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.

Gene Therapy with the Sleeping Beauty Transposon System

“…In principle, these hybrid vectors could be used as alternatives to established viral vectors, and are suitable for cell type-specific gene engineering. Viral transposon hybrids have been established for integrase deficient lentivirus (IDLV) [70][71][72], adenovirus [73,74], AAV [75], herpes simplex virus [76,77], and baculovirus [78,79], where the SB transposon provides stable gene integration. Especially for in vivo approaches, hybrid vectors could be advantageous, because they: (i) bypass the need for repeated vector administration due to stable chromosomal transgene integration and expression; and (ii) may allow reduction of the applied viral dose, thereby alleviating vectorassociated immune complications.…”

Sleeping Beauty Transposition

“…The random genomic distribution of de novo SB insertions can be observed when the transposon DNA is introduced into the nucleus by extrachromosomal gene delivery, including plasmid vectors (114,115,116,117,119,122,125), integration-deficient lentiviral vectors (IDLVs) (114), adenovirus vectors (129), herpesvirus vectors (130), and adeno-associated vectors (131). In these cases, transposition takes place from the extrachromosomal vector into the genome.…”

Sleeping Beautytransposition: from biology to applications

“…For instance, the mutagenic potential of MLVbased vectors have been reported in multiple clinical gene therapy trials: SCID-X1 (Hacein-Bey-Abina et al, 2003), (Deichmann et al, 2007;Hacein-Bey-Abina et al, 2008;Howe et al, 2008;Thrasher et al, 2006), X-CGD (Stein et al, 2010) and WAS (Braun et al, 2014). Furthermore, recent analyses also demonstrate that HIV (Yant et al, 2002) AAV/SB Recombinant AAV (Zhang et al, 2013) HSV-1 amplicon/SB HSV-1 (Bowers et al, 2006;de Silva et al, 2010a;de Silva et al, 2010b;Peterson et al, 2007) Baculo/SB Baculovirus (Luo et al, 2012;Turunen et al, 2014) IDLV/SB IDLV (Moldt et al, 2011;Staunstrup et al, 2009;Vink et al, 2009;) Adeno: adenovirus; AAV: adeno associated virus; IDLV: integrase defective lentivirus; HSV-1: herpes simplex virus 1 amplicon; baculo: baculovirus Note: The transposase (highlighted in green) and the transposon (highlighted in red) plasmids can be packaged into various recombinant viruses. A colored version is available online (www.informahealthcare.com/bmg).…”

“…Due to the stable integration, the hybrid AAV/transposon (SB100X) provides sustained gene expression (Zhang et al, 2013), that generally declines in fast dividing cells, as the simple AAV vector remains episomal (Table 2). While AAV vectors have limited cargo capacity, several serotypes are available.…”