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In vivo and in vitro studies of the deafness associated protein otoferlin

耳聋相关蛋白 otoferlin 的体内和体外研究

基本信息

批准号：
9925756
负责人：
Colin P Johnson
金额：
$ 33.57万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9925756
关键词：
Address Amino Acids Animal Model Area Award Binding Biochemical Biological Assay Biological Models Biophysics C2 Domain Calcium Calcium Binding Cell physiology Child Collaborations Coupled Couples Data Defect Disease Engineering Environment Exocytosis Foundations Gene Delivery Gene Transfer Genes Genetic Code Goals Hair Cells Hearing Hereditary Disease Human In Vitro Individual Integral Membrane Protein Kinetics Measures Mediating Membrane Membrane Lipids Membrane Proteins Missense Mutation Modeling Molecular Mutation OTOF gene Outcome Pathogenicity Pathologic Patients Phenotype Positioning Attribute Property Protein Biochemistry Protein Region Proteins Recombinant Proteins Research Role Science Scientist Sensory Hair Site Specialist Speed Study models Synaptic Transmission Synaptic Vesicles Techniques Tertiary Protein Structure Test Result Testing Therapeutic Viral Work Zebrafish Zebrafish Proteins auditory neuropathy base biophysical analysis biophysical properties congenital hearing loss deaf deafness design experimental study expression vector gene therapy hearing impairment hearing restoration human disease in vivo knock-down mechanotransduction mouse model mutant neurosensory neurotransmitter release prevent protein function public health relevance ribbon synapse sensor sound sound frequency therapeutic development unnatural amino acids zebrafish development

项目摘要

DESCRIPTION (provided by applicant): Congenital hearing loss is a common disorder, with approximately 1 out of every 600 children suffering from profound deafness. A central player in hearing is the protein otoferlin. More than 60 pathogenic mutations in otoferlin are known, and up to 8% of all forms of prelingual autosomal recessive hearing loss are due to otoferlin mutations. Currently the only known bodily function for otoferlin is in mediating neurotransmitter release from hair cells, making it a hearing specific protein. While otoferlin is essential for hearing, current approaches to study otoferlin in vivo have not elucidated the exact function of the protein or provided a molecular level explanation for mutations associated with deafness. Further, the large size of otoferlin has prevented rescue experiments and prohibited viral-based gene transfer, representing a major hurdle toward the use of gene therapy as a treatment. To screen for the effects of pathogenic mutations on otoferlin function, develop truncated forms of otoferlin that can be packaged into existing gene delivery vehicles, and determine the function of otoferlin, we will use zebrafish as a model system. A major advantage of using zebrafish as a model is the ability to easily transfect hair cells with mutant and truncated forms of otoferlin, something that cannot be achieved easily in a mouse model. This work builds off studies conducted under a K99/R00 award devoted to characterizing otoferlin using recombinant protein. Our development of zebrafish as a model for testing results of recombinant protein studies uniquely positions us to pursue a powerful two-pronged approach to probe and engineer otoferlin on both the molecular and organismal level. In specific aim 1 of this proposal we will engineer and test truncated forms of otoferlin to determine the minimal regions of the protein required for hearing. Determination of the minimal sequence of otoferlin capable of restoring hearing will be critical for the design of therapeutics, including the design of truncated forms of otoferlin small enough to be packaged for viral mediated gene therapy. Specific aim 2 will use zebrafish and recombinant proteins to study pathological missense mutations associated with deafness in human patients. The goal of this aim will be to establish the molecular basis for why certain otoferlin missense mutations result in hearing loss in humans. Specific aim 3 will characterize the biophysical properties of otoferlin, with the goal of determining the unique functional properties of otoferlin that are needed for hearing. The results of these studies will directly impact the development of therapeutics for treating deafness, explain the basis for several human pathological missense mutations, and establish the mechanisms otoferlin utilizes for the encoding of sound. The PI is well suited for the proposed work, having already established zebrafish as a model for otoferlin studies, and having carried out fundamental biophysical studies under a K99/R00 award. The excellent zebrafish facilities at OSU, coupled with collaborations and support from specialists in the area of exocytosis and hair cell physiology make for an excellent overall environment for conducting the proposed studies.

描述（申请人提供）：先天性听力损失是一种常见疾病，大约每600名儿童中就有1名患有深度耳聋。听觉的一个中心角色是蛋白质otoferlin。已知有超过60种致病性的otoferlin突变，在所有形式的语前常染色体隐性听力损失中，有高达8%是由于otoferlin突变引起的。目前，耳铁蛋白唯一已知的身体功能是介导毛细胞释放神经递质，使其成为听觉特异性蛋白质。虽然otoferlin对听力至关重要，但目前在体内研究otoferlin的方法还没有阐明该蛋白的确切功能，也没有为与耳聋相关的突变提供分子水平的解释。此外，otoferlin的大尺寸已经阻止了救援实验并禁止了基于病毒的基因转移，这代表了使用基因疗法作为治疗的主要障碍。为了筛选致病性突变对otoferlin功能的影响，开发可以包装到现有基因递送载体中的截短形式的otoferlin，并确定otoferlin的功能，我们将使用斑马鱼作为模型系统。使用斑马鱼作为模型的一个主要优点是能够容易地用突变和截短形式的otoferlin来抑制毛细胞，这在小鼠模型中是不容易实现的。这项工作建立在K99/R 00奖项下进行的研究，致力于使用重组蛋白表征otoferlin。我们开发的斑马鱼作为重组蛋白研究结果的测试模型，使我们能够在分子和生物体水平上寻求一种强有力的双管齐下的方法来探测和工程化otoferlin。在本提案的具体目标1中，我们将设计和测试截断形式的otoferlin，以确定听力所需的蛋白质的最小区域。确定能够恢复听力的otoferlin的最小序列对于治疗剂的设计将是至关重要的，包括设计截短形式的小到足以包装用于病毒介导的基因治疗的otoferlin。具体目标2将使用斑马鱼和重组蛋白研究与人类患者耳聋相关的病理性错义突变。这个目标的目标将是建立为什么某些otoferlin错义突变导致人类听力损失的分子基础。具体目标3将描述otoferlin的生物物理特性，目的是确定听力所需的otoferlin的独特功能特性。这些研究的结果将直接影响治疗耳聋的疗法的发展，解释几种人类病理性错义突变的基础，并建立otoferlin用于编码声音的机制。PI非常适合拟议的工作，已经建立了斑马鱼作为otoferlin研究的模型，并在K99/R 00奖下进行了基础生物物理研究。俄勒冈州立大学优秀的斑马鱼设施，加上胞吐和毛细胞生理学领域专家的合作和支持，为开展拟议的研究提供了良好的整体环境。