Abstract
Abstract: :
Purpose:Gene therapy restored useful vision in dogs with hereditary congenital retinal dystrophy, a disease comparable to human Leber's congenital amaurosis, caused by a 4-base pair deletion in the RPE65 gene. We did retinal electrophysiology study pre and postop in order to objectively evaluate the functional effects of gene transfer treatment. Methods:Ten dogs were included in which 6 homozygous affected animals (4-11 mo old) were divided into three age groups, and injected subretinally into one eye with 30-100 µl of rAAV.RPE65 with a titer of 6x1013 particles/µl. Same volume of rAAV.GFP was injected into the contralateral eye in 4 of the dogs. Three normal heterozygous and one dog with a normal genotype were used as controls. One of the carrier dogs and the normal dog underwent sham surgery in one eye and rAAV.GFP injection into the other. Bilateral Ganzfeld dark- and light-adapted single flash electroretinograms (ERGs) and light-adapted flicker recordings were performed before and at different time points after gene transfer, from 4 w up to 6 mo postop. Results:Before subretinal injection, dark-adapted ERG waveforms were not recordable in 6 affected dogs. However, small amplitude light-adapted responses, including single flash b-wave (1 dog), 50 Hz flicker responses (3 dogs) and 30 Hz flickers responses (5 dogs) were recorded. Follow-up ERGs showed that in all cases there were increased 30Hz flicker amplitudes in rAAV.RPE65 treated eyes. Light adapted a- and b-wave and 50Hz flicker recordings were obtained in 5 dogs. In dark-adapted ERGs, 5 dogs showed increased a-wave, and 4 had increased b-wave amplitudes. The functional effects of the gene therapy increased or sustained in 5 dogs during a 3 mo follow-up and for 6 mo in one dog. Multiple regression analysis showed that 30Hz flicker amplitude obtained were related to those found preop but neither with the different dosages used nor the ages studied at the time of treatment. Conclusion:The ERG results provide evidence that gene therapy can restore limited retinal function in the RPE65 null mutation dog up to at least 6 months. The light elicited circuit generated from the photoreceptors and 2nd order neurons showed definite functional improvement. With our methods, the recording of photopic 30 Hz flicker responses is the most sensitive test in detecting residual retinal function in this disorder and in monitoring the therapeutic effects of gene therapy.
Keywords: 419 gene transfer/gene therapy • 395 electroretinography: clinical • 316 animal model