Reply to Townes-Anderson: RPE65 gene therapy does not alter the natural history of retinal degeneration.

We appreciate the interest shown by TownesAnderson (1) in our article examining the natural history of retinal degeneration in Leber congenital amaurosis caused by retinal pigment epithelium-specific protein 65kDa (RPE65) mutations and evaluating the consequences of gene augmentation therapy (2). Townes-Anderson’s remarks focused on the final phrase of the last sentence of the Discussion of our article. In the full sentence, we suggested that in the future, agents to reduce cell death could be delivered in combination with a more advanced version of the gene augmentation therapy that reaches not only remaining rods and extrafoveal cones but also foveal cone photoreceptors. Towne-Anderson made the constructive point that consideration should be given in future versions of human RPE65 gene therapy to a specific rod photoreceptor celldeath pathway (3). We are familiar with the Townes-Anderson hypothesis involving G-protein activation by mislocalized opsin, and we have recently cited this hypothesis as potentially relevant for our work demonstrating successful arrest of retinal degeneration by gene augmentation therapy in the RPGR form of X-linked retinitis pigmentosa (4). However, disease mechanisms in the retinoid deficiency caused by RPE65 mutations and in the ciliopathy caused by RPGR mutations are likely to be different. It is important to note that chromophore in RPE65-mutant retinas is nearly undetectable and thus the majority of mislocalized opsin molecules are not likely to be bound to chromophore. The protection from bright light in Rpe65-deficient mice—as opposed to exacerbation of degeneration—argues against a primary signaling pathway involving photoactivation of mislocalized isorhodopsin or rhodopsin. Alternatively, it could be argued that mislocalized free opsin is involved. Indeed, there is evidence that constitutive activation of G protein by free opsin contributes to cell death in Rpe65-deficiency, but whether signaling originates from free opsin correctly localized to rod outer segments or mislocalized to the inner segments remains unknown. In patients who received gene augmentation therapy, there is a likely increase in 11cis-retinal chromophore availability to rods and extrafoveal cones (5). According to the Townes-Anderson hypothesis, the increase in chromophore produced by gene therapy together with ambient light should have resulted in activation of mislocalized rhodopsin (1). Such signaling would be predicted to accelerate the rate of cell loss beyond that measured in the natural history of disease. However, the rate of degeneration within the retinal regions that received gene therapy was not faster than the natural history (2), and thus did not support the TownesAnderson hypothesis. In summary, we find no specific support in our data for the hypothesis advanced by Townes-Anderson; however, we wholeheartedly agree with the sentiment that we should proceed cautiously to improve the outcomes of retinal gene augmentation therapy including RPE65 gene therapy. Artur V. Cideciyan, Samuel G. Jacobson, William A. Beltran, William W. Hauswirth, and Gustavo D. Aguirre Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104; Department of Clinical Studies, Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; and Department of Ophthalmology, University of Florida, Gainesville, FL 32610