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Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms...

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Autores principales: Li, Lina, Dimitriadis, Emilios K., Yang, Yu, Li, Juan, Yuan, Zhenhua, Qiao, Chunping, Beley, Cyriaque, Smith, Richard H., Garcia, Luis, Kotin, Robert M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731302/
https://www.ncbi.nlm.nih.gov/pubmed/23936358
http://dx.doi.org/10.1371/journal.pone.0069879
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author Li, Lina
Dimitriadis, Emilios K.
Yang, Yu
Li, Juan
Yuan, Zhenhua
Qiao, Chunping
Beley, Cyriaque
Smith, Richard H.
Garcia, Luis
Kotin, Robert M.
author_facet Li, Lina
Dimitriadis, Emilios K.
Yang, Yu
Li, Juan
Yuan, Zhenhua
Qiao, Chunping
Beley, Cyriaque
Smith, Richard H.
Garcia, Luis
Kotin, Robert M.
author_sort Li, Lina
collection PubMed
description Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×10(9) Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.
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spelling pubmed-37313022013-08-09 Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer Li, Lina Dimitriadis, Emilios K. Yang, Yu Li, Juan Yuan, Zhenhua Qiao, Chunping Beley, Cyriaque Smith, Richard H. Garcia, Luis Kotin, Robert M. PLoS One Research Article Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×10(9) Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA. Public Library of Science 2013-08-01 /pmc/articles/PMC3731302/ /pubmed/23936358 http://dx.doi.org/10.1371/journal.pone.0069879 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Li, Lina
Dimitriadis, Emilios K.
Yang, Yu
Li, Juan
Yuan, Zhenhua
Qiao, Chunping
Beley, Cyriaque
Smith, Richard H.
Garcia, Luis
Kotin, Robert M.
Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer
title Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer
title_full Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer
title_fullStr Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer
title_full_unstemmed Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer
title_short Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer
title_sort production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of dna for gene transfer
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731302/
https://www.ncbi.nlm.nih.gov/pubmed/23936358
http://dx.doi.org/10.1371/journal.pone.0069879
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