Gene Transfer Into The Cochlea Using Adenovirus And Adeno-associated Viral Vectors And Hair Cell And Supporting Cell Proliferation By Rb Deletion And ISL1Overexpression

Unlike invertebrate animals such as birds and fish, hair cells in mammalian inner ear cannot be regenerated intrinsically. Irreversible hair cell loss is a major cause of permanent sensorineural hearing loss with no effective treatment. In addition mutations in hundreds of genes cause inherited deafness for which no intervention is available. Thus the development of strategy for hair cell regeneration and the development of gene delivery have become the major focus in the researching potential therapy to restore hearing.To identify suitable vehicle for inner ear gene delivery that can be used to correct genetic deafness and to efficiently study inner ear gene function, we have systematically analyzed new AAV vectors (a total of12) with different serotypes for their potential for inner ear gene delivery. We identified multiple AAV subtypes that infect different types of inner ear sensory epithelial cells including supporting cells and hair cells in vivo, both in neonatal and adult mice. We showed that inner ear gene delivery in neonatal inner ear results in sustained expression and does not lead to significant hearing deficits when mice were examined in adult, demonstrating such a strategy is suitable in developing gene therapy to correct genetic deafness that is mostly caused by the lack of function in early inner ear development. This is the most comprehensive examination of AAVs for their capacities in infection of inner ear cell subtypes with a wide implication.It has been shown that mouse hair cells with a targeted deletion of Rbl undergo cell cycle and become highly differentiated and functional. However, the proliferation with Rb1deletion is age dependent. To finely delineate the timing of Rbl deletion and cell cycle re- entry we have used adenovirus viral vector (serotype5) that carries Cre recombinase to infect Rb1floxed/floxed inner ear. We found that Rb1deletion could result in cell cycle re-entry in neonatal inner ear up to P4, whereas at later stages such an effect is no longer observed. To examine if expression of an early inner ear pro-sensory gene, Isll, in postnatal mouse inner ear together with Rbl deletion, is sufficient to induce proliferation, we examined effect of Rbl deletion and Isll hair cell expression in P5mouse and failed to detect any proliferating inner ear cells. Thus to induce proliferation in fully mature (adult) inner ear, other mechanisms especially with function in epigenetic modifications and reprogramming will have to be studied, together with Rb1deletion.Part one:Study of adenovirus vector5that target neonatal and adult mammalian cochlear cells[Objects] To evaluate inoculation of neonatal and adult mammalian cochlear cells using adenovirus vector5through scala media by cochleostomy.[Methods] In our study, we injected adenovirus vector5carrying GFP (Ad-GFP) or artificial endolymph on p1, p4,p7,P12,P21,1-2months old mice through scala media by cochleostomy. We harvested inner ear4days after injection and did immunohistochemistry staining.[Results] In P1, P4and P7mouse cochleae injected with Ad-GFP, GFP was expressed in supporting cells of base and middle turn4days after injection. In P12, P21,1-2M mice, GFP was expressed in supporting cells and some inner hair cells of base and middle turn4days after injection. We did not see any GFP expression in endolymph group and uninjected ear.[Conclusion]Our findings indicate that efficient Ad-GFP inoculation (via the scala media) can be performed in both neonatal and adult mouse ears via the scala media. The procedure can be used to evaluate gene therapy and to study inner ear gene function.Part two:Identification of of Adeno-associate viral vectors (AAV) that target neonatal and adult mammalian cochlear cell subtypes. [Objects] To identify new AAV vectors that can be targeted to neonatal and adult mammalian cochlear cell subtypes via scala media by cochleostomy.[Methods] We have systematically tested12AAV vectors for their capacity to transduce cochlear cell subtypes in neonatal (pl/P2) and mature mouse inner ears. AAV vectors were chosen for their characteristics in non-pathogenic, minimum toxicity, sustained expression, the capacity to infect postmitotic cells, and the efficient viral production (lweek,2weeks and3months after injection for neonatal mice and1week and3months for adult mice). AAV vectors of different serotypes (serotypes1,2,5,6,6.2,7,8,9, rh8, rh10, rh39and rh43) carrying a GFP reporter were delivered into the scala media by cochleostomy. In addition, in neonatal group, ABR was performed when in adult.[Results]In both neonatal and mature cochleae injected with AAV, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as the auditory nerve and spiral ligament. Serotypes1,8and9infect mainly hair cells and supporting cells efficiently in mature mice, whereas serotypes1and2had robust transduction in neonatal mice. Infection by AAV in neonatal mice induced slight ABR shifts when in adult (less than15dB) and there were no out hair cells death.[Conclusion]Our study identified additional AAV viral vectors that can be used for a targeted delivery into specific cochlear cell subtypes in neonatal and adult animals, with long-term expression level and minimum adversary effects on hearing. These AAV vectors will be suitable for a wide range of applications including functional study of cochlear cell type specific genes and possible correction of genetic deafness by gene therapy. Part three:In vivo proliferation of hair cells and supporting cells on Rb1deletion and ISL1overexpression mice.[Objects] To assess hair cell and supporting cells proliferation with Rb1deletion and ISL1overexpression.[Methods] In our study, we use in vivo neonatal and adult Er-Cre Rb loxp/loxp and ISL1transgenic mice to investigate the possible proliferation of hair cells and supporting cells after Rbl deletion and ISL1overexpression in hair cells. We studied cell cycle re-entry in p1, p4, p5, p7, p12, p21mice inner ear by conditionally deleting floxed Rbl gene using Cre mediated strategy and ISL1overexpression and evaluated proliferation by BrdU incorporation.[Results]1). In P1mice, Rbl deletion induced massive hair cells and supporting cells proliferation.2). By deleting Rbl in p4mice, very few hair cell and supporting cells can go back to cell cycle in apex organ of corti.3). There was no proliferation in p5, p7,p12and p21mice, even by combination of Rbl deletion and ISL1overexpression.[Conclusion] Assess hair cell and supporting cells proliferation caused by deleting Rbl was age dependent. In addition, ISL1overexpression did not faciliate hair cell and supporting cells proliferation.