Transduction efficiency of adenoviral vectors in the organ of Corti cultured in vitro

Aim: Adenoviral vector is one of the widely used vectors in research of inner ear gene therapy, but the targeting of vector is not clear. The purpose of this paper is to explore the transduction efficiency of adenoviral vectors EGFP in cochlear cultures. Methods: Between August 2004 and January 2006, the experiment was conducted in Center for Hearing and Deafness of SUNY University at Buffalo. 1 Experimental materials: Fisher rats were from Laboratory Animals Facilities and Adenoviral vector (AdV/EGFP) was a gift of Dr. Lee from SUNY University at Buffalo. 2 Experimental methods: Cochleas were dissected from postnatal 3 day (P3) rats. AdV/EGFP was used to transfer EGFP reporter gene into cochlear with different concentrations of 1×10⁷, 1×10⁸, 1×10⁹ and 2×10⁹ VP/mL. Cochlear cultures were fixed 6, 12, 24, 48 hours and 3 days after transferred and transduction efficiency was observed. Corti of cochleas were removed from P3 rats. Partly cochleas was isolated and immerged in 2 mL with 1×10⁹ VP/mL viral medium containing 2 mmol/L of gentamicin for 3 hours, and then placed on collagen surface. Medium containing 2 mmol/L of gentamicin was added and cultured for 24 hours and then harvested. 3 Experimental evaluation: Immunohischemical staining of TRITC-labeled phaloidin were used. Samples were examined under a fluorescence microscope to observe EGFP or TRITC labeling in cochlear cells. Results: 1 Many AdV/EGFP transfected cells were seen in the outer sulcus and interdental cells region; Few of EGFP positive cells were seen in the hair cells and spiral ganglion neurons region in normal cochlear cultures. 2 There were titer-dependent in the range of 10⁷-10⁹ VP/mL, however high titer could result in hair cells loss and outer sulcus lesion. 3 Labeling was firstly observed at hour 6 after infected and went up to peak at hour 48. High exogenous gene expressed for 7 days. 4 Cochlear cultures were damaged with gentamicin, and there was an overall increase in the number of EGFP labeled cells in the Deiters' cell region. Efficiency of infection was higher in interdental cells than that in normal tissues. Conclusion: The transduction efficiency of adenoviral vectors in vary type of cochlear cells is evidently different. The adenovirus preferentially infects supporting cells in cochlear cultures.