in vitro restriction digestion/ligation. Minicircle vectors are replication incompetent in bacterial cells [00224] mRNA: Messenger RNA
  • mRNA vector vector utilized as in vitro transcription template for mRNA vaccines and therapeutics.
  • the in vitro transcription template is a bacterial transcription unit with a bacterial promoter, typically T7 RNA polymerase promoter, followed by a 5’ UTR, kozak, transgene coding region, 3’ UTR, polyA homopolymeric repeat typically 60 to 120 bp long, ending with a unique restriction site such as BspQI for vector linearization after the polyA run.
  • the BspQI site is positioned such that the linear DNA ends in the polyA repeat, with no ‘non-A’ bases.
  • the linear DNA is utilized in an in vitro transcription reaction to make the therapeutic or vaccine mRNA
  • mSEAP Murine secreted alkaline phosphatase
  • NanoplasmidTM vector Vector combining an RNA selectable marker with a R6K, ColE2 or ColE2 related replication origin.
  • RNA selectable marker for example, NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors and modifications described in Williams, Supra, 2014 and included herein by reference
  • NTC8385 NTC8385, NTC8485 and NTC8685 plasmids are antibiotic-free pUC origin vectors that contain a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR.
  • RNA-OUT short RNA
  • the creation and application of these RNA-OUT based antibiotic-free vectors are described in Williams, JA 2008 World Patent Application W02008153733 and included herein by reference
  • NTC8485 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR.
  • RNA-OUT short RNA
  • the creation and application of NTC8485 is described in Williams, JA 2010 US Patent Application 20100184158 and included herein by reference
  • NTC8685 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR.
  • RNA-OUT short RNA
  • the creation and application of NTC8685 is described in Williams, Supra, 2010 and included herein by reference
  • NTC9385R The NTC9385R NanoplasmidTM vector described in Williams, Supra, 2014 included herein by reference has a spacer region encoded Nhel- trpA terminator-R6K origin RNA-OUT -Kpnl bacterial region (SEQ ID NO:8) linked through the flanking Nhel and Kpnl sites to the eukaryotic region.
  • ODeoo optical density at 600 nm
  • PAS Primosomal assembly site. Priming of DNA synthesis on a single stranded DNA ssi site.
  • 0X174 type PAS DNA hairpin sequence that binds priA, which, in turn, recruits the remaining proteins to form the preprimosome ⁇ priB, dnaT, recruits dnaB (delivered by dnaC) ⁇ , which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I.
  • PAS-BH and PAS-BL from plasmid pBR322.
  • DNA hairpin binds dnaA, recruits dnaB (delivered by dnaC) which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I.
  • dnaC recruits dnaB
  • dnaG primase
  • DNA polymerase I for example, the R6K plasmid CpG free ssiA primosomal assembly site or alternative 0X174 type or ABC type primosomal assembly sites.
  • a primosomal assembly site can be for a DnaA-dependent primosome or for a PriA-dependent primosome as described in Masai, Hisao and Arai, Ken-ichi, Frontiers in Bioscience 1 (1996):d48-d58 which is incorporated herein by reference in its entirety.
  • PAS-BH Primosomal assembly site on the heavy (leading) strand of pBR322
  • PAS-BH region pBR322 origin region between ROP and PAS-BL (approximately pBR322 2067-2351)
  • PAS-BL Primosomal assembly site on the light (lagging) strand of pBR322
  • PBS Phosphate buffered Saline
  • PiggyBac Transposon PB transposon.
  • a transposon system that integrates an ITR flanked PB transposon into the genome by a simple cut and paste mechanism mediated by PB transposase.
  • the transposon vector typically contains a promoter-transgene-polyA expression cassette between the PB ITRs which is excised and integrated into the genome
  • pINT pR pL vector The pINT pR pL attHK022 integration expression vector is described in Luke et al., 2011 Mol Biotechnol $TA3 and included herein by reference.
  • the target gene to be expressed is cloned downstream of the pL promoter.
  • the vector encodes the temperature inducible cI857 repressor, allowing heat inducible target gene expression
  • PL promoter Lambda promoter left.
  • PL is a strong promoter that is repressed by the cl repressor binding to OL1, OL2 and OL3 repressor binding sites.
  • the temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30°C the cI857 repressor is functional and it represses gene expression, but at 37-42 °C the repressor is inactivated so expression of the gene ensues
  • PL (OL1 G to T) promoter Lambda promoter left.
  • PL is a strong promoter that is repressed by the cl repressor binding to OL1, OL2 and OL3 repressor binding sites.
  • the temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30°C the cI857 repressor is functional and it represses gene expression, but at 37-42 °C the repressor is inactivated so expression of the gene ensues.
  • the cl repressor binding to OL1 is reduced by the OL1 G to T mutation resulting in increased promoter activity at 30°C and 37-42 °C as described in Williams, Supra. 2014.
  • Plasmid An extra chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA
  • Plasmid copy number the number of copies of a plasmid per cell. Increases in plasmid copy number increase plasmid production yield
  • Pol I Escherichia coli DNA Polymerase I
  • Pol I dependent origin of replication A replication origin that requires Pol I, for example the pMBl, ColEl or pBR322 or derivatives such as the high copy pUC origin.
  • the RNAII primer forms an RNA: DNA R-loop that is cleaved by RNase H to create a primer for DNA pol I directed DNA synthesis. DNA synthesis then converts to DNA pol III.
  • Numerous additional Pol I dependent replication origins are known in the art, many of which are summarized in del Solar et al., 1998 Microbiol. Mol. Biol. Rev 62:434-464 which is included herein by reference
  • Pol III Escherichia coli DNA Polymerase III
  • Pol III dependent origin of replication A replication origin that doesn’t require Pol I, for example the rep protein dependent R6K gamma replication origin. Numerous additional Pol III dependent replication origins are known in the art, many of which are summarized in del Solar et al., Supra, 1998 which is included herein by reference
  • polyA Polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule.
  • the polyadenylation signal contains the sequence motif recognized by the RNA cleavage complex. Most human polyadenylation signals contain an AAUAAA motif and conserved sequences 5’ and 3’ to it. Commonly utilized polyA signals are derived from the rabbit [! globin, bovine growth hormone, SV40 early, or SV40 late polyA signals [00253] polyA repeat: simple sequence DNA repeat of polyA from a few base pairs to several hundred consecutive base pairs
  • pUC origin pBR322-derived replication origin, with G to A transition that increases copy number at elevated temperature and deletion of the ROP negative regulator
  • pUC free Plasmid that does not contain the pUC origin.
  • Non-replicative fragments of the pUC origin may be included, for example the RNAI selectable marker
  • pUC plasmid Plasmid containing the pUC origin
  • R6K plasmid NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a-01, NTC9385R2a-O2, NTC9385R2b-01, NTC9385R2b-O2, NTC9385Ra-01, NTC9385Ra-O2, NTC9385RaF, and NTC9385RbF vectors as well as modifications and alternative vectors containing a R6K replication origin that were described in Williams, Supra, 2014 and included herein by reference.
  • Alternative R6K vectors known in the art including, but not limited to, pCOR vectors (Gencell), pCpGfree vectors (Invivogen), and CpG free University of Oxford vectors including pGM169
  • R6K replication origin a region which is specifically recognized by the R6K Rep protein to initiate DNA replication. Includes but not limited to R6K gamma replication origin sequence disclosed as SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO: 18 and SEQ ID NO:24. Also includes CpG free versions (e.g. SEQ ID NO:3) as described in Drocourt et al., United States Patent 7244609 and incorporated herein by reference
  • R6K replication origin-RNA-OUT bacterial region Contains a R6K replication origin for propagation and the RNA-OUT selectable marker (e.g. SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO:13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28)
  • SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO:13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28 e.g. SEQ ID NO:8; SEQ ID NO:9;
  • Replication intermediates Linear DNA fragments resulting from premature termination of plasmid replication
  • Rep protein dependent plasmid A plasmid in which replication is dependent on a replication (Rep) protein provided in Trans.
  • Rep replication
  • R6K replication origin For example, R6K replication origin, ColE2-P9 replication origin and ColE2 related replication origin plasmids in which the Rep protein is expressed from the host strain genome.
  • Numerous additional Rep protein dependent plasmids are known in the art, many of which are summarized in del Solar etal., Supra, 1998 which is included herein by reference
  • Retroviral vector Integrative viral vector that can infect dividing cells. Also call transfer plasmid. Plasmid encodes Retroviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with envelope and packaging plasmids required to make viral particles
  • Retroviral envelope vector Plasmid encoding envelope glycoprotein
  • Retroviral packaging vector Plasmid that encodes Retroviral gag, pol genes required to package the Retroviral transfer vector
  • RNA-IN Insertion sequence 10 (IS 10) encoded RNA-IN, an RNA complementary and antisense to a portion of RNA RNA-OUT.
  • IS 10 Insertion sequence 10
  • RNA-IN regulated selectable marker A genomically expressed RNA-IN regulated selectable marker. In the presence of plasmid borne RNA-OUT antisense repressor RNA (SEQ ID NO:6), expression of a protein encoded downstream of RNA-IN is repressed.
  • An RNA-IN regulated selectable marker is configured such that RNA-IN regulates either 1) a protein that is lethal or toxic to said cell per se or by generating a toxic substance (e.g. SacB), or 2) a repressor protein that is lethal or toxic to said bacterial cell by repressing the transcription of a gene that is essential for growth of said cell (e.g.
  • RNA-IN-SacB cell lines for RNA-OUT plasmid selection/propagation are described in Williams, Supra, 2008 and included herein by reference.
  • Alternative selection markers described in the art may be substituted for SacB
  • RNA-OUT Insertion sequence 10 (IS 10) encoded RNA-OUT, an antisense RNA that hybridizes to, and reduces translation of, the transposon gene expressed downstream of RNA-IN.
  • the sequence of the RNA-OUT RNA (SEQ ID NO:6) and complementary RNA-IN SacB genomically expressed RNA-IN-SacB cell lines can be modified to incorporate alternative functional RNA-IN/RNA-OUT binding pairs such as those described in Mutalik et al., 2012 Nat Chem Biol 8:447, including, but not limited to, the RNA-OUT A08/RNA-IN S49 pair, the RNA- OUT A08/RNA-IN S08 pair, and CpG free modifications of RNA-OUT A08 that modify the CG in the RNA-OUT 5’ TTCGC sequence to a non-CpG sequence.
  • CpG free RNA-OUT selection marker in which the two CpG motifs in the RNA-OUT RNA (one of which is present in the RNA-IN complementary region) are removed, was described in Williams 2015. Replicative minicircle vectors with improved expression. US Patent Application US 2015/0275221 and included herein by reference. A multitude of alternative substitutions to remove the two CpG motifs (mutating each CpG to either CpA, CpC, CpT, ApG, GpG, or TpG) may be utilized to make a CpG free RNA-OUT
  • RNA-OUT Selectable marker An RNA-OUT selectable marker DNA fragment including E. coli transcription promoter and terminator sequences flanking an RNA-OUT RNA.
  • An RNA-OUT selectable marker utilizing the RNA-OUT promoter and terminator sequences, that is flanked by Dralll and Kpnl restriction enzyme sites, and designer genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation, are described in Williams, Supra, 2008 and included herein by reference.
  • the RNA-OUT promoter and terminator sequences in SEQ ID NO: 5 that flank the RNA-OUT RNA may be replaced with heterologous promoter and terminator sequences.
  • the RNA-OUT promoter may be substituted with a CpG free promoter known in the art, for example the I-EC2K promoter or the P5/6 5/6 or P5/6 6/6 promoters described in Williams, Supra, 2008 and included herein by reference.
  • a 2 CpG RNA-OUT selectable marker in which the two CpG motifs in the RNA-OUT promoter are removed is given as SEQ ID NO: 7.
  • An example of a CpG free RNA-OUT transcription unit, in which the two CpG motifs in the RNA-OUT RNA (one of which is present in the RNA-IN complementary region) and the two CpG motifs in the RNA-OUT promoter are removed was described in Williams, Supra, 2015 and included herein by reference.
  • Vectors incorporating CpG free RNA-OUT selectable marker may be selected for sucrose resistance using the RNA-IN-SacB cell lines for RNA-OUT plasmid propagation described in Williams, Supra, 2008.
  • the RNA-IN sequence in these cell lines can be modified to incorporate the 1 bp change needed to perfectly match the CpG free RNA-OUT region complementary to RNA- IN.
  • RNA polymerase II promoter Promoter that recruits RNA Polymerase II to synthesize mRNAs, most small nuclear RNAs and microRNAs.
  • constitutive promoters such as the human or murine CMV promoter, elongation factor 1 (EFl) promoter, the chicken [! -actin promoter, the [!
  • EFl a elongation factor- 1 a
  • PGK phosphoglycerokinase
  • RSV Rous sarcoma virus
  • SA human serum albumin
  • SFFV spleen focus-forming virus
  • AAT thyroxine binding globulin
  • TG cytochrome P450 2E1
  • the vectors may also utilize combination promoters such as the chicken [3 -actin/CMV enhancer (CAG) promoter, the human or murine CMV-derived enhancer elements combined with the elongation factor la (EFla) promoters, CpG free versions of the human or murine CMV-derived enhancer elements combined with the elongation factor la (EFla) promoters, the albumin promoter combined with an a -fetoprotein MERII enhancer, etc., or the diversity of tissue specific or inducible promoters know in the art such as the muscle specific promoters muscle creatine kinase (MCK), and C5-12 or the liverspecific promoter apolipoprotein A-I (ApoAI), etc.
  • CAG chicken [3 -actin/CMV enhancer
  • EFla elongation factor la
  • EFla elongation factor la
  • albumin promoter combined with an a -fetoprotein MERII enhancer
  • RNA polymerase III promoter Promoter that recruits RNA Polymerase III to synthesize tRNAs, 5S ribosomal RNA, and other small RNAs.
  • Class I promoters such as the 5s rRNA promoter
  • Class II promoter such as tRNA promoters
  • Class III promoters such as the U6 small nuclear RNA promoter or the Hl nuclear RNase P promoter, etc.
  • RNA selectable marker is a plasmid borne expressed nontranslated RNA that regulates a chromosomally expressed target gene to afford selection. This may be a plasmid borne nonsense suppressing tRNA that regulates a nonsense suppressible selectable chromosomal target as described by Crouzet J and Soubrier F 2005 US Patent 6,977,174 included herein by reference.
  • This may also be a plasmid bome antisense repressor RNA, a non limiting list included herein by reference includes RNA-OUT that represses RNA- IN regulated targets (Williams, Supra, 2008), pMBl plasmid origin encoded RNAI that represses RNAII regulated targets (Grabherr R, Pfaffenzeller I. 2006 US patent application US20060063232; Cranenburgh RM. 2009; US Patent 7,611,883), IncB plasmid pMU720 origin encoded RNAI that represses RNA II regulated targets (Wilson IW, Siemering KR, Praszkier J, Pittard AJ, 1997.
  • RNA selectable marker may be another natural antisense repressor RNAs known in the art such as those described in Wagner EGH, Altuvia S, Romby P. 2002. Adv Genet 46:361-98 and Franch T, and Gerdes K. 2000. Current Opin Microbiol 3: 159- 64.
  • RNA selectable marker may also be an engineered repressor RNAs such as synthetic small RNAs expressed SgrS, MicC or MicF scaffolds as described in Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. 2013. Nat Biotechnol 31: 170-4.
  • An RNA selectable marker may also be an engineered repressor RNA as part of a selectable marker that represses a target RNA fused to a target gene to be regulated such as SacB as described in Williams, Supra, 2015
  • RSM RNA selectable marker
  • SacB Structural gene encoding Bacillus subtilis levansucrase. Expression of SacB in gram negative bacteria is toxic in the presence of sucrose
  • sequence identity refers to the degree of identity between any given query sequence, e.g. SEQ ID NO: 2, and a subject sequence.
  • a subject sequence may, for example, have at least 90 percent, at least 95 percent, or at least 99 percent sequence identity to a given query sequence.
  • a query sequence e.g. a nucleic acid sequence
  • ClustalW version 2.1, default parameters
  • the sequence alignment program calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more nucleotides can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair- wise alignments of nucleic acid sequences, suitable default parameters can be selected that are appropriate for the particular alignment program.
  • the output is a sequence alignment that reflects the relationship between sequences.
  • the sequences are aligned using the alignment program, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • Selectable marker A selectable marker, for example a kanamycin resistance gene or an RNA selectable marker
  • Selection marker A selectable marker, for example a kanamycin resistance gene or an RNA selectable marker
  • SIDD supercoiling-induced DNA duplex destabilized
  • shRNA Short hairpin RNA
  • S/MAR Scaffold/matrix attached region. Eukaryotic sequences that mediate DNA attachment to the nuclear matrix
  • Sleeping Beauty Transposon SB transposon.
  • the transposon vector typically contains a promoter-transgene-polyA expression cassette between the IR/DRs which is excised and integrated into the genome
  • Spacer region is the region linking the 5’ and 3’ ends of the eukaryotic region sequences.
  • the eukaryotic region 5’ and 3’ ends are typically separated by the bacterial replication origin and bacterial selectable marker in plasmid vectors (bacterial region) so many spacer regions consist of the bacterial region. In Pol III dependent origin of replication vectors of the invention, this spacer region preferably is less than 1000 bp [00286] SR: Spacer region.
  • Structured DNA sequence As used herein, a DNA sequence that is capable of forming replication inhibiting secondary structures (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71: 13-35). This includes but is not limited to inverted repeats, palindromes, direct repeats, IR/DRs, homopolymeric repeats or repeat containing eukaryotic promoter enhancers, or repeat containing eukaryotic origin of replications.
  • SV40 origin Simian Virus 40 genomic DNA that contains the origin of replication
  • SV40 enhancer Simian Virus 40 genomic DNA that contains the 72 bp and optionally the 21 bp enhancer repeats
  • target antigen Immunogenic protein or peptide epitope, or combination of proteins and epitopes, against which an immune response can be mounted.
  • Target antigens may by derived from a pathogen for infectious disease or allergy applications or derived from a host organism for applications such as cancer, allergy, or autoimmune diseases.
  • Target antigens are well defined in the art. Some examples are described in Williams, Supra, 2008 and are included herein by reference
  • TE buffer A solution containing approximately lOmM Tris pH 8 and 1 mM EDTA
  • TetR Tetracycline resistance gene
  • Tol2 Transposon A transposon system that integrates an ITR flanked Tol2 transposon into the genome by a simple cut and paste mechanism mediated by Tol2 transposase.
  • the transposon vector typically contains a promoter-transgene-polyA expression cassette between the Tol2 ITRs which is excised and integrated into the genome
  • Transcription terminator Bacterial: A DNA sequence that marks the end of a gene or operon for transcription. This may be an intrinsic transcription terminator or a Rho-dependent transcriptional terminator. For an intrinsic terminator, such as the trpA terminator, a hairpin structure forms within the transcript that disrupts the mRNA-DNA-RNA polymerase ternary complex. Alternatively, Rho-dependent transcriptional terminators require Rho factor, an RNA helicase protein complex, to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex.
  • Eukaryotic PolyA signals are not ‘terminators’, instead internal cleavage at PolyA sites leaves an uncapped 5 ’end on the 3’UTR RNA for nuclease digestion. Nuclease catches up to RNA Pol II and causes termination. Termination can be promoted within a short region of the poly A site by introduction of RNA Pol II pause sites (eukaryotic transcription terminator). Pausing of RNA Pol II allows the nuclease introduced into the 3’ UTR mRNA after PolyA cleavage to catch up to RNA Pol II at the pause site.
  • a nonlimiting list of eukaryotic transcription terminators know in the art include the C2x4 and the gastrin terminator. Eukaryotic transcription terminators may elevate mRNA levels by enhancing proper 3'-end processing of mRNA
  • transfection Method to deliver nucleic acids into cells [e.g. poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, block copolymers, Pluronic block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleofection, piezoelectric permeabilization, sonoporation, iontophoresis, ultrasound, SQZ high speed cell deformation mediated membrane disruption, corona plasma, plasma facilitated delivery, tissue tolerable plasma, laser microporation, shock wave energy, magnetic fields, contactless magneto-permeabilization, gene gun, microneedles, microdermabrasion, hydrodynamic delivery, high pressure tail vein injection, etc] as known in the art and included herein by reference
  • Transgene Gene of interest that is cloned into a vector for expression in a target organism
  • Transposase vector A vector which encodes a transposase
  • Transposon vector A vector which encodes a transposon which is a substrate for transposase mediated gene integration
  • UTR Untranslated region of a mRNA (5’ or 3’ to the coding region)
  • Vector A gene delivery vehicle, including viral (e.g. Alphavirus, Poxvirus, Lentivirus, Retrovirus, Adenovirus, Adenovirus related virus, etc.) and non-viral (e.g. plasmid, MIDGE, transcriptionally active PCR fragment, minicircles, bacteriophage, etc.) vectors. These are well known in the art and are included herein by reference [00305] Vector backbone: Eukaryotic region and bacterial region of a vector, without the transgene or target antigen coding region
  • the current technology relates generally to short ⁇ Ikb bacterial region plasmid DNA vector methods and compositions that improve plasmid manufacture yield and quality, reduce transfection associated toxicity, and increase transgene expression.
  • the current technology can be practiced to improve expression and manufacturing of vectors such as non-viral vectors (mRNA vector, transposon vector, transposase vector, Sleeping Beauty transposon vector, Sleeping Beauty transposase vector, PiggyBac transposon vector, PiggyBac transposase vector, expression vector, etc.) and viral vectors (e.g. AAV vector, AAV rep cap vector, AAV helper vector, Ad helper vector, Lentivirus vector, Lentiviral envelope vector, Lentiviral packaging vector, Retroviral vector, Retroviral envelope vector, Retroviral packaging vector, etc.).
