Mechanisms of Cellular Transduction with AAV Vectors

描述（由申请人提供）：我建议将我在医学遗传学和基因治疗方面的职业兴趣结合起来，研究腺相关病毒（AAV）载体转导。华盛顿大学是研究遗传学和基因治疗的一个独特的地方，也许它为研究人员提供了无与伦比的机会。AAV载体目前正在9项人类基因治疗试验中使用。关于载体基因组进入细胞后的命运仍有几个问题，包括对宿主细胞染色体的影响和整合机制。我们已经证明，AAV载体可以通过与染色体序列存在或不存在同源性的不同途径整合。我们分析了非同源的载体整合连接，发现序列变化与其他研究哺乳动物双链断裂修复（DSBR）的非同源末端连接途径的结果相似，这意味着这些断裂可能是载体整合的底物。产生双链断裂的基因毒性物质增加AAV载体转导频率的观察结果也支持这一假设。我们将通过有条件诱导I-Scel核酸内切酶在人细胞中产生断裂来评估AAV载体转导中双链断裂的作用，并评估存在和不存在染色体断裂的转导频率。我们将通过在细菌中克隆修复含有I-Scel识别位点的逆转录病毒穿梭载体，确定AAV载体是否发生在断裂位点整合，并评估断裂位点的序列。最后，我们计划利用AAV载体的同源整合来破坏参与同源重组和DSBR的基因（Rad52、Rad54和Ku70），并定义参与同源和非同源AAV载体整合的细胞通路。关于AAV载体转导的机制和作用的更多信息将揭示正在进行的临床试验的安全性问题，并允许改进这些载体以继续用作基因传递载体。

DESCRIPTION (provided by applicant): I propose to combine my career interests in medical genetics and gene therapy by studying Adeno Associated Virus (AAV) vector transduction. The University of Washington is a unique place to study both genetics and gene therapy and perhaps unparalleled in its opportunities for researchers with this combined interest. AAV vectors are currently being used in 9 human gene therapy trials. Several questions remain about the fate of vector genomes after cellular entry, including the effect on the host cell chromosome and mechanisms of integration. We have shown that AAV vectors can integrate by separate pathways distinguished by the presence or absence of vector homology to chromosomal sequences. We analyzed non-homologous vector integration junctions and found sequence alterations similar to the findings of others investigating the non-homologous end joining pathway of mammalian double strand break repair (DSBR) implying that these breaks may be a substrate for vector integration. The observation that genotoxic agents that generate double strand breaks increase AAV vector transduction frequencies also supports this hypothesis. We will evaluate the role of double strand breaks in AAV vector transduction by generating breaks in human cells using conditional induction of the I-Scel endonuclease, and evaluate transduction frequencies in the presence and absence of chromosomal breaks. We will determine if AAV vector integration occurs at breakage sites by rescuing retrovirus shuttle vectors containing I-Scel recognition sites through cloning in bacteria, and evaluate the sequence at the breakage site. Finally, we plan to disrupt genes involved in homologous recombination and DSBR (Rad52, Rad54, and Ku70) using homologous integration of AAV vectors and define cellular pathways involved in homologous and non-homologous AAV vector integration. More information about the mechanism and effect of AAV vector transduction will reveal safety issues for ongoing clinical trials, as well as allow improvement of these vectors for continued use as gene delivery vehicles.