Analysis of a primate cone mosaic following targeted gene transfer using recombinant adeno-associated virus vectors

Abstract

We have successfully used gene therapy to add red-green color vision, as assessed in behavioral tests, in dichromatic squirrel monkeys that had only short- (S) and middle- (M) wavelength-sensitive cones. Expression of a human long- (L) wavelength sensitive opsin transgene was targeted to the primate M-cones through subretinal injections of a recombinant adeno-associated virus vector containing the L/M opsin enhancer and promoter. By coexpressing the L-opsin transgene in a subset of the endogenous M-cones, the treatment shifted the spectral sensitivity of the transduced M-cones to respond to long wavelength light, thus producing a retina with two distinct cone types absorbing in the middle-to-long wavelengths, as required for trichromacy. A remaining question is how well does the cone mosaic produced by gene therapy mimic the normal human L/M cone mosaic? To answer this question, a squirrel monkey was injected with a 2:1 mixture of L-opsin-coding virus and an identical virus containing a green fluorescent protein (GFP) gene. Color multifocal electroretinogram analysis under conditions that strongly favor responses from the introduced L-opsin showed elevated amplitudes in discrete areas that corresponded to locations where subretinal injections were placed, as confirmed by in vivo fluorescence imaging of GFP transgene expression. Confocal microscopy, performed approximately 2 years and 3 months post-injection, revealed a mosaic pattern of GFP transgene expression in 5–12% of the cones in treated areas. Because the GFP-coding virus was diluted to 1/3 compared to the opsin-coding virus used in the behaviorally tested monkeys, we estimate that 15–36% of cones in those animals are transduced.

Footnotes

 Supported by the National Institutes of Health grants R03EY014056, R01EY016861, R01EY11123, Research Training Program in Vision Science Grant T32EY014537, NEI Core Grant for Vision Research EY01981, the Harry J. Heeb Foundation, and Research to Prevent Blindness.