Gene Editing for DFNA9 Deafness

项目摘要/摘要 在听力和耳聋领域的基因发现方面的重大进展引起了人们对 开发遗传性耳聋的新治疗方法。听力损失(HL)，是最常见的感官 世界范围内的赤字，影响到所有年龄段的人的生活质量。尽管在认识上取得了进展 耳聋的遗传病因和分子机制，目前尚无有效的治疗方法 到目前为止，自然听力仍可用。鉴于听力问题和新出现的 基因编辑技术的格局，现在至关重要的是解决未得到满足的医疗需求，并追求新的 遗传性耳聋的治疗策略。靶向开发的临床前研究的最新报道 遗传性耳聋的治疗方法为精确听力医学开辟了一条充满希望的道路 损失。最近的报道在病毒介导的基因治疗的临床前试验中取得了令人鼓舞的结果。 遗传性耳聋。一种令人感兴趣的疾病是DFNA9，一种常染色体显性遗传性非综合征迟发性HL 可变性前庭功能障碍，由编码cochlin的Coch突变引起，是检测到最多的 内耳中的蛋白质。这种疾病模型与大多数遗传性HL相似，并具有代表性 具有显性遗传模式的疾病，具有潜在的有害功能获得/显性阴性 突变蛋白的作用机制。这个项目的目标是开发一种治疗听力的基因治疗策略。 与DFNA9相关的损失，以及一名研究人员在人类耳聋遗传学领域的培训。 为了实现这些目标，我们计划利用最先进的CRISPR-Cas9基因编辑技术来开发一种 等位基因特异性靶向方法，并测试其在阻止或减缓发育或进展方面的有效性 与显性Coch p.A449T突变相关的DFNA9敲入(Ki)小鼠模型中听力损失的研究。 初步的体外研究支持所开发的CRISPR-Cas9试剂和策略的特异性。 此外，我们的新型Coch A449T Ki小鼠模型的其他最新表型特征在 8到9个月符合迟发性听力损失。我们计划进行更深入的表型分析 建立Coch A449T Ki小鼠模型，并探讨其致病机制。我们还计划进一步评估 体内CRISPR-Cas9靶基因干扰在预防或缓解慢性粒细胞白血病中的有效性和特异性 进行性听力损失。实现项目目标将在发展方面取得实质性进展 将基因疗法转化为临床应用所需的基本步骤。该项目将建立一个 遗传性耳聋和基因治疗领域知识渊博的研究员，计划领导研究 用于治疗听力损失的精准医学。

PROJECT SUMMARY/ABSTRACT Significant advances in gene discovery in the field of hearing and deafness have raised a great interest in developing new therapeutic approaches for genetic deafness. Hearing loss (HL), is the most common sensory deficit worldwide, affecting the quality of life of people of all ages. Despite the progress in the understanding of genetic etiologies and the molecular mechanisms involved in deafness, no effective therapies in restoring the natural hearing are as yet available. Given the massive burden posed by hearing problems and the emerging landscape of gene editing technology, it is now essential to address the unmet medical need and to pursue new strategies for treatment of inherited hearing loss. Recent reports of preclinical studies for developing targeted therapeutic approaches for inherited deafness have opened promising avenues for precision medicine in hearing loss. Recent reports have yielded encouraging results in preclinical trials of virally mediated gene therapy for inherited deafness. One disorder of interest is DFNA9, an autosomal dominant nonsyndromic late-onset HL with variable vestibular dysfunction, caused by mutations in COCH, encoding cochlin, the most abundantly detected protein in the inner ear. This disease model is similar to and representative of the majority of the genetic HL disorders with a dominant mode of inheritance with an underlying deleterious gain-of-function/dominant-negative mechanism of the mutant protein. The goal of this project is to develop a gene therapy strategy to treat hearing loss associated with DFNA9, along with training of a research fellow in the fields of human genetics of deafness. To achieve these goals, we plan to utilize state-of-the-art CRISPR-Cas9 gene editing technology to develop an allele-specific targeting approach, and to test its efficacy in halting or mitigating the development or progression of hearing loss in a knock-in (KI) mouse model of DFNA9 associated with the dominant COCH p.A449T mutation. Preliminary in vitro studies support the specificity of the developed CRISPR-Cas9 reagents and strategies. Furthermore, additional very recent phenotypic characterization of our novel Coch A449T KI mouse model at eight to nine months is consistent with late-onset hearing loss. We plan to perform more in-depth phenotyping of the Coch A449T KI mouse model, and to explore the mechanism of pathogenicity. We further plan to evaluate the efficiency and specificity of in vivo CRISPR-Cas9 target gene disruption in prevention or mitigation of progressive hearing loss. Carrying out the project aims will provide substantial progress in development of the essential steps required for translation of gene therapy into clinical application. This project will establish a knowledgeable research fellow in the field of hereditary deafness and gene therapy, who plans to lead research in precision medicine for hearing loss.