RT-PCR results demonstrate that after AAV1-VGLUT3 delivery, there is also more widespread VGLUT3 mRNA transcription than in just IHCs (Figure 1C). These results suggest that there is a posttranscriptional regulatory selleck chemicals mechanism acting on VGLUT3 mRNA, which leads to selective expression of the protein only within IHCs. Several types of posttranscriptional regulation have been described within the cochlea, and whether this specific mechanism involves microRNA inactivation (Elkan-Miller et al., 2011), transcription factor regulation (Masuda et al., 2011), or

another process remains to be determined. Such a mechanism, if appropriately elucidated and exploited, could theoretically allow the expression (or conversely suppression) of a number Trametinib of different proteins within the inner ear to alter function in pursuit of hearing preservation.

Another interesting finding was the variable success with the CO as compared to the RWM delivery technique. As noted, we initially started with an apical CO delivery method but abandoned it due to the low success rate of hearing restoration (17% of animals). Subsequently, we changed to an RWM delivery technique for several reasons; this would be the most likely method of delivery in any future human studies, and it was less likely to be traumatic, as evidenced by a number of recent human studies looking at hearing preservation with round window insertion of cochlear implants (von Ilberg et al., 2011). In fact, the change in technique resulted in hearing restoration in 100% of animals attempted. We believe the likely difference in success between the two techniques relates to the degree of trauma induced by each method. With a cochleostomy, a separate hole into the scala through bone must be created, which by its nature is traumatic, despite our best efforts

to minimize trauma. In contrast, an RWM injection simply involves piercing the membrane and sealing it with fascia after viral delivery. However, we were histologically unable to see any obvious differences between the ears of animals with and without hearing rescue in the cochleostomy group (data not shown) and there may be second other reasons for the variable success between the two techniques. Further, we noted that even earlier delivery via the RWM at P1–P3, as opposed to P10–P12, resulted in hearing recovery that was more consistently long lived, with all mice followed out through 9 months showing ongoing normal ABR thresholds (Figure 3D). Transgene expression with AAV1 should theoretically last for a year or longer (Henckaerts and Linden, 2010). However, it is not entirely clear why there is a variable loss of hearing after 7 weeks, regardless of delivery technique at the later P10–P12 delivery time point (Figure 3D).