  • viral vectors e.g. AAV vector, AAV rep cap vector, AAV helper vector, Ad helper vector, Lentivirus vector, Lentiviral envelope vector, L
  • Improved plasmid expression is defined herein as improved transgene expression level and/or expression duration in vitro or in vivo compared to a transgene encoding plasmid containing a bacterial region encoding the pUC replication origin. It is to be understood that all references cited herein are incorporated by reference in their entirety,
  • compositions Comprising a Pol Ill-Dependent Origin of Replication
  • compositions comprising a DNA molecule comprising a backbone comprising a Pol Ill-dependent origin of replication are disclosed
  • a covalently closed circular recombinant DNA molecule comprising a backbone and an insert, wherein the backbone comprises a Pol Ill-dependent origin of replication, a selectable marker, and a first primosomal assembly site, wherein the first primosomal assembly site is positioned downstream of the Pol Ill-dependent origin of replication in the direction of replication, and wherein the insert comprises a structured DNA sequence, is disclosed.
  • structured DNA sequence is within 1000 bp of the Pol Ill-dependent origin of replication.
  • the backbone is less than 1000 bp.
  • the backbone comprises a bacterial replication-selection region.
  • the Pol Ill-dependent origin of replication does not require Pol I.
  • the Pol Ill-dependent origin of replication is a Pol Ill-dependent R6K origin of replication.
  • the Pol Ill-dependent R6K origin of replication has at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24.
  • the selectable marker is an RNA selectable marker.
  • the RNA selectable marker is an RNA-OUT RNA selectable marker
  • the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA- OUT functional variant with at least 80% sequence identity to a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7.
  • the RNA selectable marker comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6.
  • the first primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
  • the covalently closed circular recombinant DNA molecule further comprises a second primosomal assembly site downstream of the Pol Ill-dependent origin of replication in the direction of replication,
  • the second primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
  • the covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the Pol Ill-dependent origin of replication in the direction of replication.
  • the covalently closed circular recombinant DNA molecule of any one of claims 13-14 comprises the sequence of SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the Pol Ill-dependent origin of replication in the direction of replication.
  • the covalently closed circular recombinant DNA molecule is antibiotic marker free.
  • the backbone comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the backbone comprises a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the structured DNA sequence is selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence.
  • the insert is a transposon vector.
  • the structured DNA sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
  • the insert is a transposase vector.
  • the insert is a mRNA vector.
  • the insert is an AAV vector.
  • the structured DNA sequence is an inverted repeat sequence.
  • the AAV vector encodes AAV2 ITRs.
  • the insert comprises a 5’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 35 and a 3’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 36.
  • the insert is a lentiviral vector.
  • the structured DNA sequence is a direct repeat sequence or an eukaryotic origin of replication.
  • the structured DNA sequence is selected from an homopolymeric repeat such as a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
  • an homopolymeric repeat such as a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
  • the structured DNA sequence comprises a homopolymeric repeat.
  • the homopolymeric repeat is a polyA repeat.
  • the homopolymeric repeat comprises from about 3 to about 500 residues.
  • the selectable marker is a RNA selectable marker and is oriented to transcribe in a direction divergent from the structured DNA sequence.
  • an antibiotic marker free covalently closed circular recombinant DNA molecule is disclosed, the molecule comprising a backbone and an insert, wherein the backbone comprises an origin of replication and an RNA selectable marker, wherein the insert comprises a structured DNA sequence, and wherein the RNA selectable marker is oriented to transcribe in a direction divergent from the structured DNA sequence.
  • the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is within 1000 bp of the origin of replication, [00347] In embodiments, the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule is less than 1000 bp.
  • the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a bacterial replication-selection region.
  • the origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule is a Pol I-dependent origin of replication.
  • the origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule is a Pol Ill-dependent origin of replication.
  • the Pol Ill-dependent origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule does not require Pol I.
  • the Pol Ill-dependent origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule is a Pol Ill-dependent R6K origin of replication.
  • the Pol Ill-dependent R6K origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule has at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24.
  • the RNA selectable marker of the antibiotic marker free covalently closed circular recombinant DNA molecule is an RNA-OUT RNA selectable marker.
  • the RNA-OUT RNA selectable marker of the antibiotic marker free covalently closed circular recombinant DNA molecule is an RNA-IN regulating RNA-OUT functional variant with at least 80% sequence identity to a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7.
  • the RNA selectable marker of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6.
  • the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence.
  • the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a transposon vector.
  • the structured DNA of the antibiotic marker free covalently closed circular recombinant DNA molecule sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
  • the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a transposase vector.
  • the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a mRNA vector.
  • the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is an AAV vector.
  • the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is an inverted repeat sequence.
  • the AAV vector of the antibiotic marker free covalently closed circular recombinant DNA molecule encodes AAV2 ITRs.
  • the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a 5’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 35 and a 3’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 36.
  • the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a lentiviral vector.
  • the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is a direct repeat sequence or an eukaryotic origin of replication.
  • the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is selected from a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
  • the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a homopolymeric repeat.
  • the homopolymeric repeat of the antibiotic marker free covalently closed circular recombinant DNA molecule is a polyA repeat.
  • the homopolymeric repeat of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises from about 3 to about 500 residues.
  • the antibiotic marker free covalently closed circular recombinant DNA molecule further comprises a first primosomal assembly site downstream of the origin of replication in the direction of replication,
  • the antibiotic marker free covalently closed circular recombinant DNA molecule further comprises a second primosomal assembly site downstream of the origin of replication in the direction of replication.
  • the first primosomal assembly site of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 , and SEQ ID NO: 33.
  • the first primosomal assembly site of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 , and SEQ ID NO: 33
  • the second primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 , and SEQ ID NO: 33.
  • the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site is downstream of the origin of replication in the direction of replication.
  • the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the origin of replication in the direction of replication.
  • a method for replicating a circular recombinant DNA molecule is disclosed. In embodiments, a method for replicating a closed circular recombinant DNA molecule is disclosed. In embodiments, a method for replicating a covalently closed circular recombinant DNA molecule is disclosed.
  • the method comprises providing a cell containing the recombinant DNA molecule as disclosed herein and subjecting the cell to a fermentation process.
  • the cell is an engineered Rep protein-expressing E. coli strain.
  • the cell comprises a chromosomally-integrated arabinose inducible CI857ts gene.
  • the Rep protein comprises at least one of the following mutations: P42L; P 1061; F107S; and Pl 13S. In embodiments, the Rep protein comprises at least two of the following mutations: P42L; P106I; F107S; and P113S. In embodiments, the Rep protein comprises three of the following mutations: P42L; P106I; F107S; and P113S. In embodiments, the Rep protein comprises all four of the following mutations: P42L; P 1061; F107S; and Pl 13S. [00395] hi embodiments, the fermentation process comprises growing the cells in media containing arabinose.
  • the yield of the covalently closed circular plasmid following the fermentation process is in excess of 0.5 g/L.
  • FIGS. 1A-1F show annotated maps of: FIG. 1A) R6K origin with the locations of the 22 bp iteron repeats, DnaA boxes 1 and 2, and the regions included in the SEQ ID NO: 1, 2, 3, and 4 R6K origins; FIG. IB) SEQ ID NO: 5 RNA-OUT selectable marker with the locations of the RNA-OUT promoter -35 and -10 elements, SEQ ID NO: 6 RNA OUT antisense RNA with RNA-IN complementary homology region and RNA-OUT terminator 3’ hairpin; FIG.
  • the individual 22 bp iteron repeat sequences are shown below the origin map; and FIG. IF) R6K origin from SEQ ID NO: 18, with locations of the 7 iterons highlighted.
  • the individual 22 bp iteron repeat sequences are shown below the origin map.
  • 7 iteron vector iteron 5 has been tandemly duplication; however, a 7 iteron vector of the invention can be obtained by tandem duplication of any of iterons 1, 2, 3, 4, 5 or 6.
  • FIGS. 2A-2B show annotated maps of: FIG. 2A) Pol I-dependent pUC origin- Kanamycin selection Sleeping Beauty transposon vector pUC57-Kan SB1 (see Table 4); and FIG. 2B) Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection Sleeping Beauty transposon vector NTC9 SB1 (see Table 4). The locations of the left and right Sleeping Beauty IR/DR relative to the bacterial backbone replication origins and selection markers are shown.
  • FIGS. 3A-3C show annotated maps of: FIG. 3A) Pol I-dependent pUC origin- Ampicillin selection AAV vector pAAV (see Table 7); FIG. 3B) Pol I-dependent pUC origin- RNA-OUT antibiotic free selection AAV vector NTC8-AAV (see Table 7); and FIG. 3C) Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection AAV vector NTC9-AAV (see Table 7). The locations of the left and right AAV ITRs relative to the bacterial backbone replication origins and selection markers are shown.
  • FIGS. 4A— 41 show annotated maps of: FIG. 4A) Pol I-dependent pUC origin- Ampicillin selection A60 polyA repeat encoding mRNA vector pGEM4Z T7 A60 pA (see Table 6); FIG. 4B) Pol I-dependent pUC origin-RNA-OUT antibiotic free selection A60 polyA repeat encoding mRNA vector NTC8-T7 A60 pA (see Table 6); FIG. 4C) Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection A60 polyA repeat encoding mRNA vector NTC9-T7 A60 pA (see Table 6); FIG.
  • FIG. 4D Pol I-dependent pUC origin- Ampicillin selection A99 polyA repeat encoding mRNA vector pT3/T7 A99 pA (see Table 6);
  • FIG. 4E Pol I-dependent pUC origin-kanR selection A99 polyA repeat encoding mRNA vector NTC7-T7 A99 pA (see Table 6);
  • FIG. 4F Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection A99 polyA repeat encoding mRNA vector NTC9-T7 A99 pA (see Table 6). The location of the A60 or A99 polyA repeat relative to the bacterial backbone replication origins and selection markers are shown.
  • FIGS. 5A-5D show annotated maps of the Pol Ill-dependent R6K origin R6K-RNA- OUT bacterial backbones from SEQ ID NO: 25 (A) SEQ ID NO: 27 (B) SEQ ID NO: 28 (C) SEQ ID NO: 34 (D)
  • FIGS. 6A-6B show annotated maps of an example Pol Ill-dependent R6K origin AAV ITR encoding AAV vector (FIG. 6A) or A 100 polyA repeat encoding mRNA vector (FIG. 6B)
  • FIGS. 7A-7B are BspQl linearizations of purified plasmid DNA from fermentation harvests of (Fig. 7A) mRNA vector -NP (polyAlOO ⁇ ROUT R6K origin>) and (Fig. 7B) 2 different fermentation harvest lots of mRNA vector -NP 7 iteron PAS R6K> ROUT> (polyAlOO R6K origin>_PAS ROUT>).
  • Example 1 pUC, and R6K replication origin plasmid replication and production
  • the vast majority of therapeutic plasmids use the pUC origin which is a high copy derivative of the pMBl origin (closely related to the ColEl origin).
  • plasmid DNA synthesis is unidirectional and does not require a plasmid borne initiator protein.
  • the pUC origin is a copy -up derivative of the pMB 1 origin that deletes the accessory ROP (rom) protein and has an additional temperature sensitive mutation that destabilizes the RNAFRNAII interaction. Shifting of a culture containing these origins from 30 to 42°C leads to an increase in plasmid copy number.
  • pUC plasmids can be produced in a multitude of E. coli cell lines.
  • RNA-OUT antibiotic free selectable marker background Antibiotic-free selection is performed in E. coli strains containing phage lambda attachment site chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) as described in Williams, Supra, 2008. SacB (Bacillus subtilis levansucrase) is a counterselectable marker which is lethal to E. coli cells in the presence of sucrose. Translation of SacB from the RNA-IN-SacB transcript is inhibited by plasmid encoded RNA-OUT (Fig. IB). This facilitates plasmid selection in the presence of sucrose, by inhibition of SacB mediated lethality.
  • R6K origin vector replication and production background The R6K gamma plasmid replication origin requires a single plasmid replication protein TJ that binds as a replication initiating monomer to multiple repeated ‘heron’ sites (seven core repeats containing TGAGNG consensus) and as a replication inhibiting dimer to repressive sites (TGAGNG) and to iterons with reduced affinity.
  • Replication requires multiple host factors including IHF, DnaA, and primosomal assembly proteins DnaB, DnaC, DnaG (Abhyankar et al,, 2003 J Biol Chem 278:45476-45484),
  • the R6K core origin contains binding sites for DnaA and IHF that affect plasmid replication since ft, IHF and DnaA interact to initiate replication.
  • R6K gamma replication origin has been utilized in various eukaryotic expression vectors, for example pCOR vectors (Soubrier et al., 1999, Gene Therapy 6: 1482-88) and a CpG free version in pCpGfree vectors (Invivogen, San Diego CA), and pGM169 (University of Oxford). Incorporation of the R6K replication origin per se does not improve transgene expression levels compared to an optimized pUC origin vector (Soubrier et al., Supra, 1999). However, use of a conditional replication origin such as R6K gamma that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patient’s endogenous flora.
  • conditional replication origin such as R6K gamma that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patient’s endogenous flora.
  • a highly minimalized 6 iteron R6K gamma derived replication origin (SEQ ID NO: 1; Fig. IE) that contains core sequences required for replication (including the DnaA box and stb 1- 3 sites; Wu et al., 1995. J Bacterial. 177: 6338-6345), but with the upstream p dimer repressor binding sites and downstream n promoter deleted (by removing one copy of the iterons) was described in Williams, Supra, 2014 and incorporated herein by reference.
  • This R6K origin contains 6 tandem direct repeat iterons (Fig. IE).
  • Typical R6K production strains express from the genome the P protein derivative PIR116 that contains a P106L substitution that increases copy number (by reducing P dimerization; p monomers activate while P dimers repress). Fermentation results with pCOR (Soubrier et al., Supra, 1999) and pCpG plasmids (Hebei HL, Cai Y, Davies LA, Hyde SC, Pringle IA, Gill DR. 2008. Mol Ther 16: Si 10) were low, around 100 mg/L in PIR116 cell lines. [00412] Mutagenesis of the pir-116 replication protein and selection for increased copy number has been used to make new production strains.
  • the TEX2pir42 strain contains a combination of P106L and P42L.
  • the P42L mutation interferes with DNA looping replication repression.
  • the TEX2pir42 cell line improved copy number and fermentation yield with pCOR plasmids with reported yields of 205 mg/L (Soubrier E. 2004. World Patent Application W02004033664).
  • Fl copy number mutants that improve copy number include ‘P42L and Pl 13S’ and ‘P42L, P106L and F107S’ (Abhyankar et al., 2004. J Biol Chem 279:6711-6719).
  • Williams, Supra, 2014 describes host strains expressing phage HK022 attachment site integrated pL promoter heat inducible P P42L, P106L and F107S high copy mutant replication (Rep) protein for selection and propagation of R6K origin NanoplasmidTM vectors. This is an additional NanoplasmidTM safety factor since R6K origin vectors can only replicate within the engineered Rep protein-expressing E. coli host strain.
  • NTC821601 DH5a atW:P 5/6 6/6-RNA-IN- SacB, catR; att H K02 2 :: pL (OL1-G to T) P42L- P106L-F107S (P3-), SpecR StrepR dcm+ version ofNTC711772
  • NTC940211 DH5a att ⁇ ::P 5 /66/6-RNA-IN- SacB, catR; att H K022::pL (OL1-G to T) P42L- P106I-F107S P113S (P3-), SpecR StrepR high copy substitution of P106I for P106L combined with P113S to create a quadruple copy number increasing mutant rep protein derivative of NTC821601
  • NTC1050811 DH5a att ⁇ ::P 5 /66/6-RNA-IN- SacB, catR; att H K022::pL (OLl-G to T) P42L- P106I-F107S P113S (P3-), SpecR StrepR; att ⁇ p8o::pARA-CI857ts, tetR pARA-CI857ts derivative of NTC940211.
  • This strain contains a phage cp80 attachment site chromosomally integrated copy of a arabinose inducible CI857ts gene. Addition of arabinose to plates or media (e.g.
  • SpecR StrepR Stbl4 version of NTC66113.5 (XI ,.l Blue- dem- att>.::P5/6 6/6-RNA- IN- SacB, catR; attHK022"pR pL P42L P106L-F107S ( 1’3 - ) SpecR StrepR described in Williams, Supra, 2014
  • NanoplasmidTM production yields are improved with the quadruple mutant heat inducible pL (OL1-G to T) P42L-P106I-F107S P113S (P3-) compared to the triple mutant heat inducible pL (OL1-G to T) P42L-P106L-F107S (P3-) described in Williams, Supra, 2014. Yields in excess of 2 g/L NanoplasmidTM have been obtained with the quadruple mutant NTC 1050811 cell line (e.g. 2240 mg/L with NTC9 T7 A99 pA, Table 6)
  • conditional replication origin such as these R6K origins that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patient’s endogenous flora.
  • Shake flask production was performed using proprietary PlasmidF shake culture medium. The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 pg/mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids).
  • the plates were grown at 30-32°C; cells were resuspended in media and used to provide approximately 2.5 ODgoo inoculums for the 500 mL Plasmid+ shake flasks that contained 50 pg/mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose to select for RNA-OUT plasmids. Flasks were grown with shaking to saturation at the growth temperatures as indicated in Tables 5, 6, 7, and 9.
  • Fermentation production Fermentations were performed using proprietary fed-batch media (NTC3019, HyperGRO media) in New Brunswick BioFlo 110 bioreactors as described (Carnes and Williams, Supra, 2011). The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 pg/mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids).
  • the plates were grown at 30-32°C; cells were resuspended in media and used to provide approximately 0.1% inoculums for the fermentations that contained 50 pg/mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT plasmids. HyperGRO temperature shifts were as indicated in Tables 8 and 9.
  • Production hosts pUC origin AmpR or KanR plasmid fermentations were performed in E. coli strain DH5a [F- Q80/acZAM15 A(ZacZYA-argF) U169 recAl ewt/Al /m/R17 (rK- mK+) phoS supEA6 X- thi-1 gyr A96 re/A I ] (Invitrogen, Carlsbad CA) or Stbl4.
  • RNA-OUT plasmid fermentations were performed in E. coli strain DH5a containing phage lambda attachment site chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) as described in Williams, Supra, 2008.
  • RNA-OUT plasmid propagation and fermentations were performed using E. coli RNA-OUT selection hosts further encoding phage HK022 attachment site integrated pL promoter heat inducible TJ copy number mutant cell line lines, methods for the creation of which are described in Williams, Supra, 2014 and included herein by reference.
  • Example 3 pUC and R6K origin structured vector construction and manufacturing
  • the R6K gamma origin (SEQ ID NO:1; Fig. 1E)-RNA-OUT (SEQ ID NO:5; Fig. IB) bacterial replication-selection region (SEQ ID NO:8; Fig. 1C) was cloned into the polylinker region of a variety of pUC57 based vectors to create the pNTC-NPl, pNTC-NP2, pNTC-NP3, pNTC-NP4, pNTC-NP5, pNTC-NP6, pNTC-NP7, vectors.
  • Each vector has different flanking restriction sites that can be used to retrofit a target vector to R6K replication-RNA-OUT selection.
  • the 5’ and 3’ polylinker sequences flanking the R6K-RNA-OUT insert in the pNTC-NP 1-7 vectors are shown in Table 2.
  • a pUC57 based version of the 1 CpG R6K gamma origin- 2 CpG RNA-OUT bacterial replication-selection region (SEQ ID NO:9; Fig. ID) was also created (pNTC-3xCpG NP1) and is shown in Table 2.
  • the R6K gamma origin is an engineered 6 iteron R6K origin (Fig. IE).
  • a pUC57 based version of a 7 iteron R6K gamma origin (SEQ ID NO:18; Fig. 1F)-RNA-OUT (SEQ ID NO:5; Fig. IB) bacterial replication-selection region was also created and used to construct and evaluate the utility of additional iterons on manufacturing.
  • high quality, high yield manufacture was obtained with vectors differing only by containing either the SEQ ID NO:18 seven iteron R6K gamma origin or the six iteron R6K gamma origin (SEQ ID NO:1).
  • the following harvest production yields were obtained in 30-42°C 10 hr ramp temperature shift HyperGRO fermentations:
  • SEQ ID NO:1 6 iteron 3203 bp R6K origin vector a biomass of 120 ODeoo; plasmid titer of 1363 mg/L; plasmid specific yield of 11.3 mg plasmid/L/ODeoo
  • SEQ ID NO:18 7 iteron 3225 bp R6K origin vector a biomass of 137 ODgoo; plasmid titer of 1503 mg/L; plasmid specific yield of 11.0 mg plasmid/L/ODeoo
  • the 7 iteron R6K gamma origin in SEQ ID NO: 18 is a tandem duplication of iteron 5 (Fig. IF; SEQ ID NO:18) but the 7 iteron R6K gamma origin vectors of the invention can be tandem duplications of any of the iterons given as SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23 (Fig. IE), or random combinations of SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23 into 7 iteron R6K origin compositions, or iteron repeat variants that retain the TGAGNG consensus. Additional iteron derivatives (e.g. 8, 9 or 10 iteron vectors) are also contemplated for practice of the invention.
  • Viral and non-viral vector pUC origin-antibiotic selection bacterial backbone retrofits to R6K-RNA-OUT were performed by:
  • the R6K origin and RNA-OUT units were assembled in multi-fragment ligations from separate restriction fragments using the non-palindromic Dralll linker site (see Table 2).
