An AAV vector carrying a chimeric photopigment for evaluating gene therapy methods in Old World primates

Abstract

Purpose : Treating cone disorders by gene therapy via intravitreal injection is challenging because the inner limiting membrane and retinal thickness limit accessibility of photoreceptors to injected agents. Overcoming this requires pre-clinical testing in non-human primates. Blue cone monochromacy is a cone-based disorder with a variety of underlying mutations, a subset of which is likely amenable to gene therapy, however a suitable primate model does not exist. Thus, our goal is to develop a vector optimized for driving robust expression of a photopigment with an absorption spectrum that allows functional detection via ERG and behavioral testing in retinas expressing endogenous primate cone photopigments.

Methods : The vector contains an optimized expression cassette designed to target expression of a passenger opsin gene to primate cones packaged in the AAV-7m8 capsid for an intravitreal route of administration. The opsin gene comprises partial sequence of middle wavelength opsin cDNA of the Mongolian gerbil (Meriones unguiculatus) flanked by partial exon 1 and exon 6 sequences of the human OPN1LW gene. The chimeric photopigment is expected to peak ~493 nm, about midway between the absorption maxima of the macaque monkey S and M photopigments (see Figure). Virus was injected into the eyes of mice that had both M and S opsin genes knocked out. Expression of the transgene was evaluated using photopic ERG. Spectral sensitivity was characterized using flicker photometric ERG.

Results : Photopic ERG responses in the cone opsin knockout mice were rescued by gene therapy treatment. Treated mice gave robust photopic ERG responses compared to knockout controls which gave no detectible cone responses. The characteristics of the ERG waveform were comparable to wild-type mice. ERG flicker photometric spectral sensitivity functions were well fit to a single photopigment template with a spectral peak of 493 nm.

Conclusions : We have developed a gene therapy vector designed to be administered intravitreally expressing a photopigment that is well separated in spectral sensitivity from any of the wild-type cone pigments in Old World primates. It should be possible to isolate the responses from this photopigment if it is expressed in macaque cones using silent substitution in ERG and behavioral experiments to evaluate gene therapy success.

This is a 2021 ARVO Annual Meeting abstract.