Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Gene Ther. 2010 Mar;17(3):424-31. doi: 10.1038/gt.2009.144. Epub 2009 Oct 29.
The ability of a viral vector to safely deliver and stably integrate large transgene units (transgenons), which not only include one or several therapeutic genes, but also requisite native transcriptional regulatory elements, would be of significant benefit for diseases presently refractory to available technologies. The herpes simplex virus type-1 (HSV-1) amplicon vector has the largest known payload capacity of approximately 130 kb, but its episomal maintenance within the transduced cell nucleus and induction of host cell silencing mechanisms limits the duration of the delivered therapeutic gene(s). Our laboratory developed an integration-competent version of the HSV-1 amplicon by adaptation of the Sleeping Beauty (SB) transposon system, which significantly extends transgene expression in vivo. The maximum size limit of the amplicon-vectored transposable element remains unknown, but previously published plasmid-centric studies have established that DNA segments longer than 6-kb are inefficiently transposed. Here, we compared the transposition efficiency of SB transposase in the context of both the HSV amplicon vector as well as the HSV amplicon plasmid harboring 7 and 12-kb transposable reporter transgene units. Our results indicate that the transposition efficiency of the 12-kb transposable unit via SB transposase was significantly reduced as compared with the 7-kb transposable unit when the plasmid version of the HSV amplicon was used. However, the packaged HSV amplicon vector form provided a more amenable platform from which the 12-kb transposable unit was mobilized at efficiency similar to that of the 7-kb transposable unit via the SB transposase. Overall, our results indicate that SB is competent in stably integrating transgenon units of at least 12 kb in size within the human genome upon delivery of the platform via HSV amplicons.
病毒载体将大转基因单元（转基因）安全地递送到并稳定地整合，这不仅包括一个或几个治疗基因，还包括必需的天然转录调控元件，这对于目前无法用现有技术治疗的疾病将具有重要意义。单纯疱疹病毒 1 型（HSV-1）扩增子载体具有最大的已知载物量，约为 130kb，但它在转导细胞的核内以附加体形式维持以及诱导宿主细胞沉默机制，限制了所递送的治疗基因的持续时间。我们的实验室通过适应睡眠美人（SB）转座子系统开发了一种具有整合能力的 HSV-1 扩增子，这显著延长了体内转基因的表达。整合载体的最大尺寸限制未知，但以前发表的基于质粒的研究已经证实，长于 6kb 的 DNA 片段的转座效率较低。在这里，我们比较了 SB 转座酶在 HSV 扩增子载体以及携带 7 和 12kb 可转座报告基因单元的 HSV 扩增子质粒中的转座效率。我们的结果表明，当使用 HSV 扩增子质粒版本时，SB 转座酶的 12kb 可转座单元的转座效率与 7kb 可转座单元相比显著降低。然而，包装的 HSV 扩增子载体形式提供了一个更适合的平台，通过该平台，通过 SB 转座酶，12kb 可转座单元的移动效率类似于 7kb 可转座单元。总体而言，我们的结果表明，SB 在通过 HSV 扩增子递送至平台后，能够稳定地将至少 12kb 大小的转基因单元整合到人类基因组中。
