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A. J. Roman, S. L. Boye, T. S. Aleman, J. J. Pang, J. H. McDowell, S. E. Boye, A. V. Cideciyan, S. G. Jacobson, W. W. Hauswirth; A Bioassay for Human Gene Therapy Trials of Leber Congenital Amaurosis (LCA). Invest. Ophthalmol. Vis. Sci. 2007;48(13):3707.
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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.
The rd12 mouse model of RPE65-LCA was used. A single subretinal injection of 1µl GMP AAV2-CBSB-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.
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.
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.
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