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Deniz Dalkara, Ryan R. Klimczak, Meike Visel, Natalie Hoffmann, David Schaffer, John Flannery; Developing Photoreceptor Targeted AAV Variant by Directed Evolution. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4381.
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© ARVO (1962-2015); The Authors (2016-present)
The majority of mutations causing retinal degeneration are found in photoreceptor (PR) -specific genes. Gene therapy has great potential for treating PR diseases; however, to date all vectors that are able to target these cells require subretinal (SR) administration. SR injection of AAV vector is impractical for many patients with retinal degenerations, in which random, scattered islands of surviving PRs render selection of an ideal injection site difficult. The aim of our study is to apply a directed evolutionary approach to create AAV variants capable of transducing large numbers of PRs via a safe, intravitreal injection.
Three viral libraries were employed in this study: a random mutagenesis library of the AAV2 cap gene, a shuffled library composed of chimeric AAV cap genes from serotypes 1-9, and a 7mer library composed of randomized 7mers inserted near the three-fold axis of symmetry on the AAV2 capsid. These libraries were mixed together and injected into mice eyes expressing a rhodopsin-GFP fusion in their PRs. The PRs were then sorted by flow cytometry and successful variants were chosen through multiple iterations and subjected to a further round of error-prone PCR to create a successive generation of permissive variants. After multiple generations, variants from the resulting pool were analyzed to elucidate PR permissive mutations by cloning these variants into recombinant form and packaging them with a GFP transgene to assay PR-infectivity.
Sequence data from the final round of selections showed substantial convergence, as an initial library diversity consisting of millions resulted in a handful of variants derived from the 7mer library with 60% of the clones carrying the identical 7mer sequence. One of these variants, named 7m8 was used to package the GFP gene and was tested for retinal transduction. 7m8 led to strong, pan-retinal transgene expression in the PRs.
Directed evolution is a powerful approach to manipulate the tropism of AAV vectors in the retina. The isolated variants provide us with new information on amino acid changes that allow extended diffusion in retinal tissue as well as increases in infection capacity likely resulting from improvements in binding and endocytosis. We are now testing the feasibility of a gene replacement therapy from the vitreous using our new PR permissive variant.
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