Engineered nuclease for CCR5 gene editing

说明(由申请人提供)：说明申请的广泛、长期目标和具体目标，并提及项目与健康的相关性(即与机构使命的相关性)。简明扼要地描述实现这些目标的研究设计和方法。描述你将用来实现这些目标的基本原理和技术。此外，用两三句简单明了的话描述这项研究与公共卫生的相关性。如果申请得到资助，这一描述将成为公开信息。因此，不包括专有/机密信息。基因组工程是一个新兴的领域，在这个领域中，针对生物技术和治疗应用进行有针对性的基因组修改。特定部位的稀有切割内切酶是基因组工程的重要工具，因为它们需要在所需的基因组部位产生DNA双链断裂。内切酶诱导的双链断裂通过内源性DNA修复途径解决，如果通过非同源末端连接解决，则导致高效的基因编辑；如果通过同源重组解决，则导致靶向基因修饰。精密基因组工程公司开发了基于I-anii LAGLIDADG归巢内切酶作为支架的稀有切割内切酶的产生和分离的专有方法。这一阶段的STTR将支持这种方法的应用，以产生一种能够切割人CCR5基因的新型LAGLIDADG核酸酶。这种核酸酶可用于产生CCR5缺陷的T细胞。缺乏CCR5的T细胞对嗜CCR5的HIV毒株的感染具有抵抗力，这种毒株是美国最常见的HIV病毒。这种T细胞的产生和在艾滋病毒感染患者中的重新植入代表了一种治疗已确定的艾滋病毒感染的新方法，具有重要的前景。与公共卫生相关：该项目将支持开发一种能够切割人类CCR5基因的工程化位点特异性核酸酶。这种蛋白或精制的衍生物将适用于CCR5缺陷T细胞的产生，这是治疗HIV感染的一种新方法。

DESCRIPTION (provided by applicant): State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe the rationale and techniques you will use to pursue these goals. In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. Genome engineering is an emerging field in which targeted genome modifications are made for biotechnological and therapeutic applications. Site specific rare cutting endonucleases are a crucial tool for genome engineering, as they are required to create DNA double strand breaks at desired genomic sites. Endonuclease-induced double strand breaks are resolved by endogenous DNA repair pathways, resulting in high efficiency gene editing if resolved via non-homologous end joining, or targeted gene modification if resolved via homologous recombination. Precision Genome Engineering has developed proprietary methods for generation and isolation of rare cutting endonucleases based on the use of the I-AniI LAGLIDADG homing endonuclease as a scaffold. This phase I STTR will support the application of this approach to generate a novel LAGLIDADG nuclease capable of cleaving the human CCR5 gene. Such a nuclease can be applied for generation of CCR5- deficient T-cells. CCR5-deficient T-cells are resistant to infection by CCR5-tropic strains of HIV, the most common form of HIV in the United States. Generation of such T-cells and re-engraftment in an HIV infected patient represents a new approach to treatment of established HIV infections with significant promise. PUBLIC HEALTH RELEVANCE: This project will support development of an engineered site specific nuclease capable of cleaving the human CCR5 gene. This protein or refined derivatives will be applicable to generation of CCR5-deficient T-cells, a novel approach to therapy of HIV infections.