The improvements to adenovirus necessary for an optimal gene transfer vector include the removal of virus gene expression in transduced cells, increased transgene capacity, complete replication incompetence, and elimination of replication-competent virus that can be produced during the growth of first-generation adenovirus vectors. To achieve these aims, we have developed a vector-cell line system for complete functional complementation of both adenovirus early region 1 (E1) and E4. A library of cell lines that efficiently complement both E1 and E4 was constructed by transforming 293 cells with an inducible E4-ORF6 expression cassette. These 293-ORF6 cell lines were used to construct and propagate viruses with E1 and E4 deleted. While the construction and propagation of AdRSV␤gal.11 (an E1 ؊ /E4 ؊ vector engineered to contain a deletion of the entire E4 coding region) were possible in 293-ORF6 cells, the yield of purified virus was depressed approximately 30-fold compared with that of E1 ؊ vectors. The debilitation in AdRSV␤gal.11 vector growth was found to correlate with reduced fiber protein and mRNA accumulation. AdCFTR.11A, a modified E1 ؊ /E4 ؊ vector with a spacer sequence placed between late region 5 and the right inverted terminal repeat, efficiently expressed fiber and grew with the same kinetic profile and virus yield as did E1 ؊ vectors. Moreover, purified AdCFTR.11A yields were equivalent to E1 ؊ vector levels. Since no overlapping sequences exist in the E4 regions of E1 ؊ /E4 ؊ vectors and 293-ORF6 cell lines, replication-competent virus cannot be generated by homologous recombination. In addition, these second-generation E1 ؊ /E4 ؊ vectors have increased transgene capacity and have been rendered virus replication incompetent outside of the new complementing cell lines.

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Loss of balance is often due to loss of vestibular hair cells. In mammals, regeneration of functional hair cells in the mature sensory epithelium is limited; therefore, loss of sensory cells can lead to debilitating balance problems. Delivery of the transcription factor atonal (atoh1) after aminoglycoside ototoxicity has previously been shown to induce the transdifferentiation of supporting cells into new hair cells and restore function. A problem with mouse aminoglycoside models is that the partial loss of hair cells seen in human disease is difficult to establish consistently. In order to more closely mirror human clinical balance dysfunction, we have used systemic application of 3,3’-iminodipropionitrile (IDPN), a vestibulotoxic nitrile compound known to cause vestibular hair cell loss, to induce a consistent partial loss of vestibular hair cells. To determine if balance function could be restored we delivered atoh1 using a new adenovirus vector based on Ad28. The Ad28 adenovector is based on a human serotype with a low seroprevalence that appears to target gene delivery to vestibular supporting cells. In order to further provide cell type selectivity of gene delivery, we expressed atoh1 using the supporting cell specific glial fibrillary acid protein (GFAP) promoter. Delivery of this vector to IDPN-damaged vestibular organs resulted in a significant recovery of vestibular hair cells and restoration of balance as measured by time on rotarod compared to untreated controls.

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