Abstract
Purpose :
Virus-mediated gene transfer may be a promising approach for treating disorders associated with loss of function and/or viability of the cone photoreceptors including macular dystrophies and congenital stationary disorders such as achromatopsia, blue cone monochromacy, and protan and deutan defects. It seems desirable to restrict expression of the therapeutic genes to the cones. Also, there are advantages to an intravitreal mode of administration as opposed to treating with subretinal injections. A human long (L) / middle (M) wavelength cone-specific regulatory cassette called pR2.1, has been used to successfully transduce cones in several animal models, however, GFP expression in primate cones under the control of pR2.1 following intravitreal injection seems less than optimal. Thus, we have designed a regulatory cassette with a modified promoter, 5'UTR, intron, and polyadenylation sequences in an attempt to better optimize levels of cone-specific expression.
Methods :
Green florescent protein (GFP) transgene expression was evaluated following a single intravitreal injection of the 7m8 capsid of adeno-associated virus (AAV) in mice that either received pR2.1 or the modified regulatory cassette. GFP fluorescence was evaluated in flat mounted transduced mouse retina by wide-field and confocal microcopy in which cones were immunolabeled with L/M opsin-specific antibodies. The intensity of GFP fluorescence and the number of detectable GFP positive cones in the mouse retina were compared for the two promoters at a series of time points starting at 1-week post injection. GFP fluorescence intensity over time in the living eye was also followed using the fluorescein angiography mode of the Micron II fundus camera.
Results :
Compared to pR2.1, GFP expression directed by optimized regulatory cassette was visible at earlier time points, was of higher intensity and occurred in more cones. The difference was larger when the vector was delivered by an intravitreal injection than subretinally.
Conclusions :
Optimization of an L/M cone specific regulatory cassette may facilitate the development of therapeutic agents for treating cone-associated disorders by intravitreal injection.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.