1. ¹Cornell University, Ithaca, NY 14853, USA
2. ²University of Pennsylvania, Philadelphia, PA 19104, USA
3. ³University of Washington, Seattle, WA 98195, USA
4. ⁴University of Florida, Gainesville, FL 32611, USA

Correspondence: Gregory M. Acland, Baker Institute, Cornell University, Hungerford Hill Road, Ithaca, NY 14853, USA. Fax: +1 (607) 256 5608. E-mail: gma2@cornell.edu.

Received 27 April 2005; Revised 1 August 2005; Accepted 19 August 2005.

Abstract

The short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no detectable 11-cis-retinal, but markedly elevated retinyl esters. Subretinal AAV-RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Subretinal AAV-RPE65 vector is well tolerated and does not elicit high antibody levels to the vector or the protein in ocular fluids or serum. In long-term studies, wild-type cDNA is expressed only in target cells. Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations.