A Bioassay for Human Gene Therapy Trials of Leber Congenital Amaurosis (LCA)

Abstract

Purpose:: Viral-mediated retinal gene transfer has produced dramatic restoration of retinal function in murine and canine RPE65-deficient animals. Steady progress toward clinical trials of RPE65-LCA prompted us to initiate design of an in vivo assay to verify stability of clinical grade vector.

Methods:: The rd12 mouse model of RPE65-LCA was used. A single subretinal injection of 1µl GMP AAV2-CB^SB-hRPE65 vector was delivered to one eye of 2-5 week old mutant mice (n=41) and a series of vector doses were studied: 1.0X, 0.3X, 0.1X and 0.01X dose relative to maximum (1X10¹⁰ vg/µl). Uninjected rd12 eyes and wild-type (wt) mice served as controls. Full-field ERGs to flash stimuli (range, -4.1 to +3.6 log scot-cd.s.m^-2) were performed in anesthetized, dark-adapted mice about 6 weeks after vector delivery. B-waves were measured conventionally and leading edges of photoresponses were fit with a model of rod phototransduction activation.

Results:: Photoresponses in untreated rd12 eyes were smaller in amplitude and slower in kinetics than wt mice. B-waves were usually not detectable below an intensity of 0 log scot-cd.s.m^-2. ERG parameters did not differ in treated and untreated eyes at 0.01X dose. Treated eyes at higher doses showed a predictable dose-response relationship. There were increased photoresponse amplitudes and faster kinetics for 0.1X, 0.3X, 1.0X doses. Dose-response was evident in b-wave parameters. B-wave thresholds decreased with dose; amplitudes to a 0 log scot-cd.s.m^-2 intensity flash increased. B-wave amplitude to the 0 log scot-cd.s.m^-2 intensity provided a simple indicator for treatment success.

Conclusions:: A dark-adapted single flash ERG protocol in rd12 mice after subretinal injection of clinical grade vector can be used as an efficient bioassay to verify stability of vector function over time.

Keywords: retinal degenerations: hereditary • electroretinography: non-clinical • gene transfer/gene therapy