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

开发用于内耳基因治疗的 AAV-CRISPR 系统

基本信息

批准号：
8852593
负责人：
Jung-Bum Shin
金额：
$ 19.55万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8852593
关键词：
Adult Animals Auditory BUB/BnJ Mouse Biomedical Research Cataloging Catalogs Cell Death Cell Line Cell physiology Cells Cellular Morphology Clinical Code Complex DNA DNA Repair Dependovirus Development Embryo Engineering Face Fibroblasts Genes Genetic Genetic Materials Genetic study Genome engineering Goals Guide RNA Hair Cells Health Hearing Human Intervention Labyrinth Life Mediating Methods Modification Monitor Mus Mutation Neisseria meningitidis Neonatal Orthologous Gene Outcome Proteins Research Sensory Sensory Hair Streptococcus pyogenes System Technology Testing Time VLGR1 gene Variant Virus base cell type clinical Diagnosis deafness endonuclease functional restoration gene repair gene therapy genome editing genome sequencing hearing impairment in vivo innovation mouse model novel nuclease postnatal prevent protein expression repaired research study restoration success targeted sequencing tool vector

项目摘要

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.

描述（由申请人提供）：人类听力损失具有很强的遗传因素，开发旨在修复导致耳聋的突变的基因治疗策略是听觉研究的主要焦点。在这里，我们建议应用成簇规则间隔回文重复序列 (CRISPR)/Cas 技术来开发内耳基因治疗平台。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多个耳聋基因的效率，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，例如脱靶效应和缺乏合适的递送方法。我们预计拟议的项目将在后一个问题上取得重大进展：该提案的具体目标是 1) 设计一种新颖的、改良的 CRISPR 系统，允许包装到腺相关载体 (AAV) 中，这是目前唯一的系统能够在体内将遗传物质传递到成人感觉海细胞中，2）应用该工具修复小鼠模型中导致耳聋的突变。 CRISPR技术在基因治疗中高效、安全的应用需要对CRISPR系统进行多项创新。我们相信，我们对 Cas9 基因进行大小修饰的策略以及对来自不同原核物种的 CRISPR 组件的探索将能够设计出适用于 AAV 系统的 CRISPR 系统。该项目的成功执行将导致开发出一种有效的工具，将 CRISPR 系统传递到内耳细胞中，并通过原理验证证明 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整个生物医学研究都是一个非常重要的贡献。