Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

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.重组腺相关病毒（recombinant adeno-associated viral, AAV）载体已被证实是治疗性基因递送领域最具前景的载体之一，因其可在非分裂细胞中诱导高效且长期的转导，且副作用可忽略不计。然而，由于AAV载体大多以游离型形式存在，其载体基因组与转基因表达会在分裂细胞中丢失。因此，为实现细胞的稳定转导，我们开发了一种新型AAV/转座酶杂合载体。为实现基于睡美人（Sleeping Beauty, SB）转座酶的rAAV基因组转座，我们构建了一套系统：其中一种AAV载体携带携带有目的基因的转座子，另一种载体则递送高活性睡美人（SB）转座酶SB100X。我们将人细胞感染AAV-转座子载体，并通过瞬时及稳定质粒转染或AAV载体转导的方式反式提供转座酶。实验结果显示，接受高活性转座酶SB100X的实验组集落形成数显著升高，表明整合效率得到提升。此外，我们发现转导细胞中的转基因拷贝数呈剂量依赖性，且稳定转基因的过程主要由SB转座酶介导的转座作用所贡献。基于质粒拯救策略与线性扩增介导的PCR（linear-amplification mediated PCR, LAM-PCR）实验方案，我们分析了从AAV载体转座后SB100X介导的整合图谱。共鉴定出1840个整合事件，结果显示其整合模式接近随机整合。综上，我们首次证实AAV载体可作为SB转座酶介导的体细胞整合的模板。我们开发了该杂合载体系统的首个原型载体，经进一步优化后，该系统有望用于治疗起源于快速分裂细胞的疾病。