Development of an AAV-CRISPR system for inner ear gene therapy

描述（由申请人提供）：人类听力损失具有很强的遗传成分，开发旨在修复引起突变的耳聋的基因治疗策略是听觉研究的主要焦点。在此，我们建议将聚集规则间隔回文重复序列(CRISPR)/Cas技术应用于内耳基因治疗平台的开发。在初步研究中，我们已经证明了CRISPR系统在细胞系和小鼠模型中修饰几种耳聋基因的效率，并且是将该技术应用于耳聋研究的先驱。然而，它在基因治疗中的适用性面临着一些障碍，比如脱靶效应和缺乏合适的递送方法。我们期望所提议的项目将在后一个问题上取得重大进展：该提案的具体目标是：1)设计一种新颖的改良CRISPR系统，该系统允许包装成腺相关载体（AAV），这是目前唯一能够将遗传物质在体内传递到成人感觉海细胞中的系统；2)应用该工具修复小鼠模型中引起耳聋的突变。CRISPR技术在基因治疗中的有效和安全应用需要对CRISPR系统进行一些创新。我们相信，我们对Cas9基因进行大小修饰的策略和对来自不同原核生物物种的CRISPR成分的探索，将使CRISPR系统的工程设计能够适应AAV系统。该项目的成功实施将导致开发一种将CRISPR系统送入内耳细胞的有效工具，并在原则上证明CRISPR介导的靶向基因治疗在活体动物中是可能的。这不仅对耳聋研究，而且对一般的生物医学研究都是一个非常重要的贡献。

DESCRIPTION (provided by applicant): Hearing loss in humans has a strong genetic component, and developing gene therapy strategies aimed at repairing deafness causing mutations represents a major focus of auditory research. Here, we propose to apply the Clustered Regularly Interspaced Palindromic Repeat (CRISPR)/Cas technology towards the development of a gene therapy platform for the inner ear. In preliminary studies, we have demonstrated the efficiency of the CRISPR system in modifying several deafness genes in cell lines and mouse models, and are pioneers in applying this technology to deafness research. Its applicability for gene therapy, however, faces several hurdles, such as off-target effects and lack of suitable delivery methods. We expect that the proposed project will make significant inroads into the latter issue: The specific goals of this proposal are 1) to engineer a novel, modified CRISPR system that allows packaging into an adeno-associated vector (AAV), which presently is the only system capable of in vivo delivery of genetic material into adult sensory hai cells, and 2) to apply this tool to repair deafness-causing mutations in the mouse model. An efficient and safe application of the CRISPR technology for gene therapy requires several innovations to the CRISPR system. We are confident that our strategy of size-modifying the Cas9 gene and exploration of CRISPR components from different prokaryotic species will enable the engineering of a CRISPR system adaptable for the AAV system. The successful execution of this project will result in the development of an efficient tool for delivering the CRISPR system into inner ear cells, and in proof-of-principle demonstration that CRISPR-mediated, targeted gene therapy is possible in a living animal. This is a highly significant contribution not only for deafness research, but for biomedical research in general.