Gene Therapy for the abcr–/– Mouse–model of Stargardt’s Macular Degeneration Using Recombinant Adeno– and Adeno–associated Viral Vectors

Abstract

Abstract: : Purpose: Mutations in the human ABCA4 (ABCR) gene are responsible for several inherited retinal dystrophies including Stargardt’s macular degeneration. Similar to Stargardt’s patients, mice with a knockout mutation in the abcr gene accumulate toxic lipofuscin pigments in cells of the retinal pigment epithelium. In this study we attempted to rescue the abcr^–/– phenotype in mice by viral mediated gene therapy using recombinant adenoviral and adeno–associated viral (AAV) vectors. Methods: We assembled two DNA constructs, each containing 800 bp of upstream regulatory sequences from the mouse rhodopsin gene, followed by the 7–kb coding region of mouse ABCR, and a fragment containing the bovine growth–hormone polyadenylation signal. We cloned this ‘RhoPro–ABCR’ cassette into an adenoviral vector (serotype 5) with the E1 and E3 regions deleted. To overcome the 5–kb packaging limitation of AAV vectors, we used a dual–vector approach. We divided the RhoPro–ABCR cassette into two halves and cloned each independently into AAV vectors. These vectors contained the inverted terminal repeats (ITR’s) from AAV downstream or upstream, respectively, of the 5'– and 3'–transgene halves. These ITR’s have been shown to induce homologous recombination in vivo. After splicing the resulting synthetic intron, the mRNA from this recombined gene encodes full–length ABCR. Retinal explants from 3– and 21–day–old abcr^–/– mice were isolated and infected with the adenoviral or AAV vector. Following incubation in culture for 10 days, levels of the ABCR mRNA were measured by quantitative RT–PCR. Results: Levels of the ABCR mRNA in recombinant adenovirus–infected abcr^–/– explants were similar to the levels in explants from uninfected wild–type mice. Immunoblotting showed expression of the ABCR protein in adenovirus–infected abcr^–/– explants. The ABCR mRNA was also expressed and appropriately spliced in AAV–infected abcr^–/– explants. However, the mRNA level here was several–fold lower than in uninfected wild–type explants. Conclusions: These results demonstrate the feasibility of viral gene therapy for the abcr^–/– mouse–model of recessive Stargardt’s disease. We have begun to administer by subretinal injection recombinant adeno– and adeno–associated viral vectors to abcr^–/– mice. These animals will be analyzed for ABCR expression and rescue of the abcr^–/– phenotype.

Keywords: gene transfer/gene therapy • retinoids/retinoid binding proteins • photoreceptors