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John J. Alexander, Clark D. Witherspoon, William W. Hauswirth, Mark E. Clark, Kristen J. Sandefer, Sanford L. Boye, Vince A. Chiodo, Christopher A. Girkin, Paul D. Gamlin; A Novel Method for Delivery of Recombinant Adeno-associated Virus to the Inner Retina of Non-human Primates. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1398.
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AAV vectors have emerged as a promising therapeutic agent for outer retinal disease. However, in many retinal diseases, e.g. retinitis pigmentosa, cone-rod dystrophies, macular degeneration, most, if not all, outer retinal cells degenerate. In contrast, retinal ganglion cells (RGC) can be relatively preserved. Therefore, therapies for the recovery of vision in these diseases could utilize RGCs as a therapeutic target. Potentially, direct activation of RGCs could be used to bypass the damaged photoreceptors and produce visual sensations. However, transduction of the non-human primate (NHP) inner retina with AAV has thus far been problematic, presumably due to the inner limiting membrane (ILM). To that end, we have developed a method to directly deliver AAV to the RGC layer in the NHP by creating a sub-inner limiting membrane (sub-ILM) reservoir.
Male NHPs (Macaca nemestrina) were used in this study. All surgical procedures were preformed with sterile technique using an Alcon Accurus 800CS vitrector and Zeiss VISU 200 microscope. Access to the inner retina was accomplished using a standard 23ga. three-port pars plana vitrectomy. The sub-ILM space was cannulated with a modified needle. AAV (serotype 2) expressing GFP under the control of the CBA promoter (mixed with viscoelastic) was delivered to the sub-ILM space creating a defined reservoir. An intravitreal control injection was also performed. OCT analysis and in vivo expression of GFP was monitored using a Heidelberg Spectralis.
In vivo GFP expression was detected as early as two weeks after injection using the sub-ILM approach, with labeling in the RGC layer and their corresponding axonal tracts. The intravitreal control injection only displayed a foveal ring of labeled RGCs. In vivo GFP expression has remained stable for the duration of the study (~1 year).
We have successfully developed a novel method to deliver an AAV vector to the inner retina. We believe this method can be employed to overcome the physical barrier to inner retinal AAV delivery. This opens the possibility of delivering AAV expressed light activated, or other therapeutic, proteins directly to the RGCs in cases of retinal disease. Furthermore, it may be possible to deliver new AAV serotypes/capsid mutants with this approach to the outer retina in cases where a sub-retinal detachment is contraindicated.
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