Targeted gene correction mediated by recombinant adeno-associated virus and DNA double strand breaks

最近关于逆转录病毒基因转移临床试验中严重不良事件的新闻揭示了使用随机整合载体的内在危险。另一种方法是在染色体同源重组中校正基因突变，称为基因打靶。使用重组腺相关病毒（AAV）作为修复基质的包装载体已经导致哺乳动物基因靶向的显著改善，达到高达1%的校正频率。我们已经系统地研究了调节AAV基因靶向的变量，并表明由位点特异性核酸内切酶引入的局部DNA双链断裂（DSB）刺激了超过300倍的基因校正频率。此外，靶基因中的DSB提高了靶向率（基因校正到随机整合），从而显著降低了插入诱变的风险。该提议旨在通过研究控制AAV基因靶向离体和体内的载体和细胞参数来拓宽系统。我进一步建议设计和应用位点特异性锌指核酸酶，能够在基因组中的任何所需位点产生DSB，其长期目标是在动物疾病模型中评估该方法。通过AAV载体的修复基质和位点特异性核酸内切酶的联合收割机递送有望实现用于科学和治疗应用的高基因靶向频率。

Recent news of serious adverse events in a clinical trial involving retroviral gene transfer revealed the inherent danger of using randomly integrating vectors. An alternative approach is the correction of the genetic mutation in the chromosome throug homologous recombination, called gene targeting. The use of recombinant adeno-associated virus (AAV) as a packing vector for a repair matrix has led to a significant improvement in mammalian gene targeting, reaching correction frequencies of up to 1%. We have systematically investigated the variables that regulate AAV gene targeting and showed that a local DNA double strand break (DSB) introduced by a site-specific endonuclease stimulated the frequency of gene correction over 300-fold. Moreover, a DSB in the target gene improved the targeting ratio (gene correction to random integration), thus significantly lowering the risk of insertional mutagenesis. This proposal aims at broadening the system by investigating the vector and cell parameters that govern AAV gene targeting ex vivo and in vivo. I further propose to design and apply site-specific zinc finger nucleases capable of creating a DSB at any desired site in the genom with the long-term goal to evaluate the approach in an animal disease model. The combine delivery of a repair matrix and a site-specific endonuclease by AAV vectors holds promise for achieving high gene targeting frequencies for both scientific and therapeutic applications.