Mechanisms of Cellular Transduction with AAV Vectors

AAV 载体的细胞转导机制

• 批准号: 6607710
• 负责人: DANIEL G MILLER
• 金额: $ 12.53万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6607710
• 关键词: DNA damage; DNA repair; adeno associated virus group; cell line; endonuclease; genetic transduction; molecular cloning; transfection /expression vector; virus integration

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.

描述（由申请人提供）：我建议通过研究Adeno相关病毒（AAV）载体转导，将我在医学遗传学和基因治疗中的职业兴趣结合起来。华盛顿大学是研究遗传学和基因疗法的独特场所，也许是对这种综合兴趣的研究人员的机会。目前，AAV载体正在9种人体基因治疗试验中使用。细胞进入后载体基因组的命运仍然存在几个问题，包括对宿主细胞染色体和整合机制的影响。我们已经表明，AAV矢量可以通过通过与染色体序列的载体同源性区分的单独途径进行集成。我们分析了非同源矢量整合连接连接，发现序列改变与其他研究的发现相似，研究了哺乳动物双链断修复（DSBR）的非同源末端连接途径（DSBR），这意味着这些断裂可能是载体整合的底物。观察到产生双链断裂的遗传毒性会增加AAV载体转导频率，这也支持了这一假设。我们将使用I-SCEL核酸内切酶的条件诱导在人体细胞中产生断裂，评估双链断裂在AAV载体转导中的作用，并在存在和不存在染色体断裂的情况下评估转导频率。我们将通过在细菌中克隆含有I-SCEL识别位点的逆转录病毒穿梭媒介来确定AAV矢量积分是否发生在断裂位点，并评估断裂位点的序列。最后，我们计划使用AAV载体的同源整合并定义参与同源和非同源AAV载体矢量整合的同源整合，以破坏参与同源重组和DSBR（RAD52，RAD54和KU70）的基因。有关AAV矢量转导的机制和效果的更多信息将揭示正在进行的临床试验的安全问题，并允许改进这些向量以继续用作基因输送车。