DNA Double-Strand Break Repair Regulates rAAV-Mediated Gene Targeting

描述(申请人提供)：我们建议研究人类DNA DSB(双链断裂)修复基因功能丧失突变对rAAV(重组腺相关病毒)载体整合的影响。RAAV载体有两个强大的用途，它们在基因治疗研究中得到了广泛的应用。首先，它们可以用来将转基因输送到细胞。然而，rAAV的这一方面受到与其他载体相关的相同插入突变问题的困扰。然而，rAAV的第二个应用是使用HR(同源重组)将载体基因组与其同源同源染色体序列重组(又名基因打靶)，这对于需要工具来修改基因组的基础研究人员来说是非常理想的结果，对基因治疗师也是非常有用的。然而，由于非同源末端连接-DNA DSB修复的主要途径-在人类细胞中占主导地位，随机的rAAV整合事件通常比正确的基因靶向事件发生得更频繁。因此，NHEJ通路的治疗性失活应加强rAAV介导的基因打靶，而HR通路的相互失活应阻碍rAAV基因打靶。在最近发表的研究和未发表的数据中，我们已经证明，减少某些NHEJ因子确实会增加rAAV介导的基因靶向的频率。在本申请中，我们建议将这项工作扩展到其他NHEJ因子和HR调节的整合。此外，我们提出了模型--以及为测试它们而设计的实验--应该能阐明rAAV用于基因靶向的机制。

DESCRIPTION (provided by applicant): We propose to study the impact of loss-of-function mutations of human DNA DSB (double-strand break) repair genes on rAAV (recombinant adeno-associated virus) vector integration. rAAV vectors have two powerful applications and they are in wide use in gene therapy studies. First, they can be used to deliver transgenes to cells. This aspect of rAAV, however, is plagued by the same insertional mutagenesis problems associated with other vectors. A second application for rAAV, however, is the recombination of the vector genome with its cognate homologous chromosomal sequences (aka, gene targeting) using HR (homologous recombination), which is a highly desirable outcome for basic researchers in need of tools to modify the genome and of great utility to gene therapists. However, because NHEJ (non-homologous end joining - the major pathway for DNA DSB repair - predominates in human cells over HR, random rAAV integration events generally occur much more frequently than correct gene targeting events. Therefore, therapeutic inactivation of the NHEJ pathway should augment rAAV-mediated gene targeting and - reciprocally - inactivation of the HR pathway should hinder rAAV gene targeting. In recently published work and in unpublished data we have demonstrated that reducing certain NHEJ factors does indeed increase the frequency of rAAV-mediated gene targeting. In this application we propose to extend this work to other NHEJ factors and to HR-regulated integrations. Moreover, we propose models - and experiments designed to test them - that should illuminate the mechanism that rAAV uses for gene targeting.