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Renee C. Ryals, Sanford L. Boye, William W. Hauswirth, Shannon E. Boye; Quantifying Transduction Efficiencies Of Unmodified And Tyrosine Capsid Mutant AAV Vectors In A Cone Photoreceptor Cell Line. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5474.
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© ARVO (1962-2015); The Authors (2016-present)
With the increasing number of retinal gene-based therapies and therapeutic constructs, in vitro bioassays characterizing vector transduction efficiency and quality become increasingly important. Currently, in vitro assays quantifying vector transduction efficiency are performed predominantly in non-ocular tissues. A mouse cone photoreceptor cell line, 661W, has been well characterized and is used for many retinal metabolism and biological pathway studies. The purpose of this study is to quantify transduction efficiencies of a variety of self complimentary (sc) AAV vectors in this biologically relevant ocular cell line by high-throughput FACS analysis.
661W cells were infected with sc-smCBA-mCherry packaged in unmodified AAV capsids or capsids containing single/multiple tyrosine-phenylalanine (Y-F) mutations at MOIs ranging from 100 to 10,000. 3 days post-infection fluorescent images verified mCherry expression. Following microscopy, FACS analysis was performed to quantify the number of positive cells and the mean intensity of mCherry fluorescence, the product of which is reported as transduction efficiency for each vector. scAAV vectors containing cone-specific (sc-mCARpro-GFP), rod-specific (sc-MOPS500-eGFP), RPE-specific (sc-VMD2-GFP) or ubiquitous(sc-smCBA-GFP) promoters were used to infect the cell line at an MOI of 10,000. 3 days post infection cells were immunostained with an antibody raised against GFP and imaged.
Expression from unmodