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

描述(申请人提供)：人类的听力损失有很强的遗传成分，开发旨在修复导致突变的耳聋的基因治疗策略是听觉研究的主要重点。在这里，我们建议将聚类规则间隔回文重复(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系统在修改细胞系和小鼠模型中的几个耳聋基因方面的有效性，并是将这项技术应用于耳聋研究的先驱。然而，它在基因治疗方面的适用性面临着几个障碍，例如非靶点效应和缺乏合适的给药方法。我们预计拟议的项目将在后一个问题上取得重大进展：这项建议的具体目标是：1)设计一种新型的、经修改的CRISPR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体内输送到成人感觉HAI细胞的系统，以及2)应用该工具修复小鼠模型中导致耳聋的突变。CRISPR技术要有效和安全地应用于基因治疗，需要对CRISPR系统进行几项创新。我们相信，我们对Cas9基因进行大小调整的策略以及对不同原核生物CRISPR组分的探索将使设计出适合AAV系统的CRISPR系统成为可能。该项目的成功实施将导致开发一种将CRISPR系统导入内耳细胞的有效工具，并在原理证明中证明CRISPR介导的靶向基因治疗在活体动物中是可能的。这不仅是对耳聋研究的重大贡献，也是对整个生物医学研究的重大贡献。

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.