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.
Highlights
Gene therapy is a rapidly developing field relying on introduction of nucleic acids into mammalian cells to regulate, repair, replace, add or delete a genetic sequence
We show for the first time that associated virus (AAV) vectors can serve as template for Sleeping Beauty (SB) transposase mediated somatic integration with a close to random integration profile
For achieving stabilized transgene expression the goal of this study was to mobilize a transposon from episomal AAV vector genomes for SB transposasemediated stable integration of a transgene expression cassette into the mammalian host genome (Figure 1)
Summary
Gene therapy is a rapidly developing field relying on introduction of nucleic acids into mammalian cells to regulate, repair, replace, add or delete a genetic sequence. Monogenetic diseases like hemophilia B, Duchenne muscular dystrophy and cystic fibrosis are the three most frequent indications for clinical trials in gene therapy [1]. For life-long correction of genetic diseases, therapeutic DNA needs to be efficiently delivered to the respective target tissue and cells and transgene expression needs to be maintained at a therapeutic level. Recombinant AAV vectors lack both ORFs and combine several advantages, including efficient infectivity, stable transgene expression in quiescent cells and nonpathogenicity [3]. AAV vectors have been extensively investigated in preclinical and clinical settings [4] and they were involved in several clinical trials to treat metabolic abnormalities, hemophilia disease, Parkinson’s disease, muscular dystrophy and cystic fibrosis [2,4,5]
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