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Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent...

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Detalles Bibliográficos
Autores principales: Cervelli, Tiziana, Backovic, Ana, Galli, Alvaro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154452/
https://www.ncbi.nlm.nih.gov/pubmed/21853137
http://dx.doi.org/10.1371/journal.pone.0023474
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author Cervelli, Tiziana
Backovic, Ana
Galli, Alvaro
author_facet Cervelli, Tiziana
Backovic, Ana
Galli, Alvaro
author_sort Cervelli, Tiziana
collection PubMed
description Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.
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spelling pubmed-31544522011-08-18 Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae Cervelli, Tiziana Backovic, Ana Galli, Alvaro PLoS One Research Article Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. Public Library of Science 2011-08-10 /pmc/articles/PMC3154452/ /pubmed/21853137 http://dx.doi.org/10.1371/journal.pone.0023474 Text en Cervelli et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Cervelli, Tiziana
Backovic, Ana
Galli, Alvaro
Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae
title Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae
title_full Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae
title_fullStr Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae
title_full_unstemmed Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae
title_short Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae
title_sort formation of aav single stranded dna genome from a circular plasmid in saccharomyces cerevisiae
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154452/
https://www.ncbi.nlm.nih.gov/pubmed/21853137
http://dx.doi.org/10.1371/journal.pone.0023474
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