Zinc Finger Recombinases for Endogenous Genome Tailoring

描述(由申请人提供)：本提案寻求开发一种适用于癌症研究和治疗的基因组重组策略，是对RA-CA-126664的修改后的重新提交，以响应RFA-CA-07-002。目前，还没有针对内源性人类基因组进行靶向和定点重组的方法。这种‘基因组手术’将使癌症生物学的基因解剖成为可能，并使基因治疗的一种新方法成为可能。拟议工作的基本原理是，RecZF具有在许多序列环境中有效地切除、颠倒或整合大段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。这一策略与目前的基因疗法不同，目前的基因疗法只提供瞬时基因表达，否则就有可能导致外源DNA的非特异性基因组整合。这项拟议的研究集中在TP53基因座上，这是一种关键的肿瘤抑制基因，在许多不同类型的人类癌症中处于功能失活状态。这项研究提案的总体目标是开发工具，以TP53基因座为模型靶点，有效和选择性地整合到任何基因组位置。中心假设是，由新的锌指蛋白和进化的催化结构域构建的RecZF将能够完成这一任务。作为基因操作的强大工具，我们预计RecZF可以促进新的癌症动物模型的建立、基因治疗的应用和癌症遗传学的阐明。

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.