Abstract
Purpose: :
To describe a light sensitivity associated with expression of a T17M mutant human rhodopsin (hT17M) transgene in mice, with the goal of improving our ability to deliver rAAV–mediated retinal gene therapy.
Methods: :
We bred mice to express the hT17M rhodopsin transgene in a line that was hemizygous null for mouse rhodopsin (mrho+/–). We performed electroretinographic analysis (ERG) at 2 week intervals to follow the course of retinal degeneration. At the end of the course, histology was used to measure thickness of the outer nuclear layer (ONL). Light sensitivity of these mice was studied following short term (2.5 minute) illumination with white light at 5,000 or 10,000 lux or red light (≥ 600 nm) at 5,000 lux. The effect of fundus photography was also investigated. Impact of illumination was measured using ERG analysis 1 and 2 months following illumination and TUNEL analysis of retinal sections made 24 hours after illumination.
Results: :
Unsuccessful attempts to treat the hT17M; mrho+/– mice with subretinal injections of rAAV–expressed ribozymes led to the discovery of an hT17M–specific light sensitivity that caused attenuation of the a– and b–wave ERG amplitudes. Fundus photography also caused retinal damage in these mice. Histological analysis showed a concomitant loss of photoreceptors, and TUNEL labeling of fragmented DNA in rod photoreceptor cells demonstrated that the damage was occurring via an apoptotic pathway. Non–transgenic littermate mice were not affected by these procedures. Mice expressing a P23H mutant human rhodopsin transgene were also not sensitive. Illumination with wavelengths greater than 600nm prevented the light–induced retinal damage, allowing for non–damaging subretinal injections of the hT17M mice.
Conclusions: :
We have described an extreme light sensitivity associated with expression of the hT17M mutant rhodopsin transgene in mice. This phenotype resembles that recently reported for the T4R rhodopsin dog. Light damage was not observed in either non–transgenic littermates or mice expressing a P23H mutant human rhodopsin transgene, leading us to conclude that this type of severe light sensitivity is not common to all rhodopsin mutations. Our results suggest that human patients suffering from some types of ADRP should avoid intense light exposure.
Keywords: retinal degenerations: hereditary • apoptosis/cell death • transgenics/knock-outs