Zinc Finger Recombinases for Endogenous Genome Tailoring

描述（由申请人提供）：本提案旨在开发一种适用于癌症研究和治疗的基因组重组策略，是RA-CA-126664响应RFA-CA-07-002的修订后重新提交。目前，还没有内源性人类基因组的靶向和位点特异性重组的方法。这种“基因组手术”将使癌症生物学的基因解剖成为可能，并使基因治疗的新方法成为可能。这项工作的基本原理是，RecZF具有在许多序列背景下有效切除、反转或整合大片段DNA的能力，是引入和修复与癌症相关的遗传缺陷的独特而强大的工具。这种策略与目前的基因疗法形成对比，目前的基因疗法仅提供瞬时基因表达或风险外源DNA的非特异性基因组整合。这项研究的重点是TP 53基因座，这是一个重要的肿瘤抑制基因，在许多不同类型的人类癌症中功能失活。这项研究的总体目标是开发工具，以TP 53基因座为模型靶点，有效和选择性地整合到任何基因组位点。核心假设是，由新型锌指蛋白和进化的催化结构域构建的RecZF将能够执行这一任务。作为基因操作的有力工具，我们预计RecZF可以促进新型癌症动物模型的建立，基因治疗的应用以及癌症遗传学的阐明。

DESCRIPTION (provided by applicant): This proposal seeks to develop a strategy of genomic recombination suitable for applications in both cancer research and therapy, and is a revised resubmission of RA-CA-126664 responsive to RFA-CA-07-002. At present, there is no method for targeted and site-specific recombination of the endogenous human genome. Such `genome surgery' would enable the genetic dissection of cancer biology, as well as, empower a new approach to gene therapy. The rationale for the proposed work is that RecZFs, with their capacity for efficiently excising, inverting, or integrating large segments of DNA in many sequence contexts, are a unique and powerful tool for both introducing and repairing genetic defects related to cancer. This strategy contrasts with current gene therapies, which provide only transient gene expression or risk non-specific genomic integration of exogenous DNA. The proposed research focuses on the TP53 locus, a crucial tumor-suppressor, which is functionally inactivated in many different kinds of human cancer. The overall objective of this research proposal is to develop tools for efficient and selective integration into any genomic site, using the TP53 locus as a model target. The central hypothesis is that RecZFs, constructed from novel zinc finger proteins and evolved catalytic domains, will be able to perform this task. As powerful tools for genetic manipulation, we anticipate that RecZFs could facilitate the creation of novel animal models of cancer, the application of gene therapy, and the elucidation of cancer genetics.