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The transient expression of mRNA coding for Rep protein from AAV facilitates targeted plasmid integration
Author(s) -
Howden S. E.,
Voullaire L.,
Vadolas J.
Publication year - 2008
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1118
Subject(s) - plasmid , microbiology and biotechnology , biology , insertional mutagenesis , transfection , adeno associated virus , dna , gene , messenger rna , genome , genetics , recombinant dna , vector (molecular biology)
Background There is a risk of insertional mutagenesis when techniques that facilitate random integration of exogenous DNA into the human genome are used for gene therapy. Wild‐type adeno‐associated virus (AAV) integrates preferentially into a specific site on human chromosome 19 (AAVS1). This is mediated by the interaction of the viral Rep68/78 proteins with Rep‐binding elements in the AAV genome and AAVS1. This specificity is often lost when AAV is used as a gene therapy vector due to removal of the sequences coding for Rep. Methods Messenger RNA coding for the Rep68/78 proteins was prepared in vitro and co‐transfected with a 21 kb DNA plasmid containing the P5 integration efficiency element (P5IEE) from AAV. Single cells were seeded in plates to establish clonal cell lines that were subsequently analysed by dual colour fluorescent in situ hybridisation (FISH) to determine whether site‐specific plasmid integration had occurred on chromosome 19. Results The co‐transfection of plasmid DNA with Rep68/78 mRNA gave a 2.5‐fold increase in DNA integration when compared to transfection of cells with plasmid DNA alone. Rep68/78 mRNA expression facilitated site‐specific plasmid integration to chromosome 19 in 30% (14/44) of all analysed integration sites, while no targeted integration events were observed following transfection of cells with plasmid DNA alone. Conclusions These results demonstrate that transient expression of Rep protein using transfected mRNA facilitates site‐specific integration of plasmid DNA. This approach allows expression of Rep for only a short time, and may circumvent the toxicity and chromosome instability associated with long‐term expression of Rep. Copyright © 2007 John Wiley & Sons, Ltd.