  • the fd6 Ad helper retrofit (Table 7)
  • a 3-fragment ligation was performed using a short 500 bp synthetic gene Dralll RNA-OUT-Ad helper-Avrll to link RNA-OUT to a unique Avril site in the fd6 Ad helper eukaryotic region in a 12 kb Avril-Sall restriction fragment, and to a Sall- R6K origin-Dralll fragment from pNTC-NP4.
  • Example vector maps and vector characteristics of the original pUC origin- antibiotic selection marker vector and the retrofitted R6K origin-RNA-OUT antibiotic free selection marker vectors are shown for Sleeping Beauty (Fig 2; Table 4), AAV (Fig. 3; Table 7) and mRNA (Fig. 4; Table 6) vectors.
  • the vector characteristics of the original pUC origin- antibiotic selection marker vector and the retrofitted R6K origin-RNA-OUT antibiotic free selection marker vectors are shown for AAV helper vectors (Table 6).
  • the vector characteristics of pUC origin- RNA- OUT antibiotic free selection marker vector and the retrofitted R6K origin-RNA-OUT antibiotic free selection marker vectors are shown for Lentiviral vectors (Table 3) and AAV vectors (Table 5).
  • the bacterial backbone size was ⁇ 1 kb in the R6K origin-RNA-OUT antibiotic free selection marker retrofitted vectors (460-610 bp). This is well below the 1.1 kb bacterial backbone size limit required to improve vector expression level (Tables 1-2) and duration (Quiviger et al., Supra, 2014).
  • the original pUC origin-antibiotic selection bacterial backbone prior to retrofit was > 1.2 kb (2340-2750 bp) as were the pUC origin-RNA- OUT retrofits (1210-1500 bp).
  • these AAV, AAV helper, Sleeping Beauty, and Lentiviral R6K origin-RNA-OUT antibiotic free selection marker retrofit vectors meet the short spacer region requirement for improved expression level and duration compared to the original pUC origin-antibiotic selection marker vector. Additionally, these AAV, AAV helper, Sleeping Beauty, and Lentiviral R6K origin-RNA-OUT antibiotic free selection marker retrofit vectors have no chance of antibiotic marker gene transfer by transduction (AAV, Lentiviral vectors) or transposition (Sleeping Beauty vectors) due to removal of the KanR or ampR antibiotic resistance selection marker in the parent vector. Additionally, the vectors of the current technology do not require the complicated difficult to scale expensive additional manufacturing steps required to remove the large bacterial region between the eukaryotic polyA and promoter with minicircle vectors (Kay et al., Supra, 2010).
  • flanking direct repeat LTRs in a Lentiviral vector the eukaryotic region contains flanking direct repeat LTRs
  • flanking sequences are all structured DNA sequences.
  • replication intermediates form when any high copy number prokaryotic origin of replication is ⁇ Ikb from a structured DNA sequence such as an enhancer, LTR or IRES, but not when the high copy replication origin is >1.5 kb. Consistent with this, replication intermediates were formed in all pUC origin- RNA-OUT marker vectors in which the pUC origin was ⁇ 1 kb from a Lentiviral vector LTR (Table 3: 400 bp) or a pUC originantibiotic resistance marker vector in which the pUC origin was ⁇ 1 kb from a Sleeping Beauty IR/DR (Table 4; 280 bp).
  • the original pUC origin-antibiotic selection marker vectors have the pUC origin 0 bp from an ITR (AAV vector; Table 7) or 170 bp from a A99 repeat (mRNA vector, Table 6) which may make a replication intermediate that is too small to detect on an agarose gel.
  • production yields were very low, indicative of low plasmid copy number due to replication blockage.
  • the original pUC origin-antibiotic selection marker vector pUC origin was > 1.5 kb from a structured DNA sequence (A60 repeat)
  • high plasmid production yields were obtained (Table 6: mRNA vector pGEM4Z T7 A60).
  • RNAII primer forms an RNA:DNA R-loop that is cleaved by RNase H to create a primer for DMA Pol I directed DNA synthesis during initial leading strand synthesis. DNA synthesis then converts from slow DNA Pol I to the highly processive DNA Pol III from 400 bp to up to 1.3 kb downstream of the origin (Allen et al., 2011.
  • the R6K gamma replication origin rep protein interacts with dnaB helicase and dnaG primase which creates short RNA primers for DNA Pol III replication without requirement for DNA Pol I (Abhyankar et al., Supra, 2003).
  • the pUC origin DNA Pol I replication zone of up to 1.3 kb from the origin corresponds closely with the Levy, Supra, 2004 defined upper limit of replication intermediate formation (between 1 and 1.5 kb from the origin).
  • Pol Ill- dependent origin of replication such as the R6K origin can be used to replicate structured DNA sequences which are poorly replicated by a Pol Ldependent origin of replication such as the pUC origin.
  • the vectors of the invention are additionally useful for eliminating antibiotic resistance marker gene transfer by viral and non-viral vectors; reducing transfection associated toxicity; improving transposition from non-viral transposon vectors; improving packaging titers from viral vectors; improving expression of viral and non-viral vector encoded transgenes, etc.
  • R6K origin third generation lentiviral vectors [4 vectors: Table 53 transfer plasmid, gag pol packaging plasmid; env plasmid; REV plasmid (not shown) with R6K origin and ⁇ 1 kb bacterial backbone] of the invention showed reduced toxicity and improved viral packaging titers compared to pUC origin vector comparators with >1.5 kb bacterial backbone.
  • R6K origin mediated replication of a closely positioned structured DNA sequence could be improved by insertion of sequences between the R6K origin and the structured DNA sequence in the direction of replication was tested.
  • an AAV ITR vector with ITR structured DNA sequences flanking the bacterial region, was utilized.
  • Both vectors with two 0X174 type PAS sequences, PAS-BL and PAS-BH (SEQ ID NO: 29) inserted between the R6K origin and the ITR structured DNA sequence had improved yield compared to the standard 6 iteron R6K gamma origin (SEQ ID NO: 1) and the other tested vectors.
  • the two PAS-BL and PAS-BH 7 iteron R6K origin AAV ITR vectors were evaluated versus the standard 6 iteron R6K gamma origin AAV ITR vector in HyperGRO fermentation as described in Example 2. The results are shown in Table 10 and demonstrate both vectors have dramatically improved AAV ITR vector fermentation yields compared to the standard 6 iteron R6K gamma origin AAV ITR vector.
  • Example 6 R6K origin PAS structured mRNA vector construction and manufacturing
  • RNA-OUT vectors for mRNA production disclosed in Example 3 (Fig. 4C; Fig. 4 F) are configured to replicate away from the polyA homopolymeric run structured DNA repeat, as a polyA repeat ⁇ RNA-OUT R6K origin> orientation. This configuration has the RNA-OUT marker transcribing towards the polyA repeat. Surprisingly, with some mRNA transgene inserts, double stranded RNA that is not fully digested by RNaseA or removed in standard plasmid DNA column purification processing is formed (mRNA vector- NP Table 11; Fig. 7A).
  • RNA- OUT The basis for the problematic double stranded RNA formation is unknown but may be due to forward strand transcription from a cryptic mRNA transgene insert, annealing with reverse strand transcriptional readthrough of the mRNA terminator sequence in the RNA- OUT gene.
  • RNA-OUT> (mRNA vector-ROUT OPP-NP2)
  • Nanoplasmid backbone orientations for mRNA vectors combine orientation of both R6K origin replication and RNA- OUT transcription away from the polyA structured DNA repeat.
  • the oritenation is ⁇ — Pol III replication origin RSM— >,
  • the oritenation is ⁇ — Pol III replication origin ⁇ — RSM.
  • the oritenation is Pol III replication origin — > RSM — >.
  • the oritenation is Pol III replication origin ⁇ — RSM.
  • the oritenation is ⁇ — RSM Pol III replication origin — > .
  • the oritenation is ⁇ — RSM ⁇ — Pol III replication origin.
  • the oritenation is RSM Pol III replication origin
  • the oritenation is RSM ⁇ — Pol III replication origin.
  • an antibiotic resistance maker may be substituted for RNA-OUT, for example in the case where a simple retrofit of the pUC origin to the R6K origin is desired to improve plasmid production yield and or quality.
  • the improved Pol III dependent replication origin vectors of the current technology provide for an approach to reduce transfection associated toxicity, improve transposition from non-viral transposon vectors, improve packaging titers from viral vectors, improve expression of viral and non-viral vector encoded genes, and eliminate viral vector and non-viral vector mediated antibiotic selection marker gene transfer (i.e. through incorporation of a bacterial region preferably less than 1000 bp) while dramatically improving manufacture compared to alterative vectors such as pUC plasmids and minicircles.

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Abstract

A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid having a Pol I- dependent origin of replication, and an insert including a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication or eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also includes modifying the covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced with a Pol Ill-dependent origin of replication, whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinant DNA molecule is also provided. Recombinant DNA molecules and methods related thereto which include one or more primosomal sites and/ or a selectable marker oritented to transcribe away from a structured DNA sequence are also provided.

Description

VIRAL AND NON-VIRAL NANOPLASMID VECTORS WITH
IMPROVED PRODUCTION
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/625,050 filed on January 25, 2024, titled “Viral and Non-viral Nanoplasmid Vectors with Improved Production,” the entire content of which is incorporated herein.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 9, 2025, is named 0798038 SL and is 55,919 bytes in size.
INCORPORATION BY REFERENCE
[0003] International Patent Application Publication No. WO 2019/183248 (PCT/US2019/023209), filed March 20, 2019, is incorporated herein by reference in its entirety. All references to patents, patents applications and publications are incorporated herein by reference.
FIELD OF THE INVENTION
[0004] The present invention relates to recombinant DNA molecules, i.e., vectors, useful for viral and non-viral gene therapy, viral and non-viral cell therapy, and more particularly, for improving viral and non-viral vector manufacturing yield and quality, reducing transfection associated toxicity, improving transposition from non-viral transposon vectors, improving packaging titers from viral vectors, improving expression of viral and non-viral vector encoded genes, and for eliminating viral vector and non-viral vector mediated antibiotic selection marker gene transfer.
[0005] Such recombinant DNA molecules are useful in biotechnology, ex vivo gene therapy, transgenic organisms, gene therapy, therapeutic vaccination, agriculture and DNA vaccines. BACKGROUND
[0006] E. coli plasmids have long been an important source of recombinant DNA molecules used by researchers and by industry. Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (e.g. gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines. Plasmids are also utilized in gene therapy or gene replacement applications, wherein the desired gene product is expressed from the plasmid after administration to a patient. Plasmids are also utilized as in vitro transcription template vectors for mRNA vaccines and therapeutics. Plasmids are also utilized in non-viral transposon vectors for gene therapy or gene replacement applications, wherein the desired gene product is expressed from the genome after transposition from the plasmid and genome integration. Plasmids are also utilized in viral vectors for gene therapy or gene replacement applications, wherein the desired gene product is packaged in a transducing virus particle after transfection of a production cell line, and is then expressed from the virus in a target cell after viral transduction.
[0007] Various investigators have identified that minicircle vectors are superior to plasmid vectors for production of AAV vectors (improved transducing unit titers, see Table 1) and transposon vectors (increased transposition, see Table 1). The improved performance due to improved expression duration with short backbone minicircle vectors should also be observed with short bacterial backbone plasmid vectors up to 1.1 kb. Indeed 2-fold improved sleeping beauty transposition into human cells was reported with the 1.1 kb bacterial backbone pFAR4 SB transposon vector/SBlOOx transposase vector combination compared to a 2.8 kb bacterial backbone pT2 plasmid SB transposon vector/SB lOOx transposase vector combination (Pastor, M, Johnen S, Harmening N, Quiviger M, Pailloux J, Kropp, M, Walter P, Ivies Z, Izsvak Z, Thumann G, Scherman D, 2018 Molecular Therapy 11: 57-67).
[0008] However, viral vectors such as AAV, lentiviral and retroviral vectors, and transposon vectors contained structured DNA sequences at their termini. For example, Sleeping Beauty transposon vectors contain flanking IR/DR sequences, AAV vectors contain flanking ITRs, and Lentiviral and Retroviral vectors contain flanking LTRs.
[0009] The close proximity of the pUC origin to a structured DNA sequence results in aberrant replication termination, resulting in replication intermediates which unacceptably reduce plasmid quality (Levy J. 2004. US Patent 6709844). Levy teaches that replication intermediates form when any high copy replication origin is < Ikb from a structured DNA sequence such as an enhancer, LTR or IRES, but not when the high copy replication origin is >1.5 kb away. Since the pUC origin itself is 1 kb, there is no configuration to make a <1.1 kb bacterial region AAV, Lentiviral, Retroviral or transposon vector containing the pUC origin which is not predicted to produce replication intermediates.
[0010] Lu J, Williams JA, Luke J, Zhang E, Chu K, and Kay MA. 2017. Human Gene Therapy 28: 125-34 disclose antibiotic free Mini-Intronic Plasmid (MIP) AAV vectors and suggest that MIP intron AAV vectors could have the vector backbone removed to create a short backbone AAV vector. Attempts to create a minicircle like 6 or 10 bp spacer region in Mini-Intronic Plasmid AAV vectors were toxic (see Table 5, footnote e) presumably due to creation of a long palindrome by such close juxtaposition of the AAV ITRs. While MIP vectors with longer spacer regions < Ikb can be made, a drawback of the MIP intron strategy is that it requires cloning of a replication and selection encoding intron into the eukaryotic region, which is not possible or desired with many vectors.
[0011] A drawback of the minicircle strategy to create short bacterial region AAV, Lentiviral, Retroviral or transposon vectors, is that methods to manufacture minicircle vectors are expensive and not easily scalable. For minicircle vectors, E. coli-based manufacturing systems have been developed in which, after plasmid production, the bacterial region and the eukaryotic region are separated and circularized into a minicircle (eukaryotic region) and a bacterial region circle via the action of phage recombinases on recognition sequences in the plasmid. In some methods, a restriction enzyme is then utilized to digest the bacterial region circle at a unique site to eliminate this difficult to remove contaminant. These production procedures are very inefficient. For example, optimal manufacture of minicircle vectors yields only 5 mg of minicircle per liter culture (Kay MA, He CY, Chen ZY. 2010. Nat Biotechnol 28: 1287-1289).
[0012] Methods for high yield manufacture of pF AR vectors have not been reported; this system utilizes a plasmid borne suppressor tRNA gene to complement a TAG amber nonsense mutation of the thyA gene to complement thymidine auxotrophy and allow cell growth on minimal media (Marie et al., Supra, 2010).
[0013] A solution is needed to develop mRNA, AAV, lentiviral, retroviral or transposon vector containing short spacer regions preferably less than 1000 bp that can be efficiently manufactured without replication intermediates or poor production. In embodiments, the vectors do not encode a protein-based selection marker. In further embodiments, the vectors are minimalized to eliminate all non-essential sequences. SUMMARY
[0014] In embodiments, disclosed are vectors useful for viral and non-viral gene therapy, [0015] hi embodiments, disclosed are vectors useful for viral and non-viral cell therapy, [0016] hi embodiments, disclosed are vectors for improving viral and non-viral vector manufacturing yield and quality,
[0017] In embodiments, disclosed are vectors for reducing transfection associated toxicity.
[0018] In embodiments, disclosed are vectors for improving transposition from non-viral transposon vectors.
[0019] In embodiments, disclosed are vectors for improving packaging titers from viral vectors. [0020] In embodiments, disclosed are vectors for improving expression of viral and non-viral vector encoded transgenes.
[0021] In embodiments, disclosed are vectors for eliminating antibiotic resistance marker gene transfer by viral and non-viral vectors.
[0022] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate structured DNA sequences are disclosed.
[0023] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate inverted repeat DNA sequences are disclosed.
[0024] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate direct repeat DNA sequences are disclosed.
[0025] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate homopolymeric repeat DNA sequences are disclosed.
[0026] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate enhancer structured DNA sequences are disclosed.
[0027] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate polyA repeat DNA sequences are disclosed.
[0028] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate SV40 origin of replication DNA sequences are disclosed.
[0029] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate Lentiviral LTR DNA sequences are disclosed.
[0030] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate Retroviral LTR DNA sequences are disclosed.
[0031] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate viral LTR DNA sequences are disclosed. [0032] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate AAV ITR DNA sequences are disclosed.
[0033] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate transposon IR/DR DNA sequences are disclosed.
[0034] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate Sleeping Beauty IR/DR DNA sequences are disclosed.
[0035] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate PiggyBac ITR DNA sequences are disclosed.
[0036] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate CMV enhancer DNA sequences are disclosed.
[0037] Improved vector methods and compositions that utilize a Pol Ill-dependent origin of replication to replicate direct SV40 enhancer DNA sequences are disclosed.
[0038] Improved viral vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0039] Improved Lentiviral vector, Lentiviral envelope vector and Lentiviral packaging vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0040] Improved Retroviral vector, Retroviral envelope vector and Retroviral packaging vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0041] Improved AAV vector and AAV helper vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0042] Improved Adenoviral vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0043] Improved non-viral transposon and transposase vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0044] Improved non-viral Sleeping Beauty transposon and transposase vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0045] Improved non-viral PiggyBac transposon and transposase vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0046] Improved non-viral Tol2 transposon and transposase vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed.
[0047] Improved non-viral polyA containing mRNA vector methods and compositions that utilize a Pol Ill-dependent origin of replication are disclosed
[0048] Improved viral vector methods and compositions with improved viral transducing unit production that utilize a Pol Ill-dependent origin of replication are disclosed. [0049] Improved Lentiviral vector methods and compositions with improved viral transducing unit production that utilize a Pol Ill-dependent origin of replication are disclosed.
[0050] Improved Retroviral vector methods and compositions with improved viral transducing unit production that utilize a Pol Ill-dependent origin of replication are disclosed.
[0051] Improved AAV vector and AAV helper vector methods and compositions with improved viral transducing unit production that utilize a Pol Ill-dependent origin of replication are disclosed.
[0052] Improved non- viral transposon and transposase vector methods and compositions with improved transposition that utilize a Pol Ill-dependent origin of replication are disclosed.
[0053] Improved non-viral Sleeping Beauty transposon and transposase vector methods and compositions with improved transposition that utilize a Pol Ill-dependent origin of replication are disclosed.
[0054] Improved non-viral PiggyBac transposon and transposase vector methods and compositions with improved transposition that utilize a Pol Ill-dependent origin of replication are disclosed.
[0055] Improved non-viral Tol2 transposon and transposase vector methods and compositions with improved transposition that utilize a Pol Ill-dependent origin of replication are disclosed.
[0056] Improved viral vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
[0057] Improved Lentiviral vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
[0058] Improved Retroviral vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
[0059] Improved AAV vector and AAV helper vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
[0060] Improved non-viral transposon and transposase vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
[0061] Improved non-viral Sleeping Beauty transposon and transposase vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
[0062] Improved non-viral PiggyBac transposon and transposase vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed. [0063] Improved non-viral Tol2 transposon and transposase vector methods and compositions with improved expression that utilize a Pol Ill-dependent origin of replication are disclosed.
[0064] Improved viral vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
[0065] Improved Lentiviral vector, lentiviral envelope vector and lentiviral packaging vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
[0066] Improved Retroviral vector, retroviral envelope vector and retroviral packing vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
[0067] Improved AAV vector and AAV helper vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
[0068] Improved non-viral transposon and transposase vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
[0069] Improved non-viral Sleeping Beauty transposon and transposase vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill- dependent origin of replication are disclosed.
[0070] Improved non-viral PiggyBac transposon and transposase vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill- dependent origin of replication are disclosed.
[0071] Improved non-viral Tol2 transposon and transposase vector methods and compositions with no antibiotic resistance marker gene transfer risk that utilize a Pol Ill-dependent origin of replication are disclosed.
[0072] Improved viral vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
[0073] Improved lentiviral vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
[0074] Improved retroviral vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
[0075] Improved AAV vector and AAV helper vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed. [0076] Improved non-viral transposon and transposase vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
[0077] Improved non-viral Sleeping Beauty transposon and transposase vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
[0078] Improved non-viral PiggyBac transposon and transposase vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
[0079] Improved non-viral Tol2 transposon and transposase vector methods and compositions with reduced transfection associated toxicity that utilize a Pol Ill-dependent origin of replication are disclosed.
[0080] Improved vector methods and compositions with one or more primosomal assembly sites downstream of an origin of replication, particularly a Pol Ill-dependent origin of replication, are disclosed including with a structured DNA sequence insert.
[0081] Improved vector methods and compositions with an RNA selectable marker oriented to transcribe divergent from a structured DNA sequence are disclosed including with a structured DNA sequence insert,
[0082] Each of the above improvements and the improvements described below is, by way of example but not limitation, relative to what is achieved in similar or identical circumstances, but with a different recombinant DNA molecule that does not include a Pol-III dependet origin of replication, and/or one or more primosomal assembly sites, and/or a marker, such as an RNA selectable marker, oriented to transcribe in a direction divergent from a structured DNA sequence. [0083] In embodiments, the disclosed vectors provide improved viral and non-viral vector manufacturing yield.
[0084] In embodiments, the disclosed vectors provide improved viral and non-viral vector manufacturing quality.
[0085] In embodiments, the disclosed vectors provide viral vectors with improved packaging titers.
[0086] hi embodiments, the disclosed vectors provide non-viral transposon vectors with improved transposition.
[0087] In embodiments, the disclosed vectors provide viral and non-viral vectors with improved expression of encoded transgenes. [0088] In embodiments, the disclosed vectors provide viral and non-viral vectors that eliminate antibiotic resistance marker gene transfer.
[0089] In embodiments, the disclosed vectors provide viral and non-viral vectors with reduced transfection associated toxicity.
