Design and testing of AAV vectors for Sod2 gene augmentation therapy

Abstract

 

Purpose

 

Retinal cell death due to oxidative stress is a contributing factor for development of age Related Macular degeneration (AMD) which causes vision loss among millions of people in industrialized nations. We established a 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 of the features of geographic atrophy. It is the purpose of these experiments to determine whether 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. Retinal degeneration was monitored by electroretinography (ERG) and spectral domain optical coherence tomography over a period of 9 months. Mouse Sod2 with a Myc epitope under the control of a small chicken beta- actin promoter (smCBA) or a mouse VMD2 promoter was packaged into self-complementary AAV serotype 1 vector (ScAAV1). Ten C57BL6/J adult mice were injected subretinally either with ScAAV1-smCBA-Sod2-Myc or ScAAV1-VMD2-Sod2-Myc into right eye. 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.

 

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 (by SD-OCT) which were statistically significant by 6 months. Myc tagged MnSOD expression was detected in RPE of mice injected with vector driven by smCBA promoter and negligible expression was seen in the neural retina. However, the eyes injected with Myc tagged MnSOD driven by VMD2 promoter did not detect expression in RPE. ERG and OCT data suggests no adverse functional and structural integrity due to increased expression of MnSOD.

 

Conclusions

 

Deletion of Sod2 in the RPE leads to some of the salient features of dry AMD. ScAAV1 delivery of Sod2 led to expression in RPE. Delivery of ScAAV1-smCBA-SOD2-Myc vector can be used as an tool to reverse oxidative stress in this mouse model of dry AMD.

 

Keywords: 538 gene transfer/gene therapy • 701 retinal pigment epithelium • 412 age-related macular degeneration