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：
8765296
负责人：
Jung-Bum Shin
金额：
$ 23.7万
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 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8765296
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 Genome Genome engineering Goals Guide RNA Hair Cells 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 sequencing hearing impairment in vivo innovation mouse model novel nuclease postnatal prevent protein expression public health relevance repaired research study restoration success 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）：人类的听力损失具有强大的遗传成分，并且制定旨在修复耳聋引起突变的基因治疗策略是听觉研究的主要重点。在这里，我们建议将定期间隔间的全文重复（CRISPR）/CAS技术应用于内耳的基因治疗平台的开发。在初步研究中，我们证明了CRISPR系统在细胞系和小鼠模型中修改多个耳聋基因的效率，并且是将该技术应用于聋哑研究的先驱。然而，其适用于基因疗法，面临着几个障碍，例如脱靶效应和缺乏合适的递送方法。我们期望拟议的项目将重大介绍后一个问题：该提案的具体目标是1）设计一种新型的，修改的CRISPR系统，该系统允许将腺相关的载体（AAV）包装到目前，该系统目前是唯一能够将遗传材料输送到成人感觉HAI细胞中的唯一能够将遗传材料输送到成人感觉的系统中，以将其应用于该工具的模型，以修复型号的模型。 CRISPR技术用于基因疗法的有效且安全的应用需要对CRISPR系统进行一些创新。我们有信心，我们修改CAS9基因的尺寸策略和对不同原核种的CRISPR组件的探索将使CRISPR系统的工程适应AAV系统。该项目的成功执行将导致开发一种有效的工具将CRISPR系统传递到内耳细胞中，并在原则证明中证明了CRISPR介导的靶向基因疗法在活的动物中是可能的。这不仅对聋哑研究，而且对一般的生物医学研究做出了重要的贡献。