Figure 6

AAV-mediated gene therapy in the KO-TgAC1 USH3 model. (A) Surface preparation of the organ of Corti from KO-TgAC1 mice stained with DAPI (left panel), transfected with AAV2 -GFP (middle panel) or AAV8-GFP (right panel). AAV2-GFP and AAV8-GFP were used to assess viral delivery to the cochlea of KO-TgAC1. High titer stock (>10¹³ vg/ml) of AAV2 or 8-GFP was injected through the RWM of the KO-TgAC1 mice at P1-P3, and the organ of Corti was examined for GFP expression in the cochlea whole mount at P10 by immunofluorescence using anti-GFP antibody. Almost all IHCs were GFP positive (green), whereas a mosaic pattern of GFP positive cells was observed in the three rows of OHCs. Representative specimens from the mid-basal turn of the cochlea from mice injected with AAV2-GFP or AAV8-GFP  are shown in the middle and right panels. (B–D) Hearing restoration in the KO-TgAC1 mice. (B) Schematic representation of AAV vector constructs used in gene rescue experiments. The AAV expression cassette contains the mouse clarin-1 coding sequence (CDS) with or without its UTR sequences. Both constructs were packaged in either serotype 2 or serotype 8 AAV capsids. The Clrn1 cDNA expression in each vector is driven by the ubiquitous small CBA promoter. The expression cassette is flanked by inverted terminal repeats (TR) of AAV serotype 2; poly-A, SV40 polyadenylation signal. (C) Representative ABR traces in response to click stimulus from WT, KO-TgAC1 and rescued KO-TgAC1 mice following delivery of 2 μl AAV2 or AAV8-Clrn1-UTR construct. At P100, waveforms from WT and KO-TgAC1 mice injected with AAV2-Clrn1-UTR appear normal and similar, while non-injected KO-TgAC1 mice show no ABR responses at the sound levels tested. (D) AAV2 or AAV8 containing the Clrn1 gene, with or without the UTR sequence, were directly injected through the RWM of the KO or KO-TgAC1 mice at P1-P3 when the hair cells in KO-TgAC1 mice are comparable to the WT hair cells. To monitor preservation or loss of hearing over time, ABR thresholds were recorded from the same set of mice in each group (genotype). ABRs evoked with click stimulus recorded longitudinally from the same mice 4 to 22 weeks after birth (P27 to P150). The KO-TgAC1 mice injected with AAV2 or AAV8-Clrn1-UTR (n = 7) (red line) showed significant preservation of hearing through their adult life compared to the KO-TgAC1 mice injected with Clrn1 without UTR (n = 10) or non-injected sham surgery KO-TgAC1 (n = 6). There was no statistically significant differences in ABR thresholds over time for the same construct delivered with either AAV2 or AAV8, thus permitting the grouping of results from AAV2 and AAV8. These groupings are indicated within paranthesis in the key to the legend, where the second numeral after AAV serotype is the n value.