Restoring visual function in blind mice and dogs with an improved 2nd generation azobenzene based photoswitch

Abstract

Purpose: Inherited eye diseases lead to blindness by progressive loss of photoreceptor cells, but can leave many retinal interneurons intact and functional. Recently, we established that virus-mediated expression of a chemically and genetically engineered light-gated ion channel LiGLuR(1) in retinal ganglion cells (RGC) can restore visual function in rd mice in-vitro and in-vivo (Caporale et al. Mol Ther. 2011 Jul;19(7):1212-9). The application of this 1st generation photoswitch system as a therapeutic is limited by two properties: the spectral sensitivity of the original LiGLuR(1) chromophore is out of the visible range, peaking at 380nm and the system is bistable - requiring a second pulse of 500nm light to push the chromophore moiety back to inactivated (relaxed) state.

Methods: -

Results: To improve upon these initial results, we designed a second generation LiGluR(2) system using a red-shifted azobenzene photoswitch that can be activated with white light, relaxes spontaneously in the dark and displays fast kinetics. We expressed LiGluR(2) in RGCs and in ON-bipolar cells of the degenerating rd1 mouse retina and showed that both RGCs and ON-bipolar cells can be engineered to become light receptive and drive robust light responses in-vitro. We found that LiGluR(2) expression in RGCs leads to a robust and synchronized response to light whereas expression in ON bipolar cells leads to a more diverse output. Downstream of the retina, we were able to detect visually evoked potentials (VEP) in the visual cortex with amplitudes approaching wild type levels. We tested blind rd1 mice treated with LiGluR(2) for visually guided behavior. They showed innate light avoidance in the open field test in-vivo and learned to associate different patterns of light with reward using the morris water maze task. In a translational effort, we also expressed LiGluR(2) in a dog model of retinitis pigmentosa. We were able to record light responses from retinal explants using the multielectrode array in-vitro and showed safety and tolerance of intravitreal delivery of >10x the minimal therapeutic dose of azobenzene chromophore in-vivo.

Conclusions: In this study we show that our improved second generation LiGluR photoswitch system can restore visual responses to degenerating mouse and dog retina in-vitro, enables innate and learned visually guided behavior in mice in-vivo and is safe and well tolerated in dogs.

Keywords: 538 gene transfer/gene therapy • 608 nanomedicine • 702 retinitis