Electroretinographic analyses of Rpe65-mutant rd12 mice: developing an in vivo bioassay for human gene therapy trials of Leber congenital amaurosis.
OBJECTIVE
Dramatic restoration of retinal function has followed subretinal viral-mediated gene therapy in RPE65-deficient animal models of human Leber congenital amaurosis (LCA) caused by RPE65 mutations. Progress in early-phase clinical trials of RPE65-LCA prompted us to begin development of an in vivo bioassay of clinical grade vector stability for later-phase trials.


METHODS
Naturally-occurring Rpe65-mutant rd12 mice (2-4 mo of age) were studied with full-field electroretinograms (ERGs). Flash stimuli (range, -4.1 to 3.6 log scot-cd x s x m(-2)) were used to evoke ERGs in anesthetized, dark-adapted mice. B-wave amplitudes were measured conventionally and luminance-response functions were fit. Leading edges of photoresponses were analyzed with a model of rod phototransduction activation. A unilateral subretinal injection of AAV2-CB(SB)-hRPE65 vector was delivered and therapeutic efficacy of 4 doses spanning a 2 log unit range was studied with ERGs performed about 6 weeks after injection. Uninjected rd12 eyes and wild-type (wt) mice served as controls.


RESULTS
Rd12 mice showed substantially smaller amplitudes and lower sensitivities than wt mice for all measured ERG b-wave and photoresponse parameters. For the dose-response study, there was no difference between 0.01X-dosed mice and untreated mutants. Improved receptoral and post-receptoral function was evident for 0.1X, 0.3X, 1X doses: b-wave semi-saturation constants decreased, b-wave amplitudes increased with dose; photoresponses showed faster kinetics and higher maximum amplitudes. ERG b-wave amplitude to a selected stimulus light intensity could provide evidence of biologic activity of the vector; interocular differences in b-wave amplitude comparing treated versus untreated eyes in the same animal also revealed vector efficacy.


CONCLUSIONS
We have taken the first steps toward developing an ERG assay of biologic activity of human grade vector for future clinical trials of RPE65-LCA. Faithful murine models of treatable human disease tested with specific ERG protocols may emerge as valuable in vivo bioassays for future human clinical trials of therapy in many retinal degenerative diseases.
Roman
Alejandro J
AJ
Department of Ophthalmology, Scheie Eye Institute, Philadelphia, PA, USA.
Boye
Sanford L
SL
Aleman
Tomas S
TS
Pang
Ji-jing
JJ
McDowell
J Hugh
JH
Boye
Shannon E
SE
Cideciyan
Artur V
AV
Jacobson
Samuel G
SG
Hauswirth
William W
WW
D000818:Animals; D001681:Biological Assay; D001766:Blindness / Q000151:congenital / Q000628:therapy; D002352:Carrier Proteins / Q000235:genetics*; D002986:Clinical Trials as Topic; D004195:Disease Models, Animal; D004596:Electroretinography; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation; D029242:Optic Atrophy, Hereditary, Leber / Q000628:therapy; D010775:Photic Stimulation; D012162:Retinal Degeneration / Q000175:diagnosis / Q000235:genetics* / Q000503:physiopathology / Q000628:therapy; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002352:Carrier Proteins; D005136:Eye Proteins; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase