Efficiency of gene therapy via intravitreal injection in primate cones

Abstract

Purpose : Intravitreal delivery of gene therapy vectors to the retina carries lower risk of adverse events compared to subretinal injections, but has thus far exhibited less therapeutic potential in animal studies. We developed a new adeno-associated virus (AAV) vector for intravitreal delivery of genes to cone photoreceptors. The virus carries an expression cassette with a cDNA encoding an engineered opsin fused to the gene for green fluorescent protein (GFP) under the control of regulatory elements directing expression to long- and middle-wavelength (L/M) cone photoreceptors. The purpose of this study was to evaluate the expression pattern of the opsin-GFP fusion protein after intravitreal injection of the vector in a primate eye.

Methods : The expression cassette was packaged in the 7m8 AAV capsid and 8.5 x 10¹⁰ viral genomes in a volume of 50 microliters was injected into the vitreous of an adult macaque eye. The eye was harvested 5 weeks after the injection and retinal cryosections were labeled with antibodies against short-wavelength (S) and L/M opsin. Following confocal imaging, volumes of fovea (400 µm wide, 20 µm deep) were analyzed in TrakEM2 to count individual GFP-expressing, S, and L/M cones.

Results : GFP fluorescence was confined to the fovea. Across the fovea around 25% of cones expressed GFP, with expression highest at the center. GFP was localized primarily in the outer segments, and diffusely along the plasma membrane. While the majority of transduced cones were L/M, surprisingly a small fraction (~5%) were S cones.

Conclusions : A gene therapy vector designed for intravitreal delivery directed robust expression of an engineered opsin to foveal cones in a primate. This finding is promising for the next phase of ocular gene therapy in humans.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Confocal image of a foveal section from a primate retina 5 weeks after intravitreal injection of 7m8_AAV carrying the gene for an opsin-GFP fusion targeted to L/M cones. Transduced cones express GFP (green) and primarily L/M opsin (magenta); a small portion of transduced cones express S opsin (blue). Presented image is maximum intensity projected over a depth of 20 µm.