Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors

To understand the function of specific proteins in sensory hair cells, it i s necessary to add or inactivate those proteins in a system where their phy siological effects can be studied. Unfortunately, the usefulness of heterol ogous expression systems for the study of many hair cell proteins is limite d by the inherent difficulty of reconstituting the hair cell's exquisite cy toarchitecture. Expression of exogenous proteins within hair cells themselv es may provide an alternative approach. Because recombinant viruses were ef ficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of orga notypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the o ther hand, we found robust expression of GFP in hair cells exposed to a rec ombinant, replication-deficient adenovirus that carried the gene for GFP dr iven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were suffici ent for expression in 50% of the similar to 1,000 hair cells in the utricul ar epithelium; < 1% of the nonhair cells in the epithelium were GFP positiv e. Expression of GFP was evident as early as 12 h postinfection, was maxima l at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and trans duction currents from infected cells identified by GFP fluorescence. At lon ger times hair bundle integrity was compromised despite a cell body that ap peared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2 .1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the con trol of an ecdysone promoter. Unlike the diffuse distribution within the ce ll body we observed with Gm, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranucle ar punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude la rger than the endogenous inward rectifier. The zero-current potential in in fected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in cu lture, which can be used to study the effects of gene products on hair cell function.