Recent molecular genetic studies of the inner ear have identified several genes whose function is required for normal auditory function. Defective expression of these genes may be associated with hereditary hearing loss, and the ability to correct such genetic defects using gene therapy is a promising experimental strategy for correcting these defects. Initial studies in animals suggest that gene therapy in the cochlea may be possible; however, to date, no strategy has been proposed for limiting the expression of transgenes to specific cell types within the cochlea, such as the outer hair cell. The long term goal of this project is to develop a technique that can be used to correct gene dysfunction in the cochlea. using a delivery system that will target expression to the outer hair cells. Oncomodulin is a small acidic calcium binding protein, the expression of which has recently been found to be limited to the outer hair cells of the adult guinea pig. The promoter region of the gene that encodes this protein is thus an excellent candidate for use in targeted expression studies in the cochlea.
In Specific Aim 1, the promoter region of the guinea pig oncomodulin gene will be cloned using a PCR-based strategy, to amplify the 762 bp promoter sequence from guinea pig genomic DNA with the same primers used to isolate the promoter for the mouse gene.
In Specific Aim 2, the oncomodulin promoter will be cloned into two types of vectors for delivery into the guinea pig cochlea, so that it may be used to target the transcription of a reporter gene (beta-galactosidase or green fluorescent protein) to the outer hair cell. Using the plasmid vectors pbeta-gal-Basic or pUFP-1, the oncomodulin promoter will be cloned upstream of either reporter gene in independent clones such that the functional expression of the reporter gene is dependent on the inserted promoter. The transcriptional unit (promoter and reporter gene) also will be subcloned into an adeno-associated virus plasmid such that it will be flanked by the AAV inverted terminal repeats.
In Specific Aim 3, the recombinant oncomodulin-reporter gene vectors will be introduced into the guinea pig cochlea by direct instillation and the level and duration of gene transfer obtained with each vector will be assessed using histological methods to detect the pattern and duration of expression of the reporter proteins. The proposed strategy of targeted gene delivery to hair cells of the inner ear should provide important information for future therapeutic strategies using gene therapy in the treatment of deafness.
