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Development of Genome Editing as Treatment for Genetic Hearing Loss

基因组编辑治疗遗传性听力损失的进展

基本信息

批准号：
10542663
负责人：
Zheng-Yi Chen
金额：
$ 52.06万
依托单位：
MASSACHUSETTS EYE AND EAR INFIRMARY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10542663
关键词：
Adult Affect Animals Antisense Oligonucleotides Auditory Biomedical Research CRISPR/Cas technology Ca(2+)-Transporting ATPase Categories Cell Survival Cells Child Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex DNA Defect Development Disease Disease Progression Dominant Genetic Conditions Enzymes Formulation Foundations Gene Delivery General Population Genes Genetic Heterogeneity Genome Genomic DNA Guide RNA Hair Cells Hearing High-Throughput Nucleotide Sequencing Human Injections Intervention Labyrinth Lipids Liposomes Maps Measures Mediating Medical Membrane Methods MicroRNAs Modeling Mutation Names Newborn Infant Outcome Outer Hair Cells Patients Pattern Proteins Publishing Recovery Ribonucleoproteins Specificity Startle Reaction Structure Technology Therapeutic Time Toxic effect Translating Variant deafness delivery vehicle design dominant genetic mutation dosage endonuclease gene discovery genetic deafness genome editing hearing impairment hereditary hearing loss human model improved in vivo innovation insertion/deletion mutation mouse model nanoparticle new technology novel therapeutics nuclease preservation progressive hearing loss round window screening small hairpin RNA therapeutic genome editing therapy outcome transmission process whole genome

项目摘要

Project Summary and Relevance Genetic hearing loss affects one in 1000 newborns and contributes significantly to general populations with hearing loss. Over 130 deafness loci have been mapped and more than 80 have been cloned. Despite the tremendous progress in deafness gene discovery, there is no medical treatment for genetic hearing loss. There is an urgent and unmet medical need to develop treatment for genetic hearing loss. CRISPR/Cas9-mediated genome editing is transforming biomedical research and with a promise of becoming new treatment of disease. It enables the application of nuclease with guide RNA to pair with and modify DNA permanently, which can be developed into new therapies for wide range of diseases. We have successfully used transient and in vivo local delivery of editing agents to treat a mouse model, Beethoven, of human dominant hearing loss DFNA36. In this application, we propose two broad aims to further develop CRISPR technology for inner ear editing and to apply it to treat genetic hearing loss. First, we will characterize CRISPR nuclease variants SaCas9 and Cpf1 for hair cell editing by direct RNP (ribonucleoprotein) delivery. This study will expand deafness mutations to be targeted by genome editing due to additional PAM (protospacer adjacent motif) sequences and nuclease activities, with a possibility of improvement in editing efficiency and specificity. We will apply whole-genome application (WGA) using purified hair cells for high- throughput sequencing (HTS) and to identify insertions and deletions (indels) in hair cells, and will correlate hair cell editing efficiency with the outcome of hearing rescue. We will evaluate editing in mature hair cells by RNP delivery, which is relevant to treatment in patients as human inner ears are fully mature. We will screen new nanoparticle based liposomes to provide the options for efficient inner ear delivery and editing. In second aim, we will perform hearing rescue studies by RNP delivery of editing agents to disrupt mutations in mouse models of human dominant deafness, including 1). Hearing rescue by editing Oblivion (Obl) mutation in the Pmca2 gene that affects outer hair cells; 2). Hearing rescue by editing a mutation in the microRNA 96 (Mir96) that results in delayed onset progressive hearing loss. Hearing rescue in those models will demonstrate general therapeutic application of genome editing targeting dominant mutations of hair cell origin. The study will delineate the relationship between editing efficiency, specificity and off-target effect with the extent of hearing rescue and evaluate long-term rescue effect and the outcome of intervention at late stages. The proposal has potential to be developed as new platform for genome editing based therapy for genetic deafness. Genetic hearing loss affects large number of children and contributes to adult deafness with no treatment available. We are developing CRISPR/Cas9-mediated genome editing as a new treatment platform to target dominant mutations by transient local delivery into inner ear, with the potential to treat diverse forms of genetic hearing loss.

项目摘要和相关性遗传性听力损失影响每1000个新生儿中的一个，并对听力损失的一般人群有显着影响。目前已定位了130多个耳聋基因座，克隆了80多个。尽管取得了巨大的进步，耳聋基因的发现，目前还没有治疗遗传性听力损失的药物。有一个紧急和未满足的医疗需要开发遗传性听力损失的治疗方法。 CRISPR/Cas9介导的基因组编辑正在改变生物医学研究，并有望成为新的疾病的治疗。它使核酸酶与指导RNA的应用能够与DNA配对并永久修饰DNA，其可被开发为用于多种疾病的新疗法。我们已经成功地使用瞬时和体内局部递送编辑剂以治疗人类显性听力损失DFNA 36的小鼠模型Beethoven。在这我们提出了两个广泛的目标，进一步开发用于内耳编辑的CRISPR技术，并将其应用于治疗遗传性听力损失首先，我们将通过直接测序来表征用于毛细胞编辑的CRISPR核酸酶变体SaCas 9和Cpf 1。 RNP（核糖核蛋白）递送。这项研究将扩大耳聋突变，以通过基因组编辑进行靶向，额外的PAM（前间区序列邻近基序）序列和核酸酶活性，具有改善编辑效率和特异性。我们将使用纯化的毛细胞进行全基因组应用（WGA），通过高通量测序（HTS）和鉴定毛细胞中的插入和缺失（indels），编辑效率与听力拯救的结果。我们将通过RNP递送评估成熟毛细胞中的编辑，这与患者的治疗有关，因为人的内耳是完全成熟的。我们将筛选新的纳米粒子为基础的脂质体，以提供有效的内耳递送和编辑的选择。在第二个目标，我们将执行听证会通过RNP递送编辑剂以破坏人类显性耳聋小鼠模型中的突变的拯救研究，包括1）。通过编辑影响外毛细胞的Pmca 2基因中的遗忘（Obl）突变来拯救听力; 2）。通过编辑microRNA 96（Mir 96）中的突变来挽救听力，导致延迟发作的进行性听力损失。这些模型中的听力拯救将展示基因组编辑靶向显性遗传的一般治疗应用。毛细胞起源的突变。本研究将描述编辑效率、特异性和脱靶之间的关系对听力挽救程度的影响，并对远期挽救效果和后期干预效果进行评价。该提案有可能被开发为基于基因组编辑的遗传性耳聋治疗的新平台。遗传性听力损失影响大量儿童，并导致成人耳聋，但没有治疗方法。我们正在开发CRISPR/Cas9介导的基因组编辑作为一种新的治疗平台，通过以下方式靶向显性突变：短暂的局部传递到内耳，具有治疗各种形式的遗传性听力损失的潜力。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Reprogramming by drug-like molecules leads to regeneration of cochlear hair cell-like cells in adult mice.

DOI：
10.1073/pnas.2215253120
发表时间：
2023-04-25
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Quan, Yi-Zhou;Wei, Wei;Ergin, Volkan;Rameshbabu, Arun Prabhu;Huang, Mingqian;Tian, Chunjie;Saladi, Srinivas Vinod;Indzhykulian, Artur A.;Chen, Zheng-Yi
通讯作者：
Chen, Zheng-Yi

Otolaryngologic Manifestations of Trisomy 13 and Trisomy 18 in Pediatric Patients.

儿科患者 13 三体和 18 三体的耳鼻喉表现。