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在大多数情况下同源定向修复的效率低。 人类细胞类型。同源性定向修复的机制现在在一个非常复杂的领域得到了理解。 详细程度，以及西北基因组工程联盟（NGEC）的这一组成部分的目标 是利用这种机制的理解来开发刺激靶基因HDR的方法。我们 描述了实现这一目标的三个具体目标：（1）我们将开发一种方便的报告基因测定法， 同源性定向修复，并将其用于在细胞培养模型中优化双链断裂处的修复， 在初级造血细胞中。(2)我们会问是否有针对性的基因修复可以有效地启动在缺口; 并开发了一种用于断裂诱导的基因组不稳定性的报告测定法，以确定切口是否提供了一种 通过减少治疗中易位的可能性， 应用. (3)我们将建立染色质结构如何有助于基因标记和靶向 基因修复，并特别询问是否可以操纵染色质结构，以提高效率， 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， 实现建立单基因疾病的有效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.