[0090] In one embodiment, a covalently closed circular recombinant DNA molecule is provided that can include a backbone and an insert, where the backbone can include a Pol Ill-dependent origin of replication, a selectable marker and a first primosomal assembly site, where the first primosomal assembly site is positioned downstream of the Pol Ill-dependent origin of replication in the direction of replication, and where the insert comprises a structured DNA sequence.
[0091] In another embodiment, an antibiotic marker free covalently closed circular recombinant DNA molecule is provided that can include a backbone and an insert, where the backbone can include an origin of replication and an RNA selectable marker, where the insert can include a structured DNA sequence, and where the RNA selectable marker is oriented to transcribe in a direction divergent from the structured DNA sequence. Alternatively, the RNA selectable marker can instead be substituted by an antibiotic selectable marker and the recombinant DNA molecule can be a covalently closed circular recombinant DNA molecule, where the antibiotic selectable marker is oriented to transcribe in a direction divergent from the structured DNA sequence. It should be understood that divergent can refer to a direction away from a structured DNA sequence proximate to either end of the insert.
[0092] In any of the foregoing embodiments, the structured DNA sequence can be within 1000 bp of the origin of replication. In any of the foregoing embodiments, the backbone can be less than 1000 bp. It should be understood that, in any of the foregoing embodiments, the backbone and the insert can be operably linked such that there is no intervening sequence between the backbone and the insert. It should also be noted that, in any embodiments of the present disclosure, the position of a feature can be located on either strand of a covalently closed circular recombinant DNA molecule. By way of example, but not limitation, a first primosomal assembly site can be on one strand while a second primosomal assembly site can be on the opposite strand. For example, SEQ ID NO: 29 encodes two primosomal assembly sites, but the sequence provided has one in the sense orientation and one in the antisense orientation. It should be further understood that a backbone can, in some embodiments, be a bacterial replication-selection region of the present disclosure.
[0093] In any of the foregoing embodiments, the origin of replication can be a Pol Ill-dependent origin of replication such as, by way of example but not limitation, an R6K origin, ColE2 origin or ColE2 -related origin. In any of the foregoing embodiments, the Pol Ill-dependent origin of replication does not require Pol 1. In any of the foregoing embodiments, the Pol Ill-dependent origin of replication can be an R6K origin of replication such as, by way of example but not limitation, an R6K gamma origin. In any of the foregoing embodiments, the R6K origin of replication can have a sequence that has at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24. In any of the foregoing embodiments, the Pol Ill-dependent origin can be an R6K origin that is a 6 iteron R6K gamma origin or a 7 iteron R6K gamma origin (such as, by way of example, but not limitation, SEQ ID NO: 18) or having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity thereto. It should be understood that, in some embodiments, the iteron repeat can be selected from any one of SEQ ID NOs: 19-23.
[0094] In any of the foregoing embodiments, the selectable marker can be an RNA selectable marker. Alternatively, in embodiments where it is not an RNA selectable marker, the selectable marker can be an antibiotic selectable marker. In any of the foregoing embodiments, the RNA selectable marker can be an RNA-OUT RNA selectable marker. In any of the foregoing embodiments, the RNA-OUT RNA selectable marker can be an RNA-IN regulating RNA-OUT functional variant with at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7. In other embodiments, the RNA selectable marker can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6.
[0095] In any of the foregoing embodiments, the first primosomal assembly site can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33. In any of the foregoing embodiments, the backbone can further include a second primosomal assembly site positioned downstream of the origin of replication in the direction of replication. In any of the foregoing embodiments, the second primosomal assembly site can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33. In any of the foregoing embodiments, the backbone can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29 such that first primosomal assembly site and the second primosomal assembly site are downstream of the origin of replication in the direction of replication.
[0096] In some embodiments, the recombinant DNA molecule can be antibiotic free. [0097] In any of the foregoing embodiments, the backbone can include a sequence with at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[0098] In any of the foregoing embodiments, the structured DNA sequence can be any structured DNA sequence described in embodiments of this disclosure. By way of example, but not limitation, the structured DNA sequence can be selected from an inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and a eukaryotic promoter-enhancer sequence.
[0099] In any of the foregoing embodiment, the insert can be any type of vector described in the present disclosure. By way of example, but not limitation, the insert can be a transposon vector, a transposase vector, a mRNA vector, an AAV vector, a Lentiviral vector.
[00100] In any of the foregoing embodiments, where the insert is a transposon vector, the structured DNA sequence can be an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promoter-enhancer sequence.
[00101] In any of the foregoing embodiments, where the insert is an AAV vector, the structured DNA sequence can be an inverted repeat sequence. In any of the foregoing embodiments, where the insert is an AAV vector, the AAV vector can encode AAV ITRs. By way of example, but not limitation, the AAV vector can include a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 35 (5’ inverted terminal repeat sequence) and a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36 (3’ inverted terminal repeat sequence). In some embodiments, the insert can include one or more inverted repeats of an AAV ITR. Some embodiments, said recombinant DNA molecule is an AAV ITR containing non- viral vector
[00102] In any of the foregoing embodiments, where the insert is a lentiviral vector, the structured DNA sequence can be a direct repeat sequence or an eukaryotic origin of replication.
[00103] In any of the foregoing embodiments, the structured DNA sequence can be selected from an homopolymeric repeat such as a polyA repeat, a SV40 origin of replication, a viral LTR, a lentiviral LTR, a retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
[00104] In any of the foregoing embodiments, where the structured DNA sequence includes a homopolymeric sequence, the homopolymeric sequence can be a polyA repeat. In any of the foregoing embodiments, the homopolymeric sequence, such as, by way of example, but not limitation a polyA repeat, can include from about 3 to about 500 residues or more. In some embodiments, the polyA repeat can include the sequence of any one of SEQ ID NOs: 37-39. In some embodiments, the recombinant DNA molecule can be a mRNA vector that includes one or more homopolymeric sequences.
[00105] In any of the foregoing embodiments, the selectable marker can be an RNA selectable marker oriented to transcribe in a direction divergent from the structured DNA sequence.
[00106] In any of the foregoing embodiments, said recombinant DNA molecule is selected from viral vector, lentiviral vector, retroviral vector, AAV vector, Ad vector, non-viral transposon vector, Sleeping Beauty transposon vector, PiggyBac transposon vector, Tol2 transposon vector, and polyA containing mRNA vector.
[00107] It should be understood that the recombinant DNA molecules of the foregoing embodiments, or of any embodiment of the present disclosure, can be replicated by providing a cell containing the recombinant DNA molecule and subjecting the cell to a fermentation cell, where the cell and the fermentation process are of any embodiments described herein.
[00108] It should also be understood that an existing recombinant DNA molecule can be prepared by substituting the origin of replication and/or selectable marker to arrive at the recombinant DNA molecule of any of the embodiments of the disclosure and to have the features thereof.
[00109] In any of the foregoing embodiments, where the origin of replication is a Pol I-dependent origin of replication, it can be a pUC origin, pMBl origin, and ColEl origin.
[00110] In any of the foregoing embodiments, to the extent it apples, the primosomal assembly site(s), selectable marker and origin or replication can be in any order relative to a structured DNA sequence and within the backbone. By way of example, but not limitation, the primosomal assembly site(s) can be between the origin of replication and the selectable marker or at either end of the origin of replication and selectable marker. Similarly, the origin of replication and the selectable marker can be in either order with respect to the structured DNA sequence or insert. By way of further example, but not limitation, the following configurations represent certain embodiments where the RNA selectable marker transcribes in a direction diverent from the structured DNA sequence:
1) structured DNA sequence RNA-OUT> origin>
2) structured DNA sequence RNA-OUT> origin >PAS
3) structured DNA sequence origin> RNA-OUT>
4) structured DNA sequence origin>PAS RNA-OUT> [00111] In any of the foregoing embodiments, a R6K origin can include multiple iterons such as, by way of example, but not limitation, 6 or 7 iterons.
[00112] It should be understood that, in any embodiments of the disclosure, known equivalents and alternatives can be used and that such modifications do not deviate from the spirit of the present disclosure.
[00113] In one embodiment, the present technology provides a method for improving the replication of a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a Pol I-dependent origin of replication, and ii) an insert comprising a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication and eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication; b) modifying the covalently closed circular recombinant molecule of a) such that the Pol I-dependent origin of replication is replaced with a Pol Ill-dependent origin of replication whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has improved replication. In a further embodiment said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin. In a further embodiment said Pol Ill-dependent origin of replication is an R6K gamma replication origin. In a further embodiment said Pol Ill-dependent origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said structured DNA sequence is selected from polyA repeat, SV40 origin of replication, viral LTR, Lentiviral LTR, Retroviral LTR, transposon IR/DR repeat, Sleeping Beauty transposon IR/DR repeat, AAV ITR, CMV enhancer, and SV40 enhancer. In a further embodiment said improved replication is selected from reduced production of replication intermediates and increased plasmid copy number.
[00114] In another embodiment, the present technology provides a method for improving the replication of a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication and eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication; b) modifying the covalently closed circular recombinant molecule of a) such that the antibiotic selectable marker is replaced with an RNA selectable marker and the Pol I-dependent origin of replication is replaced with a Pol Ill-dependent origin of replication, whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has improved replication. In a further embodiment said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin. In a further embodiment said Pol Ill- dependent origin of replication is an R6K gamma replication origin. In a further embodiment said Pol Ill-dependent origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with a Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region with at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In a further embodiment said structured DNA sequence is selected from polyA repeat, SV40 origin of replication, viral LTR, Lentiviral LTR, Retroviral LTR, transposon IR/DR repeat, Sleeping Beauty transposon IRT)R repeat, AAV ITR, CMV enhancer, and SV40 enhancer. In a further embodiment said improved replication is selected from reduced production of replication intermediates, and increased plasmid copy number.
[00115] In one embodiment, the current technology provides an antibiotic marker free covalently closed circular recombinant DNA molecule comprising: a) an antibiotic marker free insert comprising a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence; b) a Pol Ill-dependent origin of replication comprising an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24; and c) an RNA-OUT RNA selectable marker comprising an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said R6K gamma replication origin and said RNA- OUT RNA selectable marker comprise a R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region with at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In a further embodiment said structured DNA sequence is selected from polyA repeat, SV40 origin of replication, viral LTR, Lentiviral LTR, Retroviral LTR, transposon IR/DR repeat, Sleeping Beauty transposon IR DR repeat, AAV ITR, CMV enhancer, and SV40 enhancer. In a further embodiment said recombinant DNA molecule is selected from viral vector, Lentiviral vector, Retroviral vector, AAV vector, Ad vector, non- viral transposon vector, Sleeping Beauty transposon vector, PiggyBac transposon vector, Tol2 transposon vector, and polyA containing mRNA vector.
[00116] In one embodiment, the present technology provides a method for improving AAV vector viral transducing unit production from a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from AAV vector, AAV rep cap vector, Ad helper vector, and Ad helper rep cap vector; b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill- dependent origin of replication covalently closed circular plasmid has improved AAV viral transducing unit production when transfected into mammalian cells. In a further embodiment said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin. In a further embodiment said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA-OUT RNA selectable marker is an RNA- IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. hi a further embodiment said RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00117] In one embodiment, the current technology provides a method for improving Retroviral or Lentiviral vector viral transducing unit production from a covalently closed circular plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from Retroviral vector, Lentiviral vector, Retroviral envelope vector, Lentiviral envelope vector, Retroviral packaging vector and Lentiviral packaging vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill- dependent origin of replication covalently closed circular plasmid has improved viral transducing unit production when transfected into mammalian cells. In a further embodiment said Pol I- dependent origin of replication is selected from the group consisting of: pUC origin, pMB 1 origin, and ColEl origin. In a further embodiment said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replicationselection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00118] In one embodiment, disclosed are methods for improving transposition from a covalently closed circular non-viral transposon plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising i) a 1 kb or larger bacterial replication- selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a non-viral eukaryotic region selected from transposon vector, Sleeping Beauty transposon vector, Sleeping Beauty transposase vector, PiggyBac transposon vector, PiggyBac transposase vector, Tol2 transposon vector, Tol2 transposase vector; b) modifying the covalently closed circular recombinant molecule of (a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has improved transposition when transfected into mammalian cells. In a further embodiment said Pol I- dependent origin of replication is selected from the group consisting of: pUC origin, pMB 1 origin, or ColEl origin. In a further embodiment said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, SEQ ID NO: 7. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replicationselection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00119] In one embodiment, current technology provides a method for improving expression from a covalently closed circular viral vector or non-viral transposon plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from Lentiviral vector, Retroviral vector, and AAV vector or non-viral transposon vector; and b) modifying the covalently closed circular recombinant molecule of (a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA- OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol III- dependent origin of replication covalently closed circular plasmid has improved expression when transfected into mammalian cells. In a further embodiment said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin. In a further embodiment said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00120] In one embodiment, the present technology provides a method for eliminating antibiotic resistant marker gene transfer from a covalently closed circular viral vector plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker, and ii) an insert comprising an antibiotic resistance marker free eukaryotic region selected from viral vector, Lentiviral vector, Lentiviral packaging vector, Lentiviral envelope vector Retroviral vector, Retroviral envelope vector, Retroviral packaging vector, AAV vector, AAV rep cap vector, Ad helper vector, and Ad helper rep cap vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA- OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill- dependent origin of replication covalently closed circular plasmid has no antibiotic resistance markers that could be packaged into Lentiviral, Retroviral or AAV transducing viral particles when transfected into mammalian cells. In a further embodiment said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin. In a further embodiment said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00121] In one embodiment, the present technology provides a method for eliminating antibiotic resistant marker gene transfer from a covalently closed circular non-viral transposon plasmid comprising the following steps: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic resistance marker, and ii) an insert comprising an antibiotic resistance marker free eukaryotic region selected from non-viral transposon vector, non-viral transposase vector, Sleeping Beauty transposon vector, Sleeping Beauty transposase vector, PiggyBac transposon vector, PiggyBac transposase vector, Tol2 transposon vector, Tol2 transposase vector; and b) modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has no antibiotic resistance markers that could be transposed into the genome when transfected into mammalian cells. In a further embodiment said Pol I-dependent origin of replication is selected from the group consisting of: pUC origin, pMBl origin, and ColEl origin. In a further embodiment said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00122] In one embodiment, the present technology provides an antibiotic marker free covalently closed circular recombinant DNA molecule comprising: a) an antibiotic marker free insert comprising a eukaryotic region selected from Lentiviral vector, Lentiviral envelope vector, Lentiviral packaging vector, Retroviral vector, Retroviral envelope vector, Retroviral packaging vector, AAV vector, AAV rep cap vector, Ad helper vector, Ad helper rep cap vector, non- viral transposon vector, and non-viral transposase vector; b) a Pol Ill-dependent origin of replication comprising an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24; and c) an RNA-OUT RNA selectable marker comprising an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said R6K gamma replication origin and said RNA-OUT RNA selectable marker comprise a R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region with at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00123] In one embodiment, the present technology provides a method for reducing transfection associated toxicity from a covalently closed circular viral vector or non-viral transposon plasmid comprising: a) providing a covalently closed circular plasmid comprising: i) a 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker, and ii) an insert comprising a eukaryotic region selected from lentiviral vector, retroviral vector, AAV vector and non-viral transposon vector; modifying the covalently closed circular recombinant molecule of a) such that the 1 kb or larger bacterial replication-selection region comprising a Pol I-dependent origin of replication and an antibiotic selectable marker is replaced with an less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replication-selection region, whereby the resultant Pol Ill- dependent origin of replication covalently closed circular plasmid has reduced toxicity when transfected by transfection associated into mammalian cells. In a farther embodiment said Pol I- dependent origin of replication is selected from the group consisting of: pUC origin, pMB 1 origin, and ColEl origin. In a further embodiment said Pol Ill-dependent R6K origin of replication is an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 24. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from SEQ ID NO: 5, and SEQ ID NO: 7. In a further embodiment said RNA-OUT RNA selectable marker is an RNA-OUT RNA selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 6. In a further embodiment said less than 1 kb Pol Ill-dependent R6K origin-RNA-OUT RNA selectable marker bacterial replicationselection region has at least 95% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00124] The resultant Pol Ill-dependent replication origin plasmids have surprisingly improved manufacturing quality and yield than the parent pMBl, ColEl or pBR322 derived replication origin expression plasmid vector.
[00125] Further objects and advantages of the invention will become apparent from a consideration of the drawings and ensuing description.
BRIEF DESCRIPTION OF THE FIGURES
[00126] FIGS. 1A-1F depict the R6K origin (FIGS. 1A, IE, and IF), RNA-OUT selectable marker (FIG. IB), and 14 and 3 CpG R6K-RNA-OUT bacterial backbones (FIGS. 1C and ID);
[00127] FIGS. 2A-2B depict a Pol I-dependent pUC origin Sleeping Beauty transposon vector (FIG. 2A) and a Pol Ill-dependent R6K origin Sleeping Beauty transposon vector (FIG. 2B);
[00128] FIGS. 3A-3C depict Pol I-dependent pUC origin AAV vectors (FIGS. 3A and 3B) and a Pol Ill-dependent R6K origin AAV vectors (FIG. 3C); and
[00129] FIGS. 4A-4F depict Pol I-dependent pUC origin A60 polyA repeat encoding mRNA vectors (FIGS. 4A^4B), a Pol Ill-dependent R6K origin A60 polyA repeat encoding mRNA vector (FIG. 4C), Pol I-dependent pUC origin A99 polyA repeat encoding mRNA vectors (FIGS. 4D-4E), and a Pol Ill-dependent R6K origin A99 polyA repeat encoding mRNA vector (FIG. 4F). [00130] FIGS. 5A-5C depict the Pol Ill-dependent R6K origin R6K-RNA-OUT bacterial backbones from SEQ ID NO: 25 (A) SEQ ID NO: 27 (B) SEQ ID NO: 28 (C) SEQ ID NO: 34.
[00131] FIGS. 6A-6B depict the Pol Ill-dependent R6K origin AAV ITR encoding AAV vector (FIG. 6A) A 100 polyA repeat encoding mRNA vector (FIG. 6B)
[00132] FIGS. 7A-7B are BspQl linearizations of purified plasmid DNA from fermentation harvests of mRNA vector -NP (polyA 100 <ROUT R6K origin>) and mRNA vector -NP 7 iteron PAS R6K> ROUT> (polyA 100 R6K origin> PAS ROUT>).
[00133] FIG 8 depicts Table 1 : Minicircle applications with various viral and non- viral vector platforms.
[00134] FIG 9 depicts Table 2: pNTC multiple cloning site flanked R6K Origin-RNA-OUT selection marker vectors.
[00135] FIG 10 depicts Table 3: SV40 origin Lentiviral vectors: pUC versus R6K origin shake flask production yields/quality.
[00136] FIG 11 depicts Table 4: Sleeping Beauty Transposon vectors: pUC versus R6K origin shake flask production yields/quality.
[00137] FIG 12 depicts Table 5: AAV vectors: pUC versus R6K origin shake flask production yields/quality.
[00138] FIG 13 depicts Table 6: mRNA vectors: pUC versus R6K origin DH5a HyperGRO fermentation yields/quality.
[00139] FIG 14 depicts Table 7 : AAV helper vectors: pUC versus R6K origin plasmid production yields/quality.
[00140] FIG 15 depicts Table 8: AAV vectors: R6K origin versus R6K origin + primosomal assembly site shake flask production yields/quality.
[00141] FIG 16 depicts Table 9: AAV vectors: R6K origin versus R6K origin + primosomal assembly site shake flask production yields/quality.
