We performed a trial of retinal gene augmentation in the Pde6a mutant dog using Pde6a delivery by capsid-mutant adeno-associated virus serotype 8, previously shown to have a rapid onset of transgene expression in the canine retina. Subretinal injections were performed in 10 dogs at 29–44 days of age, and electroretinography and vision testing were performed to assess functional outcome. Retinal structure was assessed using color fundus photography, spectral domain optical coherence tomography, and histology. Immunohistochemistry was performed to examine transgene expression and expression of other retinal genes. Treatment resulted in improvement in dim light vision and evidence of rod function on electroretinographic examination. Photoreceptor layer thickness in the treated area was preserved compared with the contralateral control vector treated or uninjected eye. Improved rod and cone photoreceptor survival, rhodopsin localization, cyclic GMP levels and bipolar cell dendrite distribution was observed in treated areas. Some adverse effects including foci of retinal separation, foci of retinal degeneration and rosette formation were identified in both AAV-Pde6a and control vector injected regions.

Three-week-old C57BL/6JRd9/Boc and Rpgr−/y mice were treated with unilateral (nRd9 = 18, nko = 24) or bilateral (treatment versus sham, nRd9 = 17, nko = 20) injections (1.5 × 109 vg in 1.5 μL). Immunohistochemistry of unfixed cryosections showed that the antibody detecting human RPGR does not cross-react with the orthologous Rpgr in C57BL/6J wild-type mice (Figure 4). More importantly, it demonstrated RPGR transgene expression and co-localization with the ciliary protein Rpgrip1 in the treated eye of C57BL/6JRd9/Boc and Rpgr−/y mice, but not the control eye. In the longitudinal ERG analyses, both animal models of XLRP showed significant rescue of the dominating rod system at 6 months, the last time point studied (Figure 5). Rpgr−/y mice also showed a significant rescue of cone function under light-adapted conditions, whereas the responses in C57BL/6JRd9/Boc mice showed a trend for efficacy, which did not reach the significance level in light-adapted conditions at this time point. Some of the measures at earlier time points also showed significant therapeutic effects or a trend for efficacy again especially at higher stimulus intensities (Figures S9 and S10). It is important to note that ERG responses from treated eyes versus untreated eyes (intra-individual comparison) showed significant rescue with the therapeutic AAV vector in both animal models at 4 and 6 months of age. However, when mice were treated with the therapeutic vector in one eye and the control vector in the other, variability increased as a result of having surgery performed on both sides. This contributed to variability in the ERG signals. Importantly, all responses from the treated eyes were still always greater than those from the control treated eyes. However, the significance threshold was only met in Rpgr−/y mice at 6 months.