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

DNA 双链断裂修复调节 rAAV 介导的基因靶向

• 批准号: 8434196
• 负责人: ERIC A HENDRICKSON
• 金额: $ 27.38万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8434196
• 关键词: Affect; Applications Grants; Assimilations; Cell Line; Cells; DNA Double Strand Break; DNA Repair Gene; Data; Development; Double Strand Break Repair; Event; Frequencies; Gene Targeting; Gene Transduction Agent; Genes; Genetic Recombination; Genetic Transcription; Genome; Grant; Human; Insertional Mutagenesis; Inverted Terminal Repeat; Mediating; Mission; Modeling; Molecular Genetics; Nonhomologous DNA End Joining; Outcome; Pathway interactions; Plague; Protein Binding; Publishing; Recombinant adeno-associated virus (rAAV); Research Personnel; Somatic Cell; Somatic Cell Genetics; System; Testing; Therapeutic; Transgenes; United States National Institutes of Health; Viral; Virus; Virus Integration; Work; adeno-associated viral vector; cell type; design; gene therapy; homologous recombination; human DNA; loss of function mutation; mutant; public health relevance; research study; site-specific integration; tool; vector; vector genome; viral DNA

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.

描述（由申请人提供）：我们拟研究人DNA DSB（双链断裂）修复基因的功能丧失突变对rAAV（重组腺相关病毒）载体整合的影响。rAAV载体具有两个强大的应用，并且它们在基因治疗研究中被广泛使用。首先，它们可用于将转基因传递到细胞。然而，rAAV的这一方面受到与其他载体相关的相同插入诱变问题的困扰。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（又名基因靶向）重组，这对于需要工具来修饰基因组的基础研究人员来说是非常理想的结果，并且对于基因治疗师来说是非常有用的。然而，由于NHEJ（非同源末端连接-DNA DSB修复的主要途径-在人细胞中比HR占主导地位，随机rAAV整合事件通常比正确的基因靶向事件更频繁地发生。因此，NHEJ途径的治疗性失活应增强rAAV介导的基因靶向，而HR途径的非治疗性失活应阻碍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-和实验设计来测试它们-应该阐明rAAV用于基因靶向的机制。