Myosin 15:Genetics, Pathology, and Therapeutic Potential

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 mode! 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, Myol5, are responsible for profound congenital deafness and vestibular dysfunction in two spontaneous mouse mutants: shaker 2 and shaker 2J, and in humans with DFNB3. During the past grant cycle we used these mouse models to demonstrate the long-term structural and functional phenotypic correction of deafness with a transgene expressing Myol5. We characterized the development of pathology in Myol5, Myo6, Myo7a, pirouette, and whir/in deficient mutants, double heterozygotes and double mutants. These studies revealed no enhanced risk for age related hearing loss in double heterozygotes, unique functions of each of the individual myosin genes, and suggested the possibility that MYO15 has other functions besides transportation of whirlin to the tips of the stereocilia. Finally, 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 Myol5 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 proline-rich 1223 amino acid 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, which has brought expertise in otolaryngology, morphology, physiology and developmental genetics. Our interdisciplinary approach is essential to fully exploit the animal models to understand the mechanisms underlying hereditary inner ear disease,