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Sanford L Boye, Shreyasi Choudhury, Damien Marsic, Christianne E Strang, John J Alexander, C. Douglas Witherspoon, Sergei Zolotukhin, Paul D Gamlin, Shannon Elizabeth Boye; Directed Evolution of Enhanced AAV Capsid Variants Following Intravitreal Injection in Macaque. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4087.
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© ARVO (1962-2015); The Authors (2016-present)
Adeno-Associated virus (AAV)- based vectors are the gold standard for retinal gene therapy. As most inherited retinal diseases are caused by photoreceptor (PR) defects, vectors that efficiently target outer retina are required. Currently, this is achieved by subretinal injection, an invasive procedure that poses risks. Efforts made to identify AAV capsids capable of outer retinal transduction following intravitreal (Ivt) injection have relied on screening in mouse. Resulting capsids are improved, but fail to transduce outer primate retina following Ivt injection to levels that warrant widespread clinical use. This is due to formidable barriers to Ivt- mediated transduction present in the primate retina, chiefly the inner limiting membrane (ILM). The purpose of our study was to identify novel AAVs that can overcome these barriers by screening capsid library in macaque (Macacca fascicularis), a species with ocular characteristics most similar to human.
Macaque PRs and retinal ganglion cells (RGC) were fluorescently labelled by subretinal injection of AAV5-GRK1-GFP and lateral geniculate injection of rhodamine dextran, respectively. Subsequently, a highly complex, AAV2-based capsid library was delivered by Ivt injection. Seven days post-injection, retina was harvested, dissociated and PRs/RGCs isolated by fluorescent activated cell sorting (FACS). AAV genomes were recovered and library was regenerated. Screening was also performed in Nrl-GFP mice, and rod PRs isolated by FACS. A total of three and four rounds of selection were performed in macaque and mice, respectively. Distribution of variants was determined at various stages by sequencing of individual clones or next generation sequencing (NGS).
AAV capsid variants containing motifs that promote interaction with the ILM were highly selected. Capsids enriched after 2 rounds of selection in macaque mirrored those arising from 3 rounds of selection in mouse. Analysis of transduction efficiencies of selected variants are underway.
As previously shown with rationally designed vectors, interaction with the ILM is a requirement for Ivt-mediated transduction by AAV. Distribution of selected variants between mouse and primate screens implies selective pressure imparted by the barriers to transduction are similar, but are significantly stronger in primate.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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