Generation of improved viral hybrid-vectors for stable transduction of mammalian cells

对于一个安全和成功的基因治疗方法的潜在风险插入诱变后，整合的病毒和非病毒载体的交付需要仔细评估。如前所述，对于主要整合到活性基因中的逆转录病毒载体，需要解决安全性问题。尽管最近开发了具有潜在较低插入诱变风险的非病毒整合载体，但基于裸DNA的整合载体要克服的一个主要挑战是将转基因递送至靶细胞并将重组DNA摄取至细胞中。为了克服这一障碍，本提案的计划是产生新的杂交载体，其可以有效地转染哺乳动物细胞并将表达盒整合到宿主基因组中。为了实现这一目标，本提案将研究[1]在病毒载体的背景下显著改进的基于DNA的转座子系统和[2]载体基因组内用于有限整合到宿主基因组中的各种改进的噬菌体衍生的DNA整合酶。本提案的最终目标[3]是评价这些改良载体在体外和体内的相对安全性和有效性，主要关注造血干细胞[4]。该项目将开发用于哺乳动物细胞稳定转导的新工具。这将是治疗遗传性疾病的重要一步，包括影响造血干细胞衍生细胞的疾病。

For a safe and successful gene therapy approach the potential risk of insertional mutagenesis after delivery of integrating viral and non-viral vectors needs to be carefully evaluated. As previously shown for retroviral vectors which predominantly integrate into active genes safety concerns need to be addressed. Although non-viral integrating vectors with a potentially lower risk of insertional mutagenesis were recently developed, one major challenge to be overcome for integrating vectors based on naked DNA is the delivery of the transgene to the target cell and uptake of the recombinant DNA into the cell. To overcome this hurdle the plan of this proposal is to generate novel hybrid-vectors that can efficiently transduce mammalian cells and integrate an expression cassette into the host genome. To accomplish this goal, this proposal will investigate [1] a significantly improved DNA based transposon system in the context of a viral vector and [2] various improved bacteriophage derived DNA integrases within the vector genome for limited integration into the host genome. The final goal [3] of this proposal is to evaluate the relative safety and efficacy of these improved vectors in vitro and in vivo with the primary focus on hematopoietic stem cells[4]. This project will develop novel tools for stable transduction of mammalian cells. It will be an important step towards treating genetic disorders including diseases affecting cells derived from hematopoietic stem cells.