Targeted Gene Repair by Homologous Recombination (Component 8 of 11)

有效的靶向基因修复的一个主要限制是大多数情况下同源定向修复的效率较低 人类细胞类型。同源定向修复的机制现在被理解为非常复杂的 详细程度，以及西北基因组工程联盟(NGEC)这一组成部分的目标 就是利用这种机械理解来开发刺激目标基因的HDR的方法。我们 描述实现这一目标的三个具体目标：(1)我们将开发一种方便的记者检测方法 同源定向修复，并使用它来优化双链断裂的修复，在细胞培养模型和 在原代造血细胞中。(2)我们将询问NICS是否能有效地启动靶向基因修复； 并开发了一种断裂诱导的基因组不稳定性的报告试验，以确定NICS是否提供了 通过降低治疗中易位的可能性而超过双链断裂的安全优势 申请。(3)我们将确定染色质结构如何对基因标记和靶向做出贡献 基因修复，并具体询问是否可以操纵染色质结构来提高 基因修复。这些计划中的实验与NGEC的其他组成部分相辅相成并相互配合 实现建立有效的单基因疾病基因治疗方法的长期目标 造血细胞。

A major limitation to effective targeted gene repair is the low efficiency of homology-directed repair in most human cell types. The mechanism of homology-directed repair is now understood at a very sophisticated level of detail, and the goal of this component of the Northwest Genome Engineering Consortium (NGEC) is to harness this mechanistic understanding to develop methods for stimulating HDR at target genes. We describe three specific aims to achieve this goal: (1) We will develop a convenient reporter assay for homology-directed repair, and use it to optimize repair at double-strand breaks, in cell culture models and in primary hematopoietic cells. (2) We will ask if targeted gene repair can be efficiently initiated at nicks; and develop a reporter assay for break-induced genomic instability to establish whether nicks provide a safety advantage over double-strand breaks by diminishing the potential for translocation in therapeutic applications. (3) We will establish how chromatin structure contributes to gene marking and to targeted gene repair, and ask specifically if chromatin structure can be manipulated to increase the efficiency of gene repair. These planned experiments complement and mesh with other components of the NGEC to achieve the long-term goal of establishing methods for effective gene therapy of monogenic disorders of hematopoietic cells.