Delaying Retinal Degeneration in a Mouse Model of Geographic Atrophy: An Antioxidant Gene Therapy Approach.

Abstract

 

Purpose

 

We established a mouse model of RPE(retinal pigment epithelium) oxidative stress by Cre-lox mediated deletion of the Sod2 gene, that codes for the protective enzyme manganese superoxide dismutase (MnSOD), leading to some features of geographic atrophy. These experiments test whether early delivery of Sod2 using adeno-associated virus (AAV) can prevent retinal degeneration seen in these mice and whether gene therapy can prevent degeneration once it has begun.

 

Methods

 

Deletion of Sod2 was induced by doxycycline treatment of mice with a “floxed” allele of Sod2 and an RPE-specific tet-transactivator controlling expression of Cre (Sod2-cre mice). Retinal degeneration was monitored by ERG and SD-OCT over a period of 9 months. Mouse Sod2 with a Myc epitope under the control of a chicken beta- actin promoter was packaged into self-complementary AAV serotype 1 vector (ScAAV1). Sod2-creadult mice were injected subretinally with ScAAV1-smCBA-Sod2-Myc or ScAAV1-smCBA-GFP. Three mice were sacrificed a month following injection to collect retina and RPE separately. Using an anti-myc antibody, western blotting was performed to detect MnSOD expression. For rest of the mice, ERG and SD-OCT were measured at different time points.

 

Results

 

Following doxycycline induction of Cre,mice demonstrated increased signs of oxidative stress in RPE and accumulation of autofluorescent material by 2 months of age. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer which were statistically significant by 6 months. Myc tagged MnSOD expression was detected in RPE of mice injected with vector and negligible expression was seen in the neural retina. ERG and OCT data suggested no adverse effects due to increased expression of MnSOD and sub retinal injection.ERG response and thinning retinal thickness was significantly delayed in Sod2-vector injected eyes compared to control eye injected with GFP vector.

 

Conclusions

 

Delivery of ScAAV1-Sod2 vector can be used as a tool to reverse oxidative stress in this mouse model of dry AMD. This result suggests that delivery of genes for antioxidant enzymes may be a useful therapeutic approach in this model and may provide a preventative approach for dry AMD.