Dose Range Finding Studies with Two RPGR Transgenes in a Canine Model of X-Linked Retinitis Pigmentosa Treated with Subretinal Gene Therapy

Recombinant adeno-associated viral (rAAV) vector-mediated gene therapy is being developed to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. In preparation for a clinical gene therapy trial, we conducted dose range finding (DRF) studies with an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF) vector administered by subretinal injection in a naturally occurringRPGR-mutant canine model (XLPRA2) to compare two different humanRPGR(hRPGR) transgenes and to establish a reasonable starting dose for a clinical trial. Different dose levels of two candidate vectors (0.15 mL at 1.2 x 10(10)-3.0 x 10(12)vg/mL of rAAV2tYF-GRK1-hRPGRcoor 4 x 10(10)-3.0 x 10(12)vg/mL of rAAV2tYF-GRK1-hRPGRstb), 6.0 x 10(11)vg/mL rAAV5-GRK1-hRPGRcoreference vector or Vehicle were subretinally administered, and the dogs were followed for 8 weeks postdose. Ophthalmic examinations, analyses of retinal structure byin vivoimaging using confocal scanning laser ophthalmoscopy (cSLO)/optical coherence tomography (OCT) in the Lower (4.0 x 10(10)vg/mL) and Lowest (1.2 x 10(10)vg/mL) Doses, immunological responses by cell based assays or enzyme-linked immunosorbent assay,RPGRtransgene expression, and reversal of opsin mislocalization by immunohistochemistry were performed. No sustained signs of ocular discomfort or ophthalmic complications were noted in any of the injected eyes except some in the High Dose group (3.0 x 10(12)vg/mL), which showed signs of retinal detachment and inflammation. A change in fundus reflectivity suggestive of a rescue effect was seen in the High, Mid (6.0 x 10(11)vg/mL), and Low (1.2 x 10(11)vg/mL) Dose groups. cSLO/OCT demonstrated qualitative and quantitative evidence of rescue effect in eyes treated with the Lower Dose. Anti-hRPGR antibodies were absent, but neutralizing antibody titers against AAV2 were detected in all animals dosed with rAAV2tYF in an apparent dose-related pattern. RPGR expression was stronger for rAAV2tYF-GRK1-hRPGRcocompared to rAAV2tYF-GRK1-hRPGRstbat all dose levels. Subretinal administration of rAAV2tYF-GRK1-hRPGRcoand rAAV2tYF-GRK1-hRPGRstbboth corrected rod and cone opsin mislocalization, two early markers of disease in the XLPRA2 canine model ofRPGR-XLRP. These results support the selection and use of rAAV2tYF-GRK1-hRPGRco(AGTC-501) and guided the initial doses in clinical studies in patients with XLRP caused byRPGRmutations.