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

 描述(申请人提供)：先天性听力损失是一种常见的疾病，大约每600名儿童中就有1名患有深度耳聋。在听力中起核心作用的是蛋白质耳铁蛋白。已知的耳铁蛋白致病突变有60多种，在所有形式的语前常染色体隐性听力损失中，高达8%是由耳铁蛋白突变引起的。目前已知的Otoferlin唯一的身体功能是调节毛细胞释放的神经递质，使其成为一种听力特异性蛋白质。虽然耳铁蛋白对听力是必不可少的，但目前在体内研究耳铁蛋白的方法还没有阐明该蛋白的确切功能，也没有为耳聋相关的突变提供分子水平的解释。此外，大尺寸的奥弗林阻止了救援实验，并禁止了基于病毒的基因转移，这是将基因疗法用作治疗的主要障碍。为了筛选致病突变对耳铁蛋白功能的影响，开发可以包装到现有基因载体中的截短形式的耳铁蛋白，并确定耳铁蛋白的功能，我们将以斑马鱼为模型系统。使用斑马鱼作为模型的一个主要优势是能够很容易地将突变和截短形式的耳铁蛋白导入毛细胞，这在小鼠模型中是不容易实现的。这项工作建立在K99/R00奖下进行的研究基础上，该奖项致力于使用重组蛋白来表征耳铁蛋白。我们开发斑马鱼作为重组蛋白研究测试结果的模型，这使我们能够寻求一种强大的双管齐下的方法，在分子和生物水平上探测和设计耳铁蛋白。在这项提案的具体目标1中，我们将设计和测试截短形式的耳铁蛋白，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最小序列将是治疗设计的关键，包括设计截断形式的 Otoferlin小到可以包装用于病毒介导的基因治疗。特定目标2将使用斑马鱼和重组蛋白来研究与人类患者耳聋相关的病理性错义突变。这个目标的目的将是建立为什么某些耳铁蛋白错义突变会导致人类听力损失的分子基础。具体目标3将描述耳铁蛋白的生物物理性质，目的是确定听力所需的耳铁蛋白的独特功能性质。这些研究的结果将直接影响治疗耳聋的疗法的发展，解释几个人类病理性错义突变的基础，并建立otoferlin用于声音编码的机制。该研究所非常适合这项拟议的工作，它已经建立了斑马鱼作为耳铁蛋白研究的模型，并根据K99/R00奖开展了基础生物物理研究。俄亥俄州立大学优良的斑马鱼设施，再加上胞吐作用和毛细胞生理学方面的专家的合作和支持，为进行拟议的研究创造了良好的整体环境。

 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.