[00142] FIG 17 depicts Table 10: AAV ITR vector: R6K origin with and without PAS HyperGRO Fermentation Yields/quality
[00143] FIG 18 depicts Table 11: mRNA polyA vector: R6K origin with and without PAS HyperGRO Fermentation Yields/quality
DESCRIPTION OF THE SEQUENCE ID NOS
[00144] SEQ ID NO:1 : R6K gamma origin
[00145] SEQ ID NO:2: 1 CpG R6K gamma origin [00146] SEQ ID NO:3: CpG free R6K gamma origin
[00147] SEQ ID NO:4 : Extended R6K gamma origin
[00148] SEQ ID NO:5: RNA-OUT Selectable Marker
[00149] SEQ ID NO:6: RNA-OUT antisense repressor RNA
[00150] SEQ ID NO:7: 2 CpG RNA-OUT Selectable Marker
[00151] SEQ ID NO:8: R6K gamma origin-RNA-OUT bacterial region flanked by Nhel and Kpnl restriction sites
[00152] SEQ ID NO:9: 1 CpG R6K gamma origin-2 CpG RNA-OUT bacterial region flanked by Nhel and Kpnl restriction sites
[00153] SEQ ID NO:10: pNTC-NPl polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoR I Hind 111
[00154] SEQ ID NO:11: pNTC-NP2 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoR I Hind 111
[00155] SEQ ID NO: 12: pNTC-NP3 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoR I Hind 111
[00156] SEQ ID NO: 13: pNTC-NP4 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRI/Hindin
[00157] SEQ ID NO:14: pNTC-NP5 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: KasEHindlll
[00158] SEQ ID NO:15: pNTC-NP6 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: EcoRESacI
[00159] SEQ ID NO:16: pNTC-NP7 polylinker trpA R6K-RNA-OUT polylinker cloning cassette: BssHII-BssHII
[00160] SEQ ID NO:17: pNTC-3xCpG NP1 polylinker R6K-RNA-OUT polylinker cloning cassette: Hindlll-EcoRI
[00161] SEQ ID NO:18: R6K gamma origin (7 iteron)
[00162] SEQ ID NO:19: R6K gamma origin 22 bp iteron repeat
[00163] SEQ ID NO:20: R6K gamma origin 22 bp iteron repeat
[00164] SEQ ID NO:21: R6K gamma origin 22 bp iteron repeat
[00165] SEQ ID NO:22: R6K gamma origin 22 bp iteron repeat
[00166] SEQ ID NO:23: R6K gamma origin 22 bp iteron repeat
[00167] SEQ ID NO:24: 1 CpG R6K gamma origin (7 iteron)
[00168] SEQ ID NO:25: R6K gamma origin (7 iteron)-RNA-OUT bacterial region [00169] SEQ ID NO:26: 1 CpG R6K gamma origin (7 iteron)-2 CpG RNA-OUT bacterial region
[00170] SEQ ID NO:27: NP 7 iteron + PAS= R6K gamma origin (7 iteron)+PAS-RNA-OUT bacterial region
[00171] SEQ ID NO:28: NP 7 iteron PAS R6K>ROUT>= R6K gamma origin (7 iteron)+PAS- RNA-OUT bacterial region
[00172] SEQ ID NO:29: PAS
[00173] SEQ ID NO:30: PAS-BH
[00174] SEQ ID NO:31 : PAS-BL
[00175] SEQ ID NO:32: TrpA terminator
[00176] SEQ ID NO :33: R6K plasmid CpG free ssiA primosomal assembly site
[00177] SEQ ID NO:34: NP 7 iteron + R6K PAS RNA-OUT bacterial region
[00178] SEQ ID NO:35: AAV2 ITR
[00179] SEQ ID NO:36: AAV2 ITR
[00180] SEQ ID NO:37: 80 bp polyA repeat
[00181] SEQ ID NO:38: 100 bp polyA repeat
[00182] SEQ ID NO:39: 120 bp polyA repeat
DEFINITION OF TERMS
[00183] AAV vector: Adeno-associated virus vector, an episomal viral vector. Includes self- complementary (sc) Adeno-associated virus vectors (scAAV) and single-stranded (ss) Adeno- associated virus vectors (ssAAV)
[00184] AF: Antibiotic-free
[00185] amp: Ampicillin
[00186] ampR: Ampicillin Resistance gene
[00187] Antibiotic selectable marker: A gene that confirs resistance to an antibiotic, e.g. ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene
[00188] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is the same or similar to a stated reference value [00189] Bacterial region: Region of a plasmid vector required for propagation and selection in the bacterial host
[00190] bp: basepairs
[00191] ccc: Covalently Closed Circular [00192] cl: Lambda repressor
[00193] cITs857: Lambda repressor further incorporating a C to T (Ala to Thr) mutation that confers temperature sensitivity. cITs857 is a functional repressor at 28-30°C but is mostly inactive at 37-42°C. Also called cI857
[00194] CatR: Chloramphenicol resistance gene
[00195] cmv: Cytomegalovirus
[00196] dem methylation: E. coli methyltransferase that methylated the sequences CC( /T)GG at the C5 position of the second cytosine
[00197] DNA replicon: A genetic element that can replicate under its own control; examples include plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof
[00198] E. coli'. Escherichia coli, a gram negative bacteria
[00199] EGFP: Enhanced green fluorescent protein
[00200] EP: Electroporation
[00201] Eukaryotic expression vector: A vector for expression of mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target eukaryotic organism using RNA Polymerase I, II or III promoters
[00202] Eukaryotic region: The region of a plasmid that encodes eukaryotic sequences and/or sequences required for plasmid function in the target organism. This includes the region of a plasmid vector required for expression of one or more transgenes in the target organism including RNA Pol II enhancers, promoters, transgenes and polyA sequences. This also includes the region of a plasmid vector required for expression of one or more transgenes in the target organism using RNA Pol I or RNA Pol III promoters, RNA Pol I or RNA Pol III expressed transgenes or RNAs. The eukaryotic region may optionally include other functional sequences, such as eukaryotic transcriptional terminators, supercoiling-induced DNA duplex destabilized (SIDD) structures, S/MARs, boundary elements, etc. In a Lentiviral or Retroviral vector, the eukaryotic region contains flanking direct repeat LTRs, in a AAV vector the eukaryotic region contains flanking inverted terminal repeats, while in a Transposon vector the eukaryotic region contains flanking transposon inverted terminal repeats or IR/DR termini (e.g. Sleeping Beauty). In genome integration vectors, the eukaryotic region may encode homology arms to direct targeted integration
[00203] Exon: A nucleotide sequence encoded by a gene that is transcribed and present within a mature mRNA product after RNA splicing to remove introns has been completed [00204] Expression vector: A vector for expression of mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target organism.
[00205] g: Gram, kg for kilogram
[00206] gene of interest: gene to be expressed in the target organism. Includes mRNA genes that encode protein or peptide antigens, protein or peptide therapeutics, and mRNA, shRNA, RNA or microRNA that encode RNA therapeutics, and mRNA, shRNA, RNA or microRNA that encode RNA vaccines, etc.
[00207] Homopolymeric repeat: simple sequence DNA repeat of polyA, polyG, polyC or polyT. Can be from a few base pairs to several hundred consecutive base pairs
[00208] Hr(s): Hour(s)
[00209] ID: Intradermal
[00210] IM: Intramuscular
[00211] immune response: Antigen reactive cellular (e.g. antigen reactive T cells) or antibody (e.g. antigen reactive IgG) responses
[00212] Intron: A nucleotide sequence encoded by a gene that is transcribed and subsequently removed from a mature mRNA product by RNA splicing
[00213] IR/DR: Inverted Repeats which are each Directly Repeated twice. For example, Sleeping Beauty transposon IR/DR repeats
[00214] Iteron: Directly repeated DNA sequences in a origin of replication that are required for replication initiation. R6K origin iteron repeats are 22 bp
[00215] ITR: Inverted Terminal Repeat
[00216] kan: Kanamycin
[00217] kanR: Kanamycin Resistance gene
[00218] Kd: Kilodalton
[00219] kozak sequence: Optimized consensus DNA sequence gccRccATG (R = G or A) immediately upstream of an ATG start codon that ensures efficient tranlation initiation. A Sall site (GTCGAC) immediately upstream of the ATG start codon (GTCGACATG) is an effective kozak sequence
[00220] Lentiviral vector: Integrative viral vector that can infect dividing and non-dividing cells. Also call Lentiviral transfer plasmid. Plasmid encodes Lentiviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with Lentiviral envelope and packaging plasmids required to make viral particles
[00221] Lentiviral envelope vector: Plasmid encoding envelope glycoprotein [00222] Lentiviral packaging vector: One or two plasmids that express gag, pol and Rev functions required to package the Lentiviral transfer vector
[00223] minicircle: Covalently closed circular plasmid derivatives in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site-specific recombination or in vitro restriction digestion/ligation. Minicircle vectors are replication incompetent in bacterial cells [00224] mRNA: Messenger RNA
[00225] mRNA vector: vector utilized as in vitro transcription template for mRNA vaccines and therapeutics. Typically the in vitro transcription template is a bacterial transcription unit with a bacterial promoter, typically T7 RNA polymerase promoter, followed by a 5’ UTR, kozak, transgene coding region, 3’ UTR, polyA homopolymeric repeat typically 60 to 120 bp long, ending with a unique restriction site such as BspQI for vector linearization after the polyA run. Ideally the BspQI site is positioned such that the linear DNA ends in the polyA repeat, with no ‘non-A’ bases. The linear DNA is utilized in an in vitro transcription reaction to make the therapeutic or vaccine mRNA
[00226] mSEAP: Murine secreted alkaline phosphatase
[00227] NA: Not Applicable
[00228] Nanoplasmid™ vector: Vector combining an RNA selectable marker with a R6K, ColE2 or ColE2 related replication origin. For example, NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors and modifications described in Williams, Supra, 2014 and included herein by reference
[00229] NTC8385: NTC8385, NTC8485 and NTC8685 plasmids are antibiotic-free pUC origin vectors that contain a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR. The creation and application of these RNA-OUT based antibiotic-free vectors are described in Williams, JA 2008 World Patent Application W02008153733 and included herein by reference
[00230] NTC8485: NTC8485 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR. The creation and application of NTC8485 is described in Williams, JA 2010 US Patent Application 20100184158 and included herein by reference
[00231] NTC8685: NTC8685 is an antibiotic-free pUC origin vector that contains a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR. The creation and application of NTC8685 is described in Williams, Supra, 2010 and included herein by reference [00232] NTC9385R: The NTC9385R Nanoplasmid™ vector described in Williams, Supra, 2014 included herein by reference has a spacer region encoded Nhel- trpA terminator-R6K origin RNA-OUT -Kpnl bacterial region (SEQ ID NO:8) linked through the flanking Nhel and Kpnl sites to the eukaryotic region.
[00233] ODeoo: optical density at 600 nm
[00234] PAS: Primosomal assembly site. Priming of DNA synthesis on a single stranded DNA ssi site. 0X174 type PAS: DNA hairpin sequence that binds priA, which, in turn, recruits the remaining proteins to form the preprimosome \priB, dnaT, recruits dnaB (delivered by dnaC)\, which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I. For example, PAS-BH and PAS-BL from plasmid pBR322. ABC type PAS: DNA hairpin binds dnaA, recruits dnaB (delivered by dnaC) which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I. For example, the R6K plasmid CpG free ssiA primosomal assembly site or alternative 0X174 type or ABC type primosomal assembly sites. By further example, a primosomal assembly site can be for a DnaA-dependent primosome or for a PriA-dependent primosome as described in Masai, Hisao and Arai, Ken-ichi, Frontiers in Bioscience 1 (1996):d48-d58 which is incorporated herein by reference in its entirety.
[00235] PAS-BH: Primosomal assembly site on the heavy (leading) strand of pBR322
[00236] PAS-BH region: pBR322 origin region between ROP and PAS-BL (approximately pBR322 2067-2351)
[00237] PAS-BL: Primosomal assembly site on the light (lagging) strand of pBR322
[00238] PBS: Phosphate buffered Saline
[00239] PCR: Polymerase Chain Reaction
[00240] pDNA: Plasmid DNA
[00241] PiggyBac Transposon: PB transposon. A transposon system that integrates an ITR flanked PB transposon into the genome by a simple cut and paste mechanism mediated by PB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the PB ITRs which is excised and integrated into the genome
[00242] pINT pR pL vector: The pINT pR pL attHK022 integration expression vector is described in Luke et al., 2011 Mol Biotechnol $TA3 and included herein by reference. The target gene to be expressed is cloned downstream of the pL promoter. The vector encodes the temperature inducible cI857 repressor, allowing heat inducible target gene expression
[00243] PL promoter: Lambda promoter left. PL is a strong promoter that is repressed by the cl repressor binding to OL1, OL2 and OL3 repressor binding sites. The temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30°C the cI857 repressor is functional and it represses gene expression, but at 37-42 °C the repressor is inactivated so expression of the gene ensues
[00244] PL (OL1 G to T) promoter: Lambda promoter left. PL is a strong promoter that is repressed by the cl repressor binding to OL1, OL2 and OL3 repressor binding sites. The temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30°C the cI857 repressor is functional and it represses gene expression, but at 37-42 °C the repressor is inactivated so expression of the gene ensues. The cl repressor binding to OL1 is reduced by the OL1 G to T mutation resulting in increased promoter activity at 30°C and 37-42 °C as described in Williams, Supra. 2014.
[00245] Plasmid: An extra chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA
[00246] Plasmid copy number: the number of copies of a plasmid per cell. Increases in plasmid copy number increase plasmid production yield
[00247] Pol: Polymerase
[00248] Pol I: Escherichia coli DNA Polymerase I
[00249] Pol I dependent origin of replication: A replication origin that requires Pol I, for example the pMBl, ColEl or pBR322 or derivatives such as the high copy pUC origin. For these origins the RNAII primer forms an RNA: DNA R-loop that is cleaved by RNase H to create a primer for DNA pol I directed DNA synthesis. DNA synthesis then converts to DNA pol III. Numerous additional Pol I dependent replication origins are known in the art, many of which are summarized in del Solar et al., 1998 Microbiol. Mol. Biol. Rev 62:434-464 which is included herein by reference
[00250] Pol III: Escherichia coli DNA Polymerase III
[00251] Pol III dependent origin of replication: A replication origin that doesn’t require Pol I, for example the rep protein dependent R6K gamma replication origin. Numerous additional Pol III dependent replication origins are known in the art, many of which are summarized in del Solar et al., Supra, 1998 which is included herein by reference
[00252] polyA: Polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The polyadenylation signal contains the sequence motif recognized by the RNA cleavage complex. Most human polyadenylation signals contain an AAUAAA motif and conserved sequences 5’ and 3’ to it. Commonly utilized polyA signals are derived from the rabbit [! globin, bovine growth hormone, SV40 early, or SV40 late polyA signals [00253] polyA repeat: simple sequence DNA repeat of polyA from a few base pairs to several hundred consecutive base pairs
[00254] pUC origin: pBR322-derived replication origin, with G to A transition that increases copy number at elevated temperature and deletion of the ROP negative regulator
[00255] pUC free: Plasmid that does not contain the pUC origin. Non-replicative fragments of the pUC origin may be included, for example the RNAI selectable marker
[00256] pUC plasmid: Plasmid containing the pUC origin
[00257] R6K plasmid: NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a-01, NTC9385R2a-O2, NTC9385R2b-01, NTC9385R2b-O2, NTC9385Ra-01, NTC9385Ra-O2, NTC9385RaF, and NTC9385RbF vectors as well as modifications and alternative vectors containing a R6K replication origin that were described in Williams, Supra, 2014 and included herein by reference. Alternative R6K vectors known in the art including, but not limited to, pCOR vectors (Gencell), pCpGfree vectors (Invivogen), and CpG free University of Oxford vectors including pGM169
[00258] R6K replication origin: a region which is specifically recognized by the R6K Rep protein to initiate DNA replication. Includes but not limited to R6K gamma replication origin sequence disclosed as SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO: 18 and SEQ ID NO:24. Also includes CpG free versions (e.g. SEQ ID NO:3) as described in Drocourt et al., United States Patent 7244609 and incorporated herein by reference
[00259] R6K replication origin-RNA-OUT bacterial region: Contains a R6K replication origin for propagation and the RNA-OUT selectable marker (e.g. SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO:13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28)
[00260] Rep: Replication
[00261] Replication intermediates: Linear DNA fragments resulting from premature termination of plasmid replication
[00262] Rep protein dependent plasmid: A plasmid in which replication is dependent on a replication (Rep) protein provided in Trans. For example, R6K replication origin, ColE2-P9 replication origin and ColE2 related replication origin plasmids in which the Rep protein is expressed from the host strain genome. Numerous additional Rep protein dependent plasmids are known in the art, many of which are summarized in del Solar etal., Supra, 1998 which is included herein by reference [00263] Retroviral vector: Integrative viral vector that can infect dividing cells. Also call transfer plasmid. Plasmid encodes Retroviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with envelope and packaging plasmids required to make viral particles
[00264] Retroviral envelope vector: Plasmid encoding envelope glycoprotein
[00265] Retroviral packaging vector: Plasmid that encodes Retroviral gag, pol genes required to package the Retroviral transfer vector
[00266] RNA-IN: Insertion sequence 10 (IS 10) encoded RNA-IN, an RNA complementary and antisense to a portion of RNA RNA-OUT. When RNA-IN is cloned in the untranslated leader of a mRNA, annealing of RNA-IN to RNA-OUT reduces translation of the gene encoded downstream of RNA-IN
[00267] RNA-IN regulated selectable marker: A genomically expressed RNA-IN regulated selectable marker. In the presence of plasmid borne RNA-OUT antisense repressor RNA (SEQ ID NO:6), expression of a protein encoded downstream of RNA-IN is repressed. An RNA-IN regulated selectable marker is configured such that RNA-IN regulates either 1) a protein that is lethal or toxic to said cell per se or by generating a toxic substance (e.g. SacB), or 2) a repressor protein that is lethal or toxic to said bacterial cell by repressing the transcription of a gene that is essential for growth of said cell (e.g. murA essential gene regulated by RNA-IN tetR repressor gene). For example, genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid selection/propagation are described in Williams, Supra, 2008 and included herein by reference. Alternative selection markers described in the art may be substituted for SacB
[00268] RNA-OUT: Insertion sequence 10 (IS 10) encoded RNA-OUT, an antisense RNA that hybridizes to, and reduces translation of, the transposon gene expressed downstream of RNA-IN. The sequence of the RNA-OUT RNA (SEQ ID NO:6) and complementary RNA-IN SacB genomically expressed RNA-IN-SacB cell lines can be modified to incorporate alternative functional RNA-IN/RNA-OUT binding pairs such as those described in Mutalik et al., 2012 Nat Chem Biol 8:447, including, but not limited to, the RNA-OUT A08/RNA-IN S49 pair, the RNA- OUT A08/RNA-IN S08 pair, and CpG free modifications of RNA-OUT A08 that modify the CG in the RNA-OUT 5’ TTCGC sequence to a non-CpG sequence. An example of a CpG free RNA- OUT selection marker, in which the two CpG motifs in the RNA-OUT RNA (one of which is present in the RNA-IN complementary region) are removed, was described in Williams 2015. Replicative minicircle vectors with improved expression. US Patent Application US 2015/0275221 and included herein by reference. A multitude of alternative substitutions to remove the two CpG motifs (mutating each CpG to either CpA, CpC, CpT, ApG, GpG, or TpG) may be utilized to make a CpG free RNA-OUT
[00269] RNA-OUT Selectable marker: An RNA-OUT selectable marker DNA fragment including E. coli transcription promoter and terminator sequences flanking an RNA-OUT RNA. An RNA-OUT selectable marker, utilizing the RNA-OUT promoter and terminator sequences, that is flanked by Dralll and Kpnl restriction enzyme sites, and designer genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation, are described in Williams, Supra, 2008 and included herein by reference. The RNA-OUT promoter and terminator sequences in SEQ ID NO: 5 that flank the RNA-OUT RNA (SEQ ID NO:6; FIG. IB) may be replaced with heterologous promoter and terminator sequences. For example, the RNA-OUT promoter may be substituted with a CpG free promoter known in the art, for example the I-EC2K promoter or the P5/6 5/6 or P5/6 6/6 promoters described in Williams, Supra, 2008 and included herein by reference. A 2 CpG RNA-OUT selectable marker in which the two CpG motifs in the RNA-OUT promoter are removed is given as SEQ ID NO: 7. An example of a CpG free RNA-OUT transcription unit, in which the two CpG motifs in the RNA-OUT RNA (one of which is present in the RNA-IN complementary region) and the two CpG motifs in the RNA-OUT promoter are removed was described in Williams, Supra, 2015 and included herein by reference. Vectors incorporating CpG free RNA-OUT selectable marker may be selected for sucrose resistance using the RNA-IN-SacB cell lines for RNA-OUT plasmid propagation described in Williams, Supra, 2008. Alternatively, the RNA-IN sequence in these cell lines can be modified to incorporate the 1 bp change needed to perfectly match the CpG free RNA-OUT region complementary to RNA- IN.
[00270] RNA polymerase II promoter: Promoter that recruits RNA Polymerase II to synthesize mRNAs, most small nuclear RNAs and microRNAs. For example, constitutive promoters such as the human or murine CMV promoter, elongation factor 1 (EFl) promoter, the chicken [! -actin promoter, the [! -actin promoter from other species, the elongation factor- 1 a (EFl a) promoter, the phosphoglycerokinase (PGK) promoter, the Rous sarcoma virus (RSV) promoter, the human serum albumin (SA) promoter, the spleen focus-forming virus (SFFV) promoter, the a -1 antitrypsin (AAT) promoter, the thyroxine binding globulin (TBG) promoter, the cytochrome P450 2E1 (CYP2E1) promoter, etc. The vectors may also utilize combination promoters such as the chicken [3 -actin/CMV enhancer (CAG) promoter, the human or murine CMV-derived enhancer elements combined with the elongation factor la (EFla) promoters, CpG free versions of the human or murine CMV-derived enhancer elements combined with the elongation factor la (EFla) promoters, the albumin promoter combined with an a -fetoprotein MERII enhancer, etc., or the diversity of tissue specific or inducible promoters know in the art such as the muscle specific promoters muscle creatine kinase (MCK), and C5-12 or the liverspecific promoter apolipoprotein A-I (ApoAI), etc.
[00271] RNA polymerase III promoter: Promoter that recruits RNA Polymerase III to synthesize tRNAs, 5S ribosomal RNA, and other small RNAs. For example, Class I promoters such as the 5s rRNA promoter, Class II promoter such as tRNA promoters, Class III promoters such as the U6 small nuclear RNA promoter or the Hl nuclear RNase P promoter, etc.
