Sensorineuronal hearing loss affects millions of people worldwide and in the U.S. Loss of the sensory hair cell in the organ of Corti is thought to be a major cause of this deficit. In animals that are capable of regenerating their hair cells after hair cell death or loss (e.g. birds), a key first step is I the renewed proliferative capacity of the non-sensory supporting cell. In birds, supporting cell proliferation gives rise to new functioning hair cells after acoustic trauma or aminoglycoside poisoning. Unfortunately, supporting cell proliferation in the mammalian organ of Corti has never been observed either after hair cell death or the addition of growth factors. The cyclin-dependent kinase inhibitor, p27Kip1, is expressed in the supporting cells of mice from birth through adulthood. Genetic deletion of p27Kip1 results in continued supporting cell proliferation after birth and into adulthood. Deletion of p27Kip1 in mice is sufficient to induce low levels of hair cell regeneration following drug-induced hair cell death. In addition, reduction of p27Kip1 levels by lipofection of antisense oligonucleotides specific to p27Kip1, can induce supporting cell proliferation in vitro and in vivo. This proliferation appears to be limited by the poor efficiency of transfection of supporting cells by lipofection reagents. We propose to compare lipofection of p27Kip1 antisense oligonucleotides to that achieved by recombinant adeno-associated virus. The most effective and least ototoxic AAV subtype will be utilized to transfect mouse organ of Corti explant cultures with a p27Kip1 antisense expression cassette or a p27Kip1 ribozyme expression cassette. The expression cassettes for p27Kip1 antisense and ribozyme will be optimized in cell lines prior to their use on explant cultures. This will facilitate the development of pharmacologic means to improve and restore hearing through regeneration of sensory cells in the cochlea over that which has been observed with antisense oligonucleotides. ? ?
