Myosin 15:Genetics, Pathology and Therapeutic Potential

描述（由申请人提供）：遗传性内耳疾病普遍存在，对生活质量有重大影响。目前临床上还没有治疗遗传性内耳疾病的方法。小鼠是了解遗传性内耳疾病和开发治疗措施的理想哺乳动物模型。小鼠模型促进了人类遗传疾病基因的发现，使研究这些基因在内耳发育和功能中的作用成为可能，并为开发遗传性内耳疾病的治疗方法提供了很大的希望。这项资助申请建立在我们的发现基础上，即非常规肌球蛋白基因Myo15的突变是导致两种自发突变小鼠（shaker 2和shaker 2J）以及DFNB3患者的严重先天性耳聋和前庭功能障碍的原因。我们使用这些小鼠模型来证明表达Myo15的转基因对耳聋的长期结构和功能表型的纠正。我们描述了Myo15、Myo6、Myo7a、pirouette和whirlin缺陷突变体、双杂合子和双突变体的病理发展。虽然双杂合子没有增加年龄相关性听力损失的风险，但这些研究揭示了每个肌球蛋白基因的独特功能，并提示MYO15除了将旋转蛋白运输到静纤毛尖端外，可能还有其他功能。我们建立了用于基因治疗的腺病毒载体，并建立了正常小鼠和Myo15突变小鼠在断奶和成年耳蜗中表现差异表达的基因数据库。这些研究为这项资助的目标奠定了坚实的基础。MYO15有多种同工异构体是由选择性剪接产生的，包括MYO15的马达结构域的一个大的富含脯氨酸的区域n末端的存在或不存在。我们假设这个富含脯氨酸的区域对听力所必需的蛋白质-蛋白质相互作用很重要。我们已经建立了一个小鼠模型，利用敲入技术再现了人类富含脯氨酸区域的突变。这些突变体有严重的先天性耳聋，毛束病理不同于shaker 2和shaker 2J小鼠，前庭功能明显正常。我们提出了一个结构-功能分析，将揭示MYO15亚型在耳蜗发育和功能中的重要性，使用突变等位基因，细胞培养和耳蜗外植体测定。我们将进行经典遗传分析，以评估突变等位基因之间的相互作用，并确定相互作用的蛋白质。我们的研究团队拥有跨学科合作的成果记录，汇集了耳鼻喉科，显微镜学，生理学和发育遗传学的专家。这个团队将使我们能够充分利用动物模型来了解内耳疾病的机制，并有可能识别正常听力所必需的新基因。

DESCRIPTION (provided by applicant): Hereditary inner ear disease is prevalent and has significant implications for quality of life. There is currently no available clinical cure for hereditary inner ear disease. The mouse serves as an ideal mammalian model for understanding genetic inner ear disease and for developing therapeutic measures. Mouse models have facilitated the discovery of genes that underlie hereditary disease in humans, have made it possible to study the role of these genes in inner ear development and function, and hold great promise as models for developing treatments for hereditary inner ear disease. This grant application builds on our discovery that mutations in the unconventional myosin gene, Myo15, are responsible for profound congenital deafness and vestibular dysfunction in two spontaneous mouse mutants: shaker 2 and shaker 2J, and in humans with DFNB3. We used these mouse models to demonstrate the long-term structural and functional phenotypic correction of deafness with a transgene expressing Myo15. We characterized the development of pathology in Myo15, Myo6, Myo7a, pirouette, and whirlin deficient mutants, double heterozygotes and double mutants. Although there is no enhanced risk of age related hearing loss in double heterozygotes, these studies revealed unique functions of each myosin gene, and suggested the possibility that MYO15 has other functions besides transportation of whirlin to the stereocilia tips. We established adenoviral vectors for gene therapy and a database of genes exhibiting differential expression in the cochlea between weaning and adulthood in normal and Myo15 mutant mice. These studies laid a sound foundation for the goals of this grant. There are multiple isoforms of MYO15 that are generated by alternative splicing, including the presence or absence of a large proline-rich region N-terminal to the motor domain of MYO15. We hypothesize that this proline-rich region is important for protein-protein interactions necessary for hearing. We have generated a mouse model that recapitulates a human mutation in the proline-rich domain using knock-in technology. These mutants have profound congenital deafness, hair bundle pathology that is distinct from shaker 2 and shaker 2J mice, and apparently normal vestibular function. We propose a structure-function analysis that will reveal the importance of MYO15 isoforms in the development and function of the cochlea using mutant alleles, cell culture and cochlear explant assays. We will conduct a classical genetic analysis to evaluate interactions between mutant alleles and identify interacting proteins. Our investigative team has a track record for accomplishments resulting from cross-disciplinary collaboration, bringing together experts in otolaryngology, microscopy, physiology, and developmental genetics. This team will enable us to exploit the animal models fully to understand the mechanisms of inner ear disease and has the potential to identify novel genes essential for normal hearing. PUBLIC HEALTH RELEVANCE: Deafness is a common birth defect, affecting about 1 birth in 2000. About half of these children are affected because of genetic reasons, and there is no known cure or prevention. Myosin 15 is a molecular motor protein that is important for normal hearing. Mutations in this gene cause deafness in humans and mice. In this grant proposal we seek to identify other proteins that Myosin 15 interacts with and to use mouse models to identify the pathological changes caused by human mutations.