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

描述（由申请人提供）：我们拟研究人类DNA双链断裂修复基因功能缺失突变对重组腺相关病毒载体整合的影响。rAAV载体具有两种强大的应用，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传递到细胞中。然而，rAAV的这一方面受到与其他载体相关的相同插入诱变问题的困扰。然而，rAAV的第二个应用是利用HR（同源重组）将载体基因组与其同源染色体序列（又名基因靶向）进行重组，这对于需要修饰基因组工具的基础研究人员和基因治疗师来说是一个非常理想的结果。然而，由于NHEJ（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rAAV整合事件通常比正确的基因靶向事件发生的频率要高得多。因此，治疗性的NHEJ通路失活会增强rAAV介导的基因靶向，而反过来，HR通路失活会阻碍rAAV基因靶向。在最近发表的工作和未发表的数据中，我们已经证明，减少某些NHEJ因子确实增加了raav介导的基因靶向的频率。在这个应用程序中，我们建议将这项工作扩展到其他NHEJ因素和hr调节的集成。此外，我们提出的模型——以及设计用来测试它们的实验——应该阐明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 In recently published work and in unpublished data we have demonstrated that reducing certain NHEJ factors does indeed increase the frequency of rAAV-mediated gene targeting.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. PUBLIC HEALTH RELEVANCE: Recombinant adeno-associated virus is one of the most promising vectors for gene therapy. We have discovered a way to enhance the utility of this virus by modifying the status of DNA repair genes within human somatic cells. The reduction in expression of certain DNA repair genes elevates the frequencies with which recombinant adeno- associated virus performs gene targeting. The enhanced ability to perform gene therapy is clearly relevant to the mission of NIH.