Abstract
Purpose::
Transgene expression targeted specifically to subclasses of retinal ganglion cells (RGCs) would be useful for a wide variety of studies, including the transfer of light-sensitive "photoswitches" to modulate neuronal activity, fluorescent physiological indicators, and reporter genes. While the human retina contains at least 18 different morphological types of ganglion cells, ON and OFF-type signal detection is the most significant subdivision among visual features extracted by RGCs. Specific transgene expression in ON or OFF-type ganglion cells should increase our understanding of signal processing and retinal circuitry. Potential therapeutic applications include restoring light sensitivity to patients with severe retinal degenerations via selective expression of light-gated "photoswitches" such as Channelrhodopsin-2 (ChR2), Synthetic Photoisomerizable Azobenzene-Regulated K+ (SPARK) channels, and Light activated Glutamate Receptors (LiGluR) in subtypes of RGCs.
Methods::
We have engineered an adeno-associated viral (AAV) vector for transgene expression in ON-type RGCs in vivo. We cloned multiple fragments of a human gap junctional protein (connexin-36) promoter and evaluated their ability to drive expression of GFP and a directly light-sensitive "photoswitch" (SPARK) in ON-type RGCs. AAV vectors were injected intravitreally in Sprague-Dawley (SD) and P23H rhodopsin mutant rats. Cell specificity of GFP reporter gene expression was evaluated by in vivo fluorescent fundus imaging and confocal microscopy.
Results::
Fluorescent fundus imaging revealed robust GFP expression in RGCs up to 2 months post injection in SD and P23H rat retinas. Confocal microscopy of flatmount and cryosectioned retinas indicated transgene expression localized to ON-type RGC bodies, axons, and dendrites. The dendrites of transduced RGCs were observed to stratify in sublamina B of the inner plexiform layer, adjacent to the ganglion cell bodies, where they make synaptic contact with axon terminals of ON-type bipolar cells signaling increments of light.
Conclusions::
Targeted transgene expression in ON-type RGCs is achievable with this novel AAV vector following a simple intravitreal injection. Directly targeting ON and OFF-type ganglion cells with light-sensitive depolarizing and hyperpolarizing ion channels may yield new insights into signal processing and potentially allow light perception and contrast discrimination in the absence of rod and cone-mediated vision.
Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • gene transfer/gene therapy • retinal connections, networks, circuitry