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K.P. Greenberg, K. Borges, D. Trauner, R.H. Kramer, J.G. Flannery; In vivo Engineering of Light Sensitive Neurons in the Diseased Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4750.
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© ARVO (1962-2015); The Authors (2016-present)
Inherited and acquired retinal degenerations cause blindness by several mechanisms, however all culminate in the apoptotic death of photoreceptor cells. While pharmacological, gene replacement, and neuroprotective therapies attempt to postpone cell death in patients with surviving photoreceptors, many individuals are not candidates for such "rescue" treatments. An alternative therapeutic strategy is to confer light sensitivity to normally light–insensitive retinal neurons. Descriptions of neuronal remodeling during retinal degenerations by Marc et al. show complete deafferentation of the neural retina, suggesting that amacrine and ganglion cells become isolated from excitatory synaptic inputs. Therefore, we chose retinal ganglion cells (RGCs) as the best class of retinal neuron to receive a light–sensitive ion channel to control signaling.
A synthetic photoisomerizable azobenzene–regulated K+ (SPARK) channel was delivered to RGCs of Sprague–Dawley and degenerating P23H–1 rat retinas by an adeno–associated viral (AAV) vector. The AAV2 vector, containing a human synapsin (hSYN) promoter driving a modified Shaker K+ ion channel fused to eGFP was injected intravitreally, followed by injection of a photoisomerizable azobenzene molecule (MAL–AZO–QA). Electroretinogram (ERG) and patch clamp recordings were used to evaluate MAL–AZO–QA toxicity and light sensitivity of RGCs, respectively.
The AAV2–hSYN vector delivered the Shaker–eGFP transgene to RGCs and amacrine cells with high efficiency, allowing identification of transduced neurons by fundus imaging and confocal microscopy. ERGs indicate that MAL–AZO–QA is non–toxic to the retina at doses required for effective photo–switching. Patch clamp recording from Shaker–eGFP positive hippocampal cultures reveals these neurons can be photo–switched by 390nm and 500nm light. We are currently measuring the photo–switching characteristics of Shaker–eGFP positive RGCs following AAV vector injection.
We demonstrate that neuronal firing and silencing of light–gated neurons is rapidly and reversibly modulated by application of light. While a small subset of naturally occurring RGCs are intrinsically light–sensitive, they do not contribute to image formation. Imparting direct light–sensitivity on ordinary RGCs may be a future therapy for those that have suffered substantial photoreceptor loss, potentially allowing light perception in the absence of rod and cone–mediated vision.
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