[00272] RNA selectable marker: An RNA selectable marker is a plasmid borne expressed nontranslated RNA that regulates a chromosomally expressed target gene to afford selection. This may be a plasmid borne nonsense suppressing tRNA that regulates a nonsense suppressible selectable chromosomal target as described by Crouzet J and Soubrier F 2005 US Patent 6,977,174 included herein by reference. This may also be a plasmid bome antisense repressor RNA, a non limiting list included herein by reference includes RNA-OUT that represses RNA- IN regulated targets (Williams, Supra, 2008), pMBl plasmid origin encoded RNAI that represses RNAII regulated targets (Grabherr R, Pfaffenzeller I. 2006 US patent application US20060063232; Cranenburgh RM. 2009; US Patent 7,611,883), IncB plasmid pMU720 origin encoded RNAI that represses RNA II regulated targets (Wilson IW, Siemering KR, Praszkier J, Pittard AJ, 1997. J Bacterial 179:742-53), ParB locus Sok of plasmid R1 that represses Hok regulated targets, Flm locus FlmB of F plasmid that represses flmA regulated targets (Morsey MA, 1999 US patent US5922583). An RNA selectable marker may be another natural antisense repressor RNAs known in the art such as those described in Wagner EGH, Altuvia S, Romby P. 2002. Adv Genet 46:361-98 and Franch T, and Gerdes K. 2000. Current Opin Microbiol 3: 159- 64. An RNA selectable marker may also be an engineered repressor RNAs such as synthetic small RNAs expressed SgrS, MicC or MicF scaffolds as described in Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. 2013. Nat Biotechnol 31: 170-4. An RNA selectable marker may also be an engineered repressor RNA as part of a selectable marker that represses a target RNA fused to a target gene to be regulated such as SacB as described in Williams, Supra, 2015
[00273] ROP: Repressor of primer
[00274] RSM: RNA selectable marker
[00275] SacB: Structural gene encoding Bacillus subtilis levansucrase. Expression of SacB in gram negative bacteria is toxic in the presence of sucrose
[00276] SD: Standard deviation
[00277] SEAP: Secreted alkaline phosphatase [00278] As used herein, the term “sequence identity” refers to the degree of identity between any given query sequence, e.g. SEQ ID NO: 2, and a subject sequence. A subject sequence may, for example, have at least 90 percent, at least 95 percent, or at least 99 percent sequence identity to a given query sequence. To determine percent sequence identity, a query sequence (e.g. a nucleic acid sequence) is aligned to one or more subject sequences using any suitable sequence alignment program that is well known in the art, for instance, the computer program ClustalW (version 2.1, default parameters), which allows alignments of nucleic acid sequences to be carried out across their entire length (global alignment). Chema et al., 2003 Nucleic Acids Res., 31:3497-500. In a preferred method, the sequence alignment program (e.g. ClustalW) calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more nucleotides can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair- wise alignments of nucleic acid sequences, suitable default parameters can be selected that are appropriate for the particular alignment program. The output is a sequence alignment that reflects the relationship between sequences. To further determine percent identity of a subject nucleic acid sequence to a query sequence, the sequences are aligned using the alignment program, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[00279] Selectable marker: A selectable marker, for example a kanamycin resistance gene or an RNA selectable marker
[00280] Selection marker: A selectable marker, for example a kanamycin resistance gene or an RNA selectable marker
[00281] SIDD: supercoiling-induced DNA duplex destabilized (SIDD) structures. These sites, when incorporated into a vector, may alter the susceptibility of other sequences within the vector to be destabilized. This can alter function. For example, addition of a SIDD site to an expression vector may reduce the helical destabilization of a promoter. This may increase or decrease promoter activity, depending on the promoter since some promoters have increased expression with promoter helical destabilization, while others will have reduced expression with promoter helical destabilization
[00282] shRNA: Short hairpin RNA
[00283] S/MAR: Scaffold/matrix attached region. Eukaryotic sequences that mediate DNA attachment to the nuclear matrix [00284] Sleeping Beauty Transposon: SB transposon. A transposon system that integrates an IR/DR flanked SB transposon into the genome by a simple cut and paste mechanism mediated by SB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the IR/DRs which is excised and integrated into the genome
[00285] Spacer region: As used herein, spacer region is the region linking the 5’ and 3’ ends of the eukaryotic region sequences. The eukaryotic region 5’ and 3’ ends are typically separated by the bacterial replication origin and bacterial selectable marker in plasmid vectors (bacterial region) so many spacer regions consist of the bacterial region. In Pol III dependent origin of replication vectors of the invention, this spacer region preferably is less than 1000 bp [00286] SR: Spacer region.
[00287] ssi: Single stranded initiation sequences
[00288] Structured DNA sequence: As used herein, a DNA sequence that is capable of forming replication inhibiting secondary structures (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71: 13-35). This includes but is not limited to inverted repeats, palindromes, direct repeats, IR/DRs, homopolymeric repeats or repeat containing eukaryotic promoter enhancers, or repeat containing eukaryotic origin of replications.
[00289] SV40 origin: Simian Virus 40 genomic DNA that contains the origin of replication [00290] SV40 enhancer: Simian Virus 40 genomic DNA that contains the 72 bp and optionally the 21 bp enhancer repeats
[00291] target antigen: Immunogenic protein or peptide epitope, or combination of proteins and epitopes, against which an immune response can be mounted. Target antigens may by derived from a pathogen for infectious disease or allergy applications or derived from a host organism for applications such as cancer, allergy, or autoimmune diseases. Target antigens are well defined in the art. Some examples are described in Williams, Supra, 2008 and are included herein by reference
[00292] TE buffer: A solution containing approximately lOmM Tris pH 8 and 1 mM EDTA [00293] TetR: Tetracycline resistance gene
[00294] Tol2 Transposon: A transposon system that integrates an ITR flanked Tol2 transposon into the genome by a simple cut and paste mechanism mediated by Tol2 transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the Tol2 ITRs which is excised and integrated into the genome
[00295] Transcription terminator: Bacterial: A DNA sequence that marks the end of a gene or operon for transcription. This may be an intrinsic transcription terminator or a Rho-dependent transcriptional terminator. For an intrinsic terminator, such as the trpA terminator, a hairpin structure forms within the transcript that disrupts the mRNA-DNA-RNA polymerase ternary complex. Alternatively, Rho-dependent transcriptional terminators require Rho factor, an RNA helicase protein complex, to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex. Eukaryotic: PolyA signals are not ‘terminators’, instead internal cleavage at PolyA sites leaves an uncapped 5 ’end on the 3’UTR RNA for nuclease digestion. Nuclease catches up to RNA Pol II and causes termination. Termination can be promoted within a short region of the poly A site by introduction of RNA Pol II pause sites (eukaryotic transcription terminator). Pausing of RNA Pol II allows the nuclease introduced into the 3’ UTR mRNA after PolyA cleavage to catch up to RNA Pol II at the pause site. A nonlimiting list of eukaryotic transcription terminators know in the art include the C2x4 and the gastrin terminator. Eukaryotic transcription terminators may elevate mRNA levels by enhancing proper 3'-end processing of mRNA
[00296] transfection: Method to deliver nucleic acids into cells [e.g. poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, block copolymers, Pluronic block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleofection, piezoelectric permeabilization, sonoporation, iontophoresis, ultrasound, SQZ high speed cell deformation mediated membrane disruption, corona plasma, plasma facilitated delivery, tissue tolerable plasma, laser microporation, shock wave energy, magnetic fields, contactless magneto-permeabilization, gene gun, microneedles, microdermabrasion, hydrodynamic delivery, high pressure tail vein injection, etc] as known in the art and included herein by reference
[00297] Transgene: Gene of interest that is cloned into a vector for expression in a target organism
[00298] Transposase vector: A vector which encodes a transposase
[00299] Transposon vector: A vector which encodes a transposon which is a substrate for transposase mediated gene integration
[00300] ts: Temperature sensitive
[00301] pg: Microgram
[00302] pl: Micro liter
[00303] UTR: Untranslated region of a mRNA (5’ or 3’ to the coding region)
[00304] Vector: A gene delivery vehicle, including viral (e.g. Alphavirus, Poxvirus, Lentivirus, Retrovirus, Adenovirus, Adenovirus related virus, etc.) and non-viral (e.g. plasmid, MIDGE, transcriptionally active PCR fragment, minicircles, bacteriophage, etc.) vectors. These are well known in the art and are included herein by reference [00305] Vector backbone: Eukaryotic region and bacterial region of a vector, without the transgene or target antigen coding region
DETAILED DESCRIPTION
[00306] The current technology relates generally to short < Ikb bacterial region plasmid DNA vector methods and compositions that improve plasmid manufacture yield and quality, reduce transfection associated toxicity, and increase transgene expression. The current technology can be practiced to improve expression and manufacturing of vectors such as non-viral vectors (mRNA vector, transposon vector, transposase vector, Sleeping Beauty transposon vector, Sleeping Beauty transposase vector, PiggyBac transposon vector, PiggyBac transposase vector, expression vector, etc.) and viral vectors (e.g. AAV vector, AAV rep cap vector, AAV helper vector, Ad helper vector, Lentivirus vector, Lentiviral envelope vector, Lentiviral packaging vector, Retroviral vector, Retroviral envelope vector, Retroviral packaging vector, etc.).
[00307] Improved plasmid expression is defined herein as improved transgene expression level and/or expression duration in vitro or in vivo compared to a transgene encoding plasmid containing a bacterial region encoding the pUC replication origin. It is to be understood that all references cited herein are incorporated by reference in their entirety,
[00308] The methods of plasmid modification of the present current technology have been surprisingly found to provide a solution to provide short spacer region vectors containing structured DNA sequences with efficient high yield manufacture.
Compositions Comprising a Pol Ill-Dependent Origin of Replication
[00309] In embodiments, compositions comprising a DNA molecule comprising a backbone comprising a Pol Ill-dependent origin of replication are disclosed
[00310] In embodiments, a covalently closed circular recombinant DNA molecule comprising a backbone and an insert, wherein the backbone comprises a Pol Ill-dependent origin of replication, a selectable marker, and a first primosomal assembly site, wherein the first primosomal assembly site is positioned downstream of the Pol Ill-dependent origin of replication in the direction of replication, and wherein the insert comprises a structured DNA sequence, is disclosed.
[00311] In embodiments, structured DNA sequence is within 1000 bp of the Pol Ill-dependent origin of replication.
[00312] In embodiments, the backbone is less than 1000 bp.
[00313] In embodiments, the backbone comprises a bacterial replication-selection region.
[00314] In embodiments, the Pol Ill-dependent origin of replication does not require Pol I. [00315] In embodiments, the Pol Ill-dependent origin of replication is a Pol Ill-dependent R6K origin of replication.
[00316] In embodiments, the Pol Ill-dependent R6K origin of replication has at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24.
[00317] In embodiments, the selectable marker is an RNA selectable marker.
[00318] In embodiments, the RNA selectable marker is an RNA-OUT RNA selectable marker, [00319] In embodiments, the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA- OUT functional variant with at least 80% sequence identity to a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7.
[00320] In embodiments, the RNA selectable marker comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6.
[00321] In embodiments, the first primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
[00322] In embodiments, the covalently closed circular recombinant DNA molecule further comprises a second primosomal assembly site downstream of the Pol Ill-dependent origin of replication in the direction of replication,
[00323] hi embodiments, the second primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
[00324] In embodiments, the covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the Pol Ill-dependent origin of replication in the direction of replication.
[00325] In embodiments, the covalently closed circular recombinant DNA molecule of any one of claims 13-14, comprises the sequence of SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the Pol Ill-dependent origin of replication in the direction of replication.
[00326] hi embodiments, the covalently closed circular recombinant DNA molecule is antibiotic marker free.
[00327] In embodiments, the backbone comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00328] In embodiments, the backbone comprises a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00329] In embodiments, the structured DNA sequence is selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence.
[00330] In embodiments, the insert is a transposon vector.
[00331] In embodiments, the structured DNA sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
[00332] In embodiments, the insert is a transposase vector.
[00333] In embodiments, the insert is a mRNA vector.
[00334] In embodiments, the insert is an AAV vector.
[00335] In embodiments, the structured DNA sequence is an inverted repeat sequence.
[00336] In embodiments, the AAV vector encodes AAV2 ITRs.
[00337] In embodiments, the insert comprises a 5’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 35 and a 3’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 36.
[00338] In embodiments, the insert is a lentiviral vector.
[00339] In embodiments, the structured DNA sequence is a direct repeat sequence or an eukaryotic origin of replication.
[00340] In embodiments, the structured DNA sequence is selected from an homopolymeric repeat such as a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
[00341] In embodiments, the structured DNA sequence comprises a homopolymeric repeat.
[00342] In embodiments, the homopolymeric repeat is a polyA repeat.
[00343] In embodiments, the homopolymeric repeat comprises from about 3 to about 500 residues.
[00344] In embodiments, the selectable marker is a RNA selectable marker and is oriented to transcribe in a direction divergent from the structured DNA sequence. [00345] In embodiments, an antibiotic marker free covalently closed circular recombinant DNA molecule is disclosed, the molecule comprising a backbone and an insert, wherein the backbone comprises an origin of replication and an RNA selectable marker, wherein the insert comprises a structured DNA sequence, and wherein the RNA selectable marker is oriented to transcribe in a direction divergent from the structured DNA sequence.
[00346] In embodiments, the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is within 1000 bp of the origin of replication, [00347] In embodiments, the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule is less than 1000 bp.
[00348] In embodiments, the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a bacterial replication-selection region.
[00349] In embodiments, the origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule is a Pol I-dependent origin of replication.
[00350] In embodiments, the origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule is a Pol Ill-dependent origin of replication.
[00351] In embodiments, the Pol Ill-dependent origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule does not require Pol I.
[00352] In embodiments, the Pol Ill-dependent origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule is a Pol Ill-dependent R6K origin of replication.
[00353] In embodiments, the Pol Ill-dependent R6K origin of replication of the antibiotic marker free covalently closed circular recombinant DNA molecule has at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24.
[00354] In embodiments, the RNA selectable marker of the antibiotic marker free covalently closed circular recombinant DNA molecule is an RNA-OUT RNA selectable marker.
[00355] In embodiments, the RNA-OUT RNA selectable marker of the antibiotic marker free covalently closed circular recombinant DNA molecule is an RNA-IN regulating RNA-OUT functional variant with at least 80% sequence identity to a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7.
[00356] fri embodiments, the RNA selectable marker of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6. [00357] In embodiments, the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00358] In embodiments, the backbone of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
[00359] In embodiments, the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence.
[00360] In embodiments, the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a transposon vector.
[00361] In embodiments, the structured DNA of the antibiotic marker free covalently closed circular recombinant DNA molecule sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
[00362] hi embodiments, the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a transposase vector.
[00363] \In embodiments, the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a mRNA vector.
[00364] In embodiments, the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is an AAV vector.
[00365] In embodiments, the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is an inverted repeat sequence.
[00366] In embodiments, the AAV vector of the antibiotic marker free covalently closed circular recombinant DNA molecule encodes AAV2 ITRs.
[00367] hi embodiments, the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a 5’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 35 and a 3’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 36. [00368] In embodiments, the insert of the antibiotic marker free covalently closed circular recombinant DNA molecule is a lentiviral vector.
[00369] In embodiments, the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is a direct repeat sequence or an eukaryotic origin of replication.
[00370] In embodiments, the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule is selected from a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer. [00371] In embodiments, the structured DNA sequence of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a homopolymeric repeat.
[00372] In embodiments, the homopolymeric repeat of the antibiotic marker free covalently closed circular recombinant DNA molecule is a polyA repeat.
[00373] In embodiments, the homopolymeric repeat of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises from about 3 to about 500 residues.
[00374] In embodiments, the antibiotic marker free covalently closed circular recombinant DNA molecule further comprises a first primosomal assembly site downstream of the origin of replication in the direction of replication,
[00375] hi embodiments, the antibiotic marker free covalently closed circular recombinant DNA molecule further comprises a second primosomal assembly site downstream of the origin of replication in the direction of replication.
[00376] In embodiments, the first primosomal assembly site of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 , and SEQ ID NO: 33.
[00377] In embodiments, the first primosomal assembly site of the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 , and SEQ ID NO: 33, and wherein the second primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31 , and SEQ ID NO: 33.
[00378] In embodiments, the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site is downstream of the origin of replication in the direction of replication.
[00379] In embodiments, the antibiotic marker free covalently closed circular recombinant DNA molecule comprises a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the origin of replication in the direction of replication.
Methods for Replicating a DNA Molecule
[00380] In embodiments, methods for replicating structured DNA sequences that are poorly replicated by a Pol I-dependent origin of replication such as the pUC orgin are disclosed.
[00381] hi embodiments, methods for improving viral vector manufacturing are disclosed.
[00382] In embodiments, methods for improving non-viral vector manufacturing are disclosed.
[00383] In embodiments, methods for improving yield of viral and/or non-viral vector manufacturing yield is disclosed.
[00384] In embodiments, methods for improving yield of viral and/or non-viral vector manufacturing quality is disclosed.
[00385] In embodiments, methods for eliminating antibiotic resistance marker gene transfer by non-viral and/or viral vectors are disclosed.
[00386] In embodiments, methods for reducing transfection associated toxicity are disclosed.
[00387] hi embodiments, methods for improving transposition from non-viral transposon vectors are disclosed.
[00388] hi embodiments, methods for improving packaging titers from viral vectors; improving expression of viral and non-viral vector encoded transgenes are disclosed.
[00389] In embodiments, methods for improving R6K origin mediated replication of a closely positioned structured DNA sequence are disclosed.
[00390] In embodiments, a method for replicating a circular recombinant DNA molecule is disclosed. In embodiments, a method for replicating a closed circular recombinant DNA molecule is disclosed. In embodiments, a method for replicating a covalently closed circular recombinant DNA molecule is disclosed.
[00391] In embodiments, the method comprises providing a cell containing the recombinant DNA molecule as disclosed herein and subjecting the cell to a fermentation process.
[00392] hi embodiments, the cell is an engineered Rep protein-expressing E. coli strain. [00393] In embodiments, the cell comprises a chromosomally-integrated arabinose inducible CI857ts gene.
[00394] In embodiments, the Rep protein comprises at least one of the following mutations: P42L; P 1061; F107S; and Pl 13S. In embodiments, the Rep protein comprises at least two of the following mutations: P42L; P106I; F107S; and P113S. In embodiments, the Rep protein comprises three of the following mutations: P42L; P106I; F107S; and P113S. In embodiments, the Rep protein comprises all four of the following mutations: P42L; P 1061; F107S; and Pl 13S. [00395] hi embodiments, the fermentation process comprises growing the cells in media containing arabinose.
[00396] In embodiments, the yield of the covalently closed circular plasmid following the fermentation process is in excess of 0.5 g/L.
[00397] Turning now to the drawings, FIGS. 1A-1F show annotated maps of: FIG. 1A) R6K origin with the locations of the 22 bp iteron repeats, DnaA boxes 1 and 2, and the regions included in the SEQ ID NO: 1, 2, 3, and 4 R6K origins; FIG. IB) SEQ ID NO: 5 RNA-OUT selectable marker with the locations of the RNA-OUT promoter -35 and -10 elements, SEQ ID NO: 6 RNA OUT antisense RNA with RNA-IN complementary homology region and RNA-OUT terminator 3’ hairpin; FIG. 1C) 14 CpG R6K-RNA-OUT bacterial backbone composed of SEQ ID NO: 1 R6K replication origin and SEQ ID NO: 5 RNA-OUT selectable marker including the trpA bacterial terminator upstream of the R6K origin and flanked byNhel and Kpnl cloning sites; FIG. ID) 3 CpG R6K-RNA-OUT bacterial backbone composed of SEQ ID NO: 2 lx CpG R6K replication origin and SEQ ID NO: 7 2x CpG RNA-OUT selectable marker flanked by Nhel and Kpnl cloning sites; FIG. IE) R6K origin from SEQ ID NO: 1, with locations of the 6 iterons highlighted. The individual 22 bp iteron repeat sequences are shown below the origin map; and FIG. IF) R6K origin from SEQ ID NO: 18, with locations of the 7 iterons highlighted. The individual 22 bp iteron repeat sequences are shown below the origin map. In this example 7 iteron vector iteron 5 has been tandemly duplication; however, a 7 iteron vector of the invention can be obtained by tandem duplication of any of iterons 1, 2, 3, 4, 5 or 6.
[00398] FIGS. 2A-2B show annotated maps of: FIG. 2A) Pol I-dependent pUC origin- Kanamycin selection Sleeping Beauty transposon vector pUC57-Kan SB1 (see Table 4); and FIG. 2B) Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection Sleeping Beauty transposon vector NTC9 SB1 (see Table 4). The locations of the left and right Sleeping Beauty IR/DR relative to the bacterial backbone replication origins and selection markers are shown.
[00399] FIGS. 3A-3C show annotated maps of: FIG. 3A) Pol I-dependent pUC origin- Ampicillin selection AAV vector pAAV (see Table 7); FIG. 3B) Pol I-dependent pUC origin- RNA-OUT antibiotic free selection AAV vector NTC8-AAV (see Table 7); and FIG. 3C) Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection AAV vector NTC9-AAV (see Table 7). The locations of the left and right AAV ITRs relative to the bacterial backbone replication origins and selection markers are shown.
[00400] FIGS. 4A— 41 show annotated maps of: FIG. 4A) Pol I-dependent pUC origin- Ampicillin selection A60 polyA repeat encoding mRNA vector pGEM4Z T7 A60 pA (see Table 6); FIG. 4B) Pol I-dependent pUC origin-RNA-OUT antibiotic free selection A60 polyA repeat encoding mRNA vector NTC8-T7 A60 pA (see Table 6); FIG. 4C) Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection A60 polyA repeat encoding mRNA vector NTC9-T7 A60 pA (see Table 6); FIG. 4D) Pol I-dependent pUC origin- Ampicillin selection A99 polyA repeat encoding mRNA vector pT3/T7 A99 pA (see Table 6); FIG. 4E) Pol I-dependent pUC origin-kanR selection A99 polyA repeat encoding mRNA vector NTC7-T7 A99 pA (see Table 6); and FIG. 4F) Pol Ill-dependent R6K origin-RNA-OUT antibiotic free selection A99 polyA repeat encoding mRNA vector NTC9-T7 A99 pA (see Table 6). The location of the A60 or A99 polyA repeat relative to the bacterial backbone replication origins and selection markers are shown.
