title: Hybrid adeno-associated viral vectors utilizing transposase-mediated somatic integration for stable transgene expression in human cells.
abstract: Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.
authors.affiliation: Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University Munich, Munich, Germany ; Institute of Virology and Microbiology, Center for Biomedical Education and Research, Department of Human Medicine, Faculty of Health, University Witten/Herdecke, Witten, Germany.
authors.forename: Wenli
authors.initials: W
authors.lastname: Zhang
authors.forename: Manish
authors.initials: M
authors.lastname: Solanki
authors.forename: Nadine
authors.initials: N
authors.lastname: Müther
authors.forename: Melanie
authors.initials: M
authors.lastname: Ebel
authors.forename: Jichang
authors.initials: J
authors.lastname: Wang
authors.forename: Chuanbo
authors.initials: C
authors.lastname: Sun
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvak
authors.forename: Anja
authors.initials: A
authors.lastname: Ehrhardt
mesh_terms: D001665:Binding Sites / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics; D004274:DNA, Recombinant; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics*; D019895:Transposases / Q000235:genetics* / Q000378:metabolism; D016662:Virus Integration / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019895:Transposases