[00401] FIGS. 5A-5D show annotated maps of the Pol Ill-dependent R6K origin R6K-RNA- OUT bacterial backbones from SEQ ID NO: 25 (A) SEQ ID NO: 27 (B) SEQ ID NO: 28 (C) SEQ ID NO: 34 (D)
[00402] FIGS. 6A-6B show annotated maps of an example Pol Ill-dependent R6K origin AAV ITR encoding AAV vector (FIG. 6A) or A 100 polyA repeat encoding mRNA vector (FIG. 6B) [00403] FIGS. 7A-7B are BspQl linearizations of purified plasmid DNA from fermentation harvests of (Fig. 7A) mRNA vector -NP (polyAlOO <ROUT R6K origin>) and (Fig. 7B) 2 different fermentation harvest lots of mRNA vector -NP 7 iteron PAS R6K> ROUT> (polyAlOO R6K origin>_PAS ROUT>). One microgram of purified DNA was digested with BspQl overnight at 50°C and resolved on an agarose gel and post stained with SybrII which recognizes DNA and RNA. Units BspQl per microgram DNA are shown above each lane. The mRNA vector -NP purified plasmid DNA prep (Fig. 7A) has RNA contamination (diffuse low molecular weight band on gel). Since concentration is determined by A260, the actual total amount of plasmid DNA in these reactions is less than the intended one microgram per lane. EXAMPLES
[00404] The methods of the current technology are further illustrated by the following examples. These are provided by way of illustration and are not intended in any way to limit the scope of the disclosure.
Example 1: pUC, and R6K replication origin plasmid replication and production
[00405] pUC origin vector replication and production background:
[00406] The vast majority of therapeutic plasmids use the pUC origin which is a high copy derivative of the pMBl origin (closely related to the ColEl origin). For pMBl replication, plasmid DNA synthesis is unidirectional and does not require a plasmid borne initiator protein. The pUC origin is a copy -up derivative of the pMB 1 origin that deletes the accessory ROP (rom) protein and has an additional temperature sensitive mutation that destabilizes the RNAFRNAII interaction. Shifting of a culture containing these origins from 30 to 42°C leads to an increase in plasmid copy number. pUC plasmids can be produced in a multitude of E. coli cell lines.
[00407] RNA-OUT antibiotic free selectable marker background: Antibiotic-free selection is performed in E. coli strains containing phage lambda attachment site chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) as described in Williams, Supra, 2008. SacB (Bacillus subtilis levansucrase) is a counterselectable marker which is lethal to E. coli cells in the presence of sucrose. Translation of SacB from the RNA-IN-SacB transcript is inhibited by plasmid encoded RNA-OUT (Fig. IB). This facilitates plasmid selection in the presence of sucrose, by inhibition of SacB mediated lethality.
[00408] R6K origin vector replication and production background: The R6K gamma plasmid replication origin requires a single plasmid replication protein TJ that binds as a replication initiating monomer to multiple repeated ‘heron’ sites (seven core repeats containing TGAGNG consensus) and as a replication inhibiting dimer to repressive sites (TGAGNG) and to iterons with reduced affinity. Replication requires multiple host factors including IHF, DnaA, and primosomal assembly proteins DnaB, DnaC, DnaG (Abhyankar et al,, 2003 J Biol Chem 278:45476-45484), The R6K core origin contains binding sites for DnaA and IHF that affect plasmid replication since ft, IHF and DnaA interact to initiate replication.
[00409] Different versions of the R6K gamma replication origin have been utilized in various eukaryotic expression vectors, for example pCOR vectors (Soubrier et al., 1999, Gene Therapy 6: 1482-88) and a CpG free version in pCpGfree vectors (Invivogen, San Diego CA), and pGM169 (University of Oxford). Incorporation of the R6K replication origin per se does not improve transgene expression levels compared to an optimized pUC origin vector (Soubrier et al., Supra, 1999). However, use of a conditional replication origin such as R6K gamma that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patient’s endogenous flora.
[00410] A highly minimalized 6 iteron R6K gamma derived replication origin (SEQ ID NO: 1; Fig. IE) that contains core sequences required for replication (including the DnaA box and stb 1- 3 sites; Wu et al., 1995. J Bacterial. 177: 6338-6345), but with the upstream p dimer repressor binding sites and downstream n promoter deleted (by removing one copy of the iterons) was described in Williams, Supra, 2014 and incorporated herein by reference. This R6K origin contains 6 tandem direct repeat iterons (Fig. IE). The NTC9385R Nanoplasmid™ vector including this minimalized R6K origin and the RNA-OUT AF selectable marker in the spacer region, was described in Williams, Supra, 2014 and included herein by reference.
[00411] Typical R6K production strains express from the genome the P protein derivative PIR116 that contains a P106L substitution that increases copy number (by reducing P dimerization; p monomers activate while P dimers repress). Fermentation results with pCOR (Soubrier et al., Supra, 1999) and pCpG plasmids (Hebei HL, Cai Y, Davies LA, Hyde SC, Pringle IA, Gill DR. 2008. Mol Ther 16: Si 10) were low, around 100 mg/L in PIR116 cell lines. [00412] Mutagenesis of the pir-116 replication protein and selection for increased copy number has been used to make new production strains. Lor example, the TEX2pir42 strain contains a combination of P106L and P42L. The P42L mutation interferes with DNA looping replication repression. The TEX2pir42 cell line improved copy number and fermentation yield with pCOR plasmids with reported yields of 205 mg/L (Soubrier E. 2004. World Patent Application W02004033664).
[00413] Other combinations of Fl copy number mutants that improve copy number include ‘P42L and Pl 13S’ and ‘P42L, P106L and F107S’ (Abhyankar et al., 2004. J Biol Chem 279:6711-6719). [00414] Williams, Supra, 2014 describes host strains expressing phage HK022 attachment site integrated pL promoter heat inducible P P42L, P106L and F107S high copy mutant replication (Rep) protein for selection and propagation of R6K origin Nanoplasmid™ vectors. This is an additional Nanoplasmid™ safety factor since R6K origin vectors can only replicate within the engineered Rep protein-expressing E. coli host strain.
[00415] RNA-OUT selectable marker-R6K plasmid propagation and fermentations described in Williams, Supra, 2014 were performed using heat inducible ‘P42L, P106L and F107S’ P copy number mutant cell lines such as DH5a host strain NTC711772 = DH5a dem- att, ::P 66/6-RNA- IN- SacB, catR; attHK022::pL (0L1-G to T) P42L-P106L-F107S (P3-), SpecR StrepR. Production yields up to 695 mg/L were reported.
[00416] Additional cell lines were created and disclosed herein including:
[00417] NTC821601 DH5a atW:P5/66/6-RNA-IN- SacB, catR; attHK022:: pL (OL1-G to T) P42L- P106L-F107S (P3-), SpecR StrepR = dcm+ version ofNTC711772
[00418] NTC940211 DH5a attλ::P5/66/6-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L- P106I-F107S P113S (P3-), SpecR StrepR = high copy substitution of P106I for P106L combined with P113S to create a quadruple copy number increasing mutant rep protein derivative of NTC821601
[00419] NTC1050811 DH5a attλ::P5/66/6-RNA-IN- SacB, catR; attHK022::pL (OLl-G to T) P42L- P106I-F107S P113S (P3-), SpecR StrepR; att<p8o::pARA-CI857ts, tetR = pARA-CI857ts derivative of NTC940211. This strain contains a phage cp80 attachment site chromosomally integrated copy of a arabinose inducible CI857ts gene. Addition of arabinose to plates or media (e.g. to 0.2-0.4% final concentration) induces pARA mediated CI857ts repressor expression which reduces copy number at 30° C through CI857ts mediated downregulation of the Rep protein expressing pL promoter [i.e. additional CI857ts mediates more effective downregulation of the pL (OL1-G to T) promoter at 30°C], Copy number induction after temperature shift to 37- 42°C is not impaired since the CI857ts repressor is inactivated at these elevated temperatures. A dem- derivative (NTC 1050811 dem-) is used in cases where dem methylation is undesirable.
[00420] NTC1011641; Stbl4 attλ ::P5/6 6/6-RNA-IN- SacB, catR; attHIco22::pL P42L-P106L- F107S (P3-) SpecR StrepR = Stbl4 version of NTC66113.5 (XI ,.l Blue- dem- att>.::P5/6 6/6-RNA- IN- SacB, catR; attHK022"pR pL P42L P106L-F107S ( 1’3 - ) SpecR StrepR described in Williams, Supra, 2014
[00421] Nanoplasmid™ production yields are improved with the quadruple mutant heat inducible pL (OL1-G to T) P42L-P106I-F107S P113S (P3-) compared to the triple mutant heat inducible pL (OL1-G to T) P42L-P106L-F107S (P3-) described in Williams, Supra, 2014. Yields in excess of 2 g/L Nanoplasmid™ have been obtained with the quadruple mutant NTC 1050811 cell line (e.g. 2240 mg/L with NTC9 T7 A99 pA, Table 6)
[00422] Use of a conditional replication origin such as these R6K origins that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patient’s endogenous flora.
Example 2: pUC and R6K origin vector production [00423] Shake flask production : Shake flask production was performed using proprietary PlasmidF shake culture medium. The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 pg/mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates were grown at 30-32°C; cells were resuspended in media and used to provide approximately 2.5 ODgoo inoculums for the 500 mL Plasmid+ shake flasks that contained 50 pg/mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose to select for RNA-OUT plasmids. Flasks were grown with shaking to saturation at the growth temperatures as indicated in Tables 5, 6, 7, and 9.
[00424] Fermentation production: Fermentations were performed using proprietary fed-batch media (NTC3019, HyperGRO media) in New Brunswick BioFlo 110 bioreactors as described (Carnes and Williams, Supra, 2011). The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 pg/mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates were grown at 30-32°C; cells were resuspended in media and used to provide approximately 0.1% inoculums for the fermentations that contained 50 pg/mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT plasmids. HyperGRO temperature shifts were as indicated in Tables 8 and 9.
[00425] Production hosts: pUC origin AmpR or KanR plasmid fermentations were performed in E. coli strain DH5a [F- Q80/acZAM15 A(ZacZYA-argF) U169 recAl ewt/Al /m/R17 (rK- mK+) phoS supEA6 X- thi-1 gyr A96 re/A I ] (Invitrogen, Carlsbad CA) or Stbl4.
[00426] Antibiotic-free pUC origin RNA-OUT plasmid fermentations were performed in E. coli strain DH5a containing phage lambda attachment site chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) as described in Williams, Supra, 2008. The production strain is NTC4862 = DH5a attX::P5/6 6/6-RNA-IN- SacB, catR.
[00427] Antibiotic-free R6K gamma origin RNA-OUT plasmid propagation and fermentations were performed using E. coli RNA-OUT selection hosts further encoding phage HK022 attachment site integrated pL promoter heat inducible TJ copy number mutant cell line lines, methods for the creation of which are described in Williams, Supra, 2014 and included herein by reference.
[00428] Production strains:
[00429] pUC origin-AmpR or KanR antibiotic selection hosts
[00430] DH5a
[00431] Stbl4
[00432] pUC origin-RNA-OUT sucrose selection hosts [00433] NTC4862 DH5a atfi::P5/66/6-RNA-IN- SacB, catR
[00434] NTC1011592 Stbl4 attk::P5/6 6/6-RNA-IN- SacB, catR
[00435] R6K origin-RNA-OUT sucrose selection Nanoplasmid™ hosts
[00436] NTC1050811 DH5a atfi::Ps/66/6-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L- P106I-F107S P113S (P3-), SpecR StrepR; att<p8o::pARA-CI857ts, tetR
[00437] NTC1011641 Stbl4 attk::P5/6 6/6-RNA-IN- SacB, catR; attHK022::pL P42L-P106L- F107S (P3-) SpecR StrepR
[00438] Analytical Methods: Culture samples were taken at key points and at regular intervals during all fermentations. Samples were analyzed immediately for biomass (ODeoo) and for plasmid yield. Plasmid yield was determined by quantification of plasmid obtained from Qiagen Spin Miniprep Kit preparations as described (Carnes and Williams, Supra, 2011). Briefly, cells were alkaline lysed, clarified, plasmid was column purified, and eluted prior to quantification. Plasmid quality was determined by agarose gel electrophoresis analysis (AGE) and was performed on 0.8-1% Tris/acetate/EDTA (TAE) gels as described in Carnes and Williams, Supra, 2011.
Example 3: pUC and R6K origin structured vector construction and manufacturing
[00439] The R6K gamma origin (SEQ ID NO:1; Fig. 1E)-RNA-OUT (SEQ ID NO:5; Fig. IB) bacterial replication-selection region (SEQ ID NO:8; Fig. 1C) was cloned into the polylinker region of a variety of pUC57 based vectors to create the pNTC-NPl, pNTC-NP2, pNTC-NP3, pNTC-NP4, pNTC-NP5, pNTC-NP6, pNTC-NP7, vectors. Each vector has different flanking restriction sites that can be used to retrofit a target vector to R6K replication-RNA-OUT selection. The 5’ and 3’ polylinker sequences flanking the R6K-RNA-OUT insert in the pNTC-NP 1-7 vectors are shown in Table 2. A pUC57 based version of the 1 CpG R6K gamma origin- 2 CpG RNA-OUT bacterial replication-selection region (SEQ ID NO:9; Fig. ID) was also created (pNTC-3xCpG NP1) and is shown in Table 2.
[00440] The R6K gamma origin (SEQ ID NO:1) is an engineered 6 iteron R6K origin (Fig. IE). A pUC57 based version of a 7 iteron R6K gamma origin (SEQ ID NO:18; Fig. 1F)-RNA-OUT (SEQ ID NO:5; Fig. IB) bacterial replication-selection region was also created and used to construct and evaluate the utility of additional iterons on manufacturing. Similarly, high quality, high yield manufacture was obtained with vectors differing only by containing either the SEQ ID NO:18 seven iteron R6K gamma origin or the six iteron R6K gamma origin (SEQ ID NO:1). For example, the following harvest production yields were obtained in 30-42°C 10 hr ramp temperature shift HyperGRO fermentations:
[00441] SEQ ID NO:1 6 iteron 3203 bp R6K origin vector: a biomass of 120 ODeoo; plasmid titer of 1363 mg/L; plasmid specific yield of 11.3 mg plasmid/L/ODeoo
[00442] SEQ ID NO:18 7 iteron 3225 bp R6K origin vector: a biomass of 137 ODgoo; plasmid titer of 1503 mg/L; plasmid specific yield of 11.0 mg plasmid/L/ODeoo
[00443] The 7 iteron R6K gamma origin in SEQ ID NO: 18 is a tandem duplication of iteron 5 (Fig. IF; SEQ ID NO:18) but the 7 iteron R6K gamma origin vectors of the invention can be tandem duplications of any of the iterons given as SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23 (Fig. IE), or random combinations of SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23 into 7 iteron R6K origin compositions, or iteron repeat variants that retain the TGAGNG consensus. Additional iteron derivatives (e.g. 8, 9 or 10 iteron vectors) are also contemplated for practice of the invention.
[00444] Viral and non-viral vector pUC origin-antibiotic selection bacterial backbone retrofits to R6K-RNA-OUT were performed by:
[00445] 1) selecting restriction sites that flank the pUC origin and antibiotic selection marker region in the target viral and non-viral vector;
[00446] 2) Identifying a pNTC-NP compatible polylinker -R6K-RNA-OUT polylinker cassette (either pNTC-NP 1, 2, 3, 4, 5, 6, or 7; Table 2);
[00447] 3) Excising the pUC origin antibiotic selection marker region and replacing with the selected R6K origin RNA-OUT region using the selected restriction digestion approach and standard ligase mediated cloning.
[00448] In some cases, the R6K origin and RNA-OUT units were assembled in multi-fragment ligations from separate restriction fragments using the non-palindromic Dralll linker site (see Table 2). In the case of the fd6 Ad helper retrofit (Table 7), a 3-fragment ligation was performed using a short 500 bp synthetic gene Dralll RNA-OUT-Ad helper-Avrll to link RNA-OUT to a unique Avril site in the fd6 Ad helper eukaryotic region in a 12 kb Avril-Sall restriction fragment, and to a Sall- R6K origin-Dralll fragment from pNTC-NP4.
[00449] Example vector maps and vector characteristics of the original pUC origin- antibiotic selection marker vector and the retrofitted R6K origin-RNA-OUT antibiotic free selection marker vectors are shown for Sleeping Beauty (Fig 2; Table 4), AAV (Fig. 3; Table 7) and mRNA (Fig. 4; Table 6) vectors. The vector characteristics of the original pUC origin- antibiotic selection marker vector and the retrofitted R6K origin-RNA-OUT antibiotic free selection marker vectors are shown for AAV helper vectors (Table 6). The vector characteristics of pUC origin- RNA- OUT antibiotic free selection marker vector and the retrofitted R6K origin-RNA-OUT antibiotic free selection marker vectors are shown for Lentiviral vectors (Table 3) and AAV vectors (Table 5).
[00450] In all cases, the bacterial backbone size was < 1 kb in the R6K origin-RNA-OUT antibiotic free selection marker retrofitted vectors (460-610 bp). This is well below the 1.1 kb bacterial backbone size limit required to improve vector expression level (Tables 1-2) and duration (Quiviger et al., Supra, 2014). In all cases, the original pUC origin-antibiotic selection bacterial backbone prior to retrofit was > 1.2 kb (2340-2750 bp) as were the pUC origin-RNA- OUT retrofits (1210-1500 bp). Thus, these AAV, AAV helper, Sleeping Beauty, and Lentiviral R6K origin-RNA-OUT antibiotic free selection marker retrofit vectors meet the short spacer region requirement for improved expression level and duration compared to the original pUC origin-antibiotic selection marker vector. Additionally, these AAV, AAV helper, Sleeping Beauty, and Lentiviral R6K origin-RNA-OUT antibiotic free selection marker retrofit vectors have no chance of antibiotic marker gene transfer by transduction (AAV, Lentiviral vectors) or transposition (Sleeping Beauty vectors) due to removal of the KanR or ampR antibiotic resistance selection marker in the parent vector. Additionally, the vectors of the current technology do not require the complicated difficult to scale expensive additional manufacturing steps required to remove the large bacterial region between the eukaryotic polyA and promoter with minicircle vectors (Kay et al., Supra, 2010).
[00451] However, in a Lentiviral vector the eukaryotic region contains flanking direct repeat LTRs, in an AAV vector the eukaryotic region contains flanking inverted terminal repeats, while in a Sleeping Beauty transposon vector the eukaryotic region contains flanking transposon IR/DR termini. These flanking sequences are all structured DNA sequences.
[00452] Levy, Supra, 2004 teaches that replication intermediates form when any high copy number prokaryotic origin of replication is < Ikb from a structured DNA sequence such as an enhancer, LTR or IRES, but not when the high copy replication origin is >1.5 kb. Consistent with this, replication intermediates were formed in all pUC origin- RNA-OUT marker vectors in which the pUC origin was <1 kb from a Lentiviral vector LTR (Table 3: 400 bp) or a pUC originantibiotic resistance marker vector in which the pUC origin was <1 kb from a Sleeping Beauty IR/DR (Table 4; 280 bp). For AAV and mRNA vectors, the original pUC origin-antibiotic selection marker vectors have the pUC origin 0 bp from an ITR (AAV vector; Table 7) or 170 bp from a A99 repeat (mRNA vector, Table 6) which may make a replication intermediate that is too small to detect on an agarose gel. However, in these cases production yields were very low, indicative of low plasmid copy number due to replication blockage. By contrast, as expected, in the case where the original pUC origin-antibiotic selection marker vector pUC origin was > 1.5 kb from a structured DNA sequence (A60 repeat), high plasmid production yields were obtained (Table 6: mRNA vector pGEM4Z T7 A60).
[00453] Williams, Supra, 2017 reported that pUC origin vector production yield is improved with a PAS-BH extended pUC origin when the pUC origin is >1.5 kb from a homopolymeric A64C31 repeat. However, production yields were low when a PAS-BH extended pUC origin is orientated <400 bp from the A64C31 repeat (Table 6, see footnotes d and e). This teaches that addition of a PAS-BH primosomal assembly site does not overcome the poor pUC origin directed replication of closely positioned structured DNA sequences.
[00454] Since the pUC origin itself is 1 kb, there is no configuration to make a <1. 1 kb bacterial region AAV, Lentiviral, Retroviral or transposon vector containing the pUC origin which is not predicted to produce replication intermediates as seen above and predicted by Levy, Supra, 2004 and poor plasmid yields as reported herein.
[00455] Surprisingly, replication intermediates were not observed in any R6K origin-RNA-OUT antibiotic-free selection marker retrofitted vectors, including those in which the R6K origin was <1 kb from a Lentiviral vector LTR (Table 3: 400 bp) or a Sleeping Beauty IR/DR (Table 4; <40 bp). Further, for AAV vectors, while the original pUC origin-antibiotic selection marker vectors with the pUC origin 0 bp from the ITR had very poor production yields, the two R6K origin- RNA-OUT antibiotic free selection marker retrofitted vectors with the R6K origin 40 bp from the ITR had much higher production yields (T ble 5). This improved production is specific to R6K and not RNA-OUT, since the two AAV pUC-RNA-OUT retrofits with the pUC origin 50 bp from the ITR had equally poor plasmid production yields as the original pUC antibiotic marker vector (Table 5); as well the direct comparison of pUC-RNA-OUT with R6K-RNA-OUT retrofits positioned 400 bp from an LTR repeat in a Lentiviral backbone showed replication intermediates with all three pUC-RNA-OUT backbones but none of the three R6K-RNA-OUT backbones (Table 3).
[00456] Without intending to be limited by theory, this surprising improvement in plasmid copy number (plasmid production yields) and quality (eliminated replication intermediates) with the R6K origin vector implies that the R6K origin can replicate through a structured DNA sequence more effectively than the pUC origin. While Levy, Supra, 2004 teaches that replication intermediates form when any high copy number prokaryotic origin of replication is < Ikb from a structured DNA sequence such as an enhancer, LTR or IRES, but not when the high copy replication origin is >1.5 kb away, the examples provided by Levy, Supra, 2004 were all with pUC origin plasmids. [00457] A fundamental difference between these replication origins is that the pUC origin is a Pol I dependent origin of replication while the R6K origin is a Pol III dependent origin of replication. With the pUC origin the RNAII primer forms an RNA:DNA R-loop that is cleaved by RNase H to create a primer for DMA Pol I directed DNA synthesis during initial leading strand synthesis. DNA synthesis then converts from slow DNA Pol I to the highly processive DNA Pol III from 400 bp to up to 1.3 kb downstream of the origin (Allen et al., 2011. Nucleic Acids Research 39:7020-33), The R6K gamma replication origin rep protein interacts with dnaB helicase and dnaG primase which creates short RNA primers for DNA Pol III replication without requirement for DNA Pol I (Abhyankar et al., Supra, 2003). The pUC origin DNA Pol I replication zone of up to 1.3 kb from the origin corresponds closely with the Levy, Supra, 2004 defined upper limit of replication intermediate formation (between 1 and 1.5 kb from the origin). Without intending to be limited by theory, it is proposed that the observed surprisingly improved replication of structured DNA when in close proximity to the R6K but not the pUC origin is due to an unexpected improvement of replication of structured DNA sequences by DNA Pol III compared to DNA Pol I.
[00458] The vector methods and compositions disclosed herein demonstrate that a Pol Ill- dependent origin of replication such as the R6K origin can be used to replicate structured DNA sequences which are poorly replicated by a Pol Ldependent origin of replication such as the pUC origin.
[00459] These results demonstrate the vectors of the invention are useful for improving viral and non- viral vector manufacturing yield and quality.
Example 4: Improved performance of R6K origin structured vector
[00460] The vectors of the invention are additionally useful for eliminating antibiotic resistance marker gene transfer by viral and non-viral vectors; reducing transfection associated toxicity; improving transposition from non-viral transposon vectors; improving packaging titers from viral vectors; improving expression of viral and non-viral vector encoded transgenes, etc.
[00461] As an example, R6K origin third generation lentiviral vectors [4 vectors: Table 53 transfer plasmid, gag pol packaging plasmid; env plasmid; REV plasmid (not shown) with R6K origin and < 1 kb bacterial backbone] of the invention showed reduced toxicity and improved viral packaging titers compared to pUC origin vector comparators with >1.5 kb bacterial backbone. Transfection of Lenti-X 293 T cell line (Takara Bio Mountain View, CA) with Table 3 R6K origin third generation lentiviral vectors with <1 kb bacterial backbone or original pUC origin-antibiotic selection marker vector with >1.5 kb bacterial backbone control in 24 well plates using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) as recommended by the manufacturer resulted in higher titer lentivirus production (>1.5 kb bacterial backbone pUC origin control vectors: l.OOx ± 0.32; < 1 kb bacterial backbone R6K origin vectors 1.45x ± 0.42) as measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio Mountain View, CA). Transfection of Lenti-X 293 T cell line in 24 well plates with Table 3 third generation lentiviral vectors (>1.5 kb bacterial backbone pUC origin control or < 1 kb bacterial backbone R6K origin) using Calcium Phosphate transfection as described (Marino MP, Luce MJ, Reiser J. 2003, Methods Mol Biol 229:43-55) resulted in higher titer lentivirus production (>1.5 kb bacterial backbone pUC origin control: l.OOx ± 0.30; < 1 kb bacterial backbone R6K origin vectors: 1.32x ± 0.19) as measured using the Lenti-X p24 Rapid Titer Kit (Takara Bio Mountain View, CA). Significantly, Calcium Phosphate transfection of the >1.5 kb bacterial backbone pUC origin third generation Lentiviral vectors resulted in extensive transfection associated toxicity (>80% cell death) compared to low toxicity with the R6K origin < 1 kb bacterial backbone R6K origin third generation Lentiviral vectors in this 24 well plate transfection. This reduced transfection associated toxicity should result in dramatically improved viral titers in larger manufacturing scale transfections. These results demonstrate the < 1 kb bacterial backbone R6K origin Nanoplasmid vectors of the invention reduce transfection associated toxicity and improve packaging titers from viral vectors compared to >1.5 kb bacterial backbone vectors.
Example 5: R6K origin PAS structured AAV vector construction and manufacturing
[00462] Whether R6K origin mediated replication of a closely positioned structured DNA sequence could be improved by insertion of sequences between the R6K origin and the structured DNA sequence in the direction of replication was tested. For this evaluation, an AAV ITR vector, with ITR structured DNA sequences flanking the bacterial region, was utilized.
[00463] The standard 6 iteron R6K gamma origin (SEQ ID NO: 1) in an AAV ITR vector was replaced with the extended R6K gamma origin (7 iteron) (SEQ ID NO: 4; Fig. 1A). This region contains Gyrase binding sites (see Fig. 1A) that may improve R6K origin activity. The 7 iteron R6K gamma origin without the extension (SEQ ID NO: 18) was also constructed as a control (Fig. 5A).
[00464] While the addition of a PAS-BH primosomal assembly site as described by Williams, Supra, 2017 did not overcome the poor pUC origin directed replication of closely positioned structured DNA sequences (see Example 3) the effect of addition of primosomal assembly sites was tested here in the context of the R6K origin as follows.
[00465] Two 0X174 type Primosomal assembly site sequences, PAS-BL (SEQ ID NO: 31) and PAS-BH (SEQ ID NO: 30) from plasmid pBR322 (SEQ ID NO: 29) was inserted between the 7 iteron R6K gamma origin without the extension (SEQ ID NO: 18) and the ITR structured DNA sequence with each of the PAS on different strands of the vector (Fig. 5B).
[00466] Finally, the ABC type PAS R6K plasmid CpG free ssiA primosomal assembly site (SEQ ID NO: 33) was inserted between the 7 iteron R6K gamma origin without the extension (SEQ ID NO: 18) and the ITR structured DNA sequence (Fig. 5D).
[00467] The vectors above were tested for shake flask production yield and quality as described in Example 2. The results are shown in Table 8. The vector with two 0X174 type PAS sequences, PAS-BL and PAS-BH (SEQ ID NO: 29) inserted between the R6K origin and the ITR structured DNA sequence had improved yield compared to the standard 6 iteron R6K gamma origin (SEQ ID NO: 1) and the other tested vectors.
[00468] An additional vector with 0X174 type PAS sequences, PAS-BL (SEQ ID NO: 31) and PAS-BH (SEQ ID NO: 30) (SEQ ID NO: 29) inserted between the 7 iteron R6K gamma origin without the extension (SEQ ID NO: 18) and the ITR structured DNA sequence was constructed, with different orientations of the bacterial region elements (ITR R6K> PAS R-OUT> ITR; Fig. 5C) compared to the original PAS vector (ITR PAS <R6K R-OUT> ITR; Fig. 5B). This new vector along with the other vectors above were tested for shake flask production yield and quality as described in Example 2. The results from independent production runs are shown in Table 9. Both vectors with two 0X174 type PAS sequences, PAS-BL and PAS-BH (SEQ ID NO: 29) inserted between the R6K origin and the ITR structured DNA sequence had improved yield compared to the standard 6 iteron R6K gamma origin (SEQ ID NO: 1) and the other tested vectors. [00469] The two PAS-BL and PAS-BH 7 iteron R6K origin AAV ITR vectors were evaluated versus the standard 6 iteron R6K gamma origin AAV ITR vector in HyperGRO fermentation as described in Example 2. The results are shown in Table 10 and demonstrate both vectors have dramatically improved AAV ITR vector fermentation yields compared to the standard 6 iteron R6K gamma origin AAV ITR vector. This demonstrates inclusion of the PAS region between the R6K origin and a structured DNA repeat in configurations SEQ ID NO: 27, and SEQ ID NO: 28 improves shake flask and fermentation production of vectors containing structured DNA sequences flanking the bacterial region. This region introduces 0X174 type primosomal assembly sites on the heavy (leading) strand (PAS-BH) and on the light (lagging) strand (PAS-BL) either of which may surprisingly improve replication mediated by the R6K origin. Interestingly, inclusion of an ABC type primosomal assembly site between the R6K origin and a structured DNA repeat did not improve production.
Example 6: R6K origin PAS structured mRNA vector construction and manufacturing
[00470] The antibiotic free R6K origin RNA-OUT vectors for mRNA production disclosed in Example 3 (Fig. 4C; Fig. 4 F) are configured to replicate away from the polyA homopolymeric run structured DNA repeat, as a polyA repeat <RNA-OUT R6K origin> orientation. This configuration has the RNA-OUT marker transcribing towards the polyA repeat. Surprisingly, with some mRNA transgene inserts, double stranded RNA that is not fully digested by RNaseA or removed in standard plasmid DNA column purification processing is formed (mRNA vector- NP Table 11; Fig. 7A). The basis for the problematic double stranded RNA formation is unknown but may be due to forward strand transcription from a cryptic mRNA transgene insert, annealing with reverse strand transcriptional readthrough of the mRNA terminator sequence in the RNA- OUT gene.
[00471] It was hypothesized that the production of problematic double stranded RNA could be eliminated by reversing the orientation of the RNA-OUT transcription unit, such that it did not transcribe towards the polyA repeat. This was tested by replacing the polyA repeat <RNA-OUT R6K origin> backbone in mRNA vector-NP with (constructs 1-3 below, constructs 4-8 provide alternate configurations):
1) polyA repeat RNA-OUT> R6K origin> (mRNA vector-ROUT OPP-NP)
2) polyA repeat R6K origin> RNA-OUT> (mRNA vector-ROUT OPP-NP2)
3) polyA repeat R6K origin (7 iteron)>PAS RNA-OUT> (mRNA vector-NP 7 iteron PAS R6K> ROUT>) Fig. 6B = SEQ ID NO: 28 bacterial backbone configuration RNA-OUT> R6K origin> PAS
4) polyA repeat R6K origin >PAS RNA-OUT>
5) polyA repeat RNA-OUT> R6K origin (7 iteron)>
6) polyA repeat R6K origin (7 iteron)> RNA-OUT>
7) polyA repeat RNA-OUT> R6K origin (7 iteron)>PAS
8) polyA repeat RNA-OUT> R6K origin>PAS
[00472] All 3 configurations (#1-3) combined high yield production with elimination of double stranded RNA (Table 11; Fig. 7B). This demonstrated that optimal Nanoplasmid backbone orientations for mRNA vectors combine orientation of both R6K origin replication and RNA- OUT transcription away from the polyA structured DNA repeat. Exemplary compositions for Nanoplasmid backbone mRNA vectors are: polyA repeat R6K origin> RNA-OUT>; polyA repeat R6K origin>PAS RNA-OUT> (Fig. 6B) = SEQ ID NO: 28 R6K origin>PAS RNA- OUT> bacterial backbone configuration polyA repeat RNA-OUT> R6K origin>; polyA repeat RNA-OUT> R6K origin> PAS;
[00473] The configurations above can be with either 6 or 7 iteron R6K origins.
[00474] While the above description contains many examples, these should not be construed as limitations on the scope of the disclosure, but rather should be viewed as an exemplification of preferred embodiments thereof. Many other variations are possible.
[00475] For example, in the vectors of the current technology various orientations of the Pol III dependent replication origin, and the RNA selectable marker, may be utilized. For example, any of the eight orientations of the Pol III dependent replication origin, and the RNA selectable marker in vectors of the current technology may be used. For example, in an embodiment, the oritenation is <— Pol III replication origin RSM— >, For example, in an embodiment, the oritenation is <— Pol III replication origin <— RSM. For example, in an embodiment, the oritenation is Pol III replication origin — > RSM — >. For example, in an embodiment, the oritenation is Pol III replication origin <— RSM. For example, in an embodiment, the oritenation is <— RSM Pol III replication origin — > . For example, in an embodiment, the oritenation is <— RSM <— Pol III replication origin. For example, in an embodiment, the oritenation is RSM Pol III replication origin For example, in an embodiment, the oritenation is RSM <— Pol III replication origin. [00476] Further, a variety of RNA selectable markers known in the art may be substituted for RNA-OUT.
[00477] Further, an antibiotic resistance maker may be substituted for RNA-OUT, for example in the case where a simple retrofit of the pUC origin to the R6K origin is desired to improve plasmid production yield and or quality.
[00478] Thus, the reader will see that the improved Pol III dependent replication origin vectors of the current technology provide for an approach to reduce transfection associated toxicity, improve transposition from non-viral transposon vectors, improve packaging titers from viral vectors, improve expression of viral and non-viral vector encoded genes, and eliminate viral vector and non-viral vector mediated antibiotic selection marker gene transfer (i.e. through incorporation of a bacterial region preferably less than 1000 bp) while dramatically improving manufacture compared to alterative vectors such as pUC plasmids and minicircles.
[00479] Accordingly, the scope of the disclosure should be determined not by the embodiments illustrated, but by the appended claims.

Claims

CLAIMS What is claimed is:
1. A covalently closed circular recombinant DNA molecule comprising a backbone and an insert, wherein the backbone comprises a Pol Ill-dependent origin of replication, a selectable marker, and a first primosomal assembly site, wherein the first primosomal assembly site is positioned downstream of the Pol Ill-dependent origin of replication in the direction of replication, and wherein the insert comprises a structured DNA sequence.
2. The covalently closed circular recombinant DNA molecule of claim 1 , wherein the structured DNA sequence is within 1000 bp of the Pol Ill-dependent origin of replication,
3. The covalently closed circular recombinant DNA molecule of any one of claims 1-2, wherein the backbone is less than 1000 bp.
4. The covalently closed circular recombinant DNA molecule of any one of claims 1-3, wherein the backbone comprises a bacterial replication-selection region.
5. The covalently closed circular recombinant DNA molecule of any one of claims 1-4, wherein the Pol Ill-dependent origin of replication does not require Pol I.
6. The covalently closed circular recombinant DNA molecule of any one of claims 1-5, wherein the Pol Ill-dependent origin of replication is a Pol Ill-dependent R6K origin of replication.
7. The covalently closed circular recombinant DNA molecule of claim 6, wherein the Pol Ill-dependent R6K origin of replication has at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24.
8. The covalently closed circular recombinant DNA molecule of any one of claims 1-7, wherein the selectable marker is an RNA selectable marker.
9. The covalently closed circular recombinant DNA molecule of claim 8, wherein the RNA selectable marker is an RNA-OUT RNA selectable marker.
10. The covalently closed circular recombinant DNA molecule of claim 9, wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 80% sequence identity to a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7.
11. The covalently closed circular recombinant DNA molecule of claim 9, wherein the RNA selectable marker comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6.
12. The covalently closed circular recombinant DNA molecule of any one of claims 1-11, wherein the first primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
13. The covalently closed circular recombinant DNA molecule of any one of claims 1-12, further comprising a second primosomal assembly site downstream of the Pol Ill-dependent origin of replication in the direction of replication.
14. The recombinant DNA molecule of claim 13, wherein the second primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
15. The covalently closed circular recombinant DNA molecule of claim any one of claims 13-14, comprising a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the Pol Ill-dependent origin of replication in the direction of replication.
16. The covalently closed circular recombinant DNA molecule of any one of claims 13- 14, comprising the sequence of SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the Pol Ill-dependent origin of replication in the direction of replication.
17. The covalently closed circular recombinant DNA molecule of any one of claims 1-16, wherein the covalently closed circular recombinant DNA molecule is antibiotic marker free.
18. The covalently closed circular recombinant DNA molecule of any one of claims 1-3, wherein the backbone comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
19. The covalently closed circular recombinant DNA molecule of any one of claims 1-3, wherein the backbone comprises a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
20. The covalently closed circular recombinant DNA molecule of any one of claims 1-19, wherein the structured DNA sequence is selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence.
21. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is a transposon vector.
22. The covalently closed circular recombinant DNA molecule of claim 21 , wherein the structured DNA sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
23. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is a transposase vector.
24. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is a mRNA vector.
25. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is an AAV vector.
26. The covalently closed circular recombinant DNA molecule of claim 25, wherein the structured DNA sequence is an inverted repeat sequence.
27. The covalently closed circular recombinant DNA molecule of any one of claims 25- 26, wherein the AAV vector encodes AAV2 ITRs.
28. The covalently closed circular recombinant DNA molecule of any one of claims 25- 26, wherein the insert comprises a 5’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 35 and a 3’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 36.
29. The covalently closed circular recombinant DNA molecule of claim 20, wherein the insert is a lentiviral vector.
30. The covalently closed circular recombinant DNA molecule of claim 29, wherein the structured DNA sequence is a direct repeat sequence or an eukaryotic origin of replication.
31. The covalently closed circular recombinant DNA molecule of claim 20, wherein the structured DNA sequence is selected from a homopolymeric repeat such as a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
32. The covalently closed circular recombinant DNA molecule of any one of claims 1-19, wherein the structured DNA sequence comprises a homopolymeric repeat.
33. The covalently closed circular recombinant DNA molecule of claim 32, wherein the homopolymeric repeat is a polyA repeat.
34. The covalently closed circular recombinant DNA molecule of any one of claims 32- 33, wherein the homopolymeric repeat comprises from about 3 to about 500 residues.
35. The covalently closed circular recombinant DNA molecule of any one of claims 1-34, wherein the selectable marker is a RNA selectable marker and is oriented to transcribe in a direction divergent from the structured DNA sequence.
36. An antibiotic marker free covalently closed circular recombinant DNA molecule comprising a backbone and an insert, wherein the backbone comprises an origin of replication and an RNA selectable marker, wherein the insert comprises a structured DNA sequence, and wherein the RNA selectable marker is oriented to transcribe in a direction divergent from the structured DNA sequence.
37. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 36, wherein the structured DNA sequence is within 1000 bp of the origin of replication.
38. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 36-37, wherein the backbone is less than 1000 bp.
39. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 36-38, wherein the backbone comprises a bacterial replication-selection region.
40. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 36-39, wherein the origin of replication is a Pol I-dependent origin of replication.
41. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 36-39, wherein the origin of replication is a Pol Ill-dependent origin of replication.
42. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 41, wherein the Pol Ill-dependent origin of replication does not require Pol I.
43. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 41-42, wherein the Pol Ill-dependent origin of replication is a Pol Ill- dependent R6K origin of replication.
44. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 43, wherein the Pol Ill-dependent R6K origin of replication has at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18, and SEQ ID NO: 24.
45. The covalently closed circular recombinant DNA molecule of any one of claims 36-
45. wherein the RNA selectable marker is an RNA-OUT RNA selectable marker.
46. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 45, wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA- OUT functional variant with at least 80% sequence identity to a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 7.
47. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 45, wherein the RNA selectable marker comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6.
48. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 36, wherein the backbone comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
49. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim
36, wherein the backbone comprises a sequence selected from SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
50. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 36-49, wherein the structured DNA sequence is selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication, and eukaryotic promoter enhancer sequence.
51. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is a transposon vector,
52. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 51 , wherein the structured DNA sequence is an inverted repeat sequence, a direct repeat sequence, or an eukaryotic promotor enhancer sequence.
53. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is a transposase vector.
54. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is a mRNA vector.
55. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is an AAV vector.
56. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 55, wherein the structured DNA sequence is an inverted repeat sequence.
57. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 55-56, wherein the AAV vector encodes AAV2 ITRs.
58. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 55-56, wherein the insert comprises a 5’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 35 and a 3’ inverted terminal repeat sequence having a sequence with at least 80% sequence identity to SEQ ID NO: 36.
59. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 50, wherein the insert is a lentiviral vector.
60. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 59, wherein the structured DNA sequence is a direct repeat sequence or an eukaryotic origin of replication.
61. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 50, wherein the structured DNA sequence is selected from a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer.
62. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 36-49, wherein the structured DNA sequence comprises a homopolymeric repeat.
63. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 62, wherein the homopolymeric repeat is a polyA repeat.
64. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 62-63, wherein the homopolymeric repeat comprises from about 3 to about 500 residues.
65. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 36-64, further comprising a first primosomal assembly site downstream of the origin of replication in the direction of replication.
66. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 65, further comprising a second primosomal assembly site downstream of the origin of replication in the direction of replication.
67. The antibiotic marker free covalently closed circular recombinant DNA molecule of any one of claims 65-66, wherein the first primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
68. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 66, wherein the first primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, and wherein the second primosomal assembly site comprises a sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33.
69. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 65, comprising a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site is downstream of the origin of replication in the direction of replication.
70. The antibiotic marker free covalently closed circular recombinant DNA molecule of claim 66, comprising a sequence with at least 80% sequence identity to SEQ ID NO: 29 such that the first primosomal assembly site and the second primosomal assembly site are downstream of the origin of replication in the direction of replication.
71. A method for replicating a covalently closed circular recombinant DNA molecule comprising the following steps: a. providing a cell containing the recombinant DNA molecule of any one of claims 1-70; and b. subjecting the cell to a fermentation process.
72. The method of claim 71, wherein the cell is an engineered Rep protein-expressing E. coli strain.
73. The method of any one of claims 71-72, wherein the cell comprises a chromosomally- integrated arabinose inducible CI857ts gene.
74. The method of any one of claims 71-73, wherein Rep protein includes the following mutations: P42L; P 1061; F107S; and Pl 13S.
75. The method of any one of claims 71-74, wherein the fermentation process comprises growing the cells in media containing arabinose.
76. The method of any one of claims 71-75, wherein the yield of the covalently closed circular plasmid following the fermentation process is in excess of 0.5 g/L.
PCT/US2025/012704 2024-01-25 2025-01-23 Viral and non-viral nanoplasmid vectors with improved production Pending WO2025160245A1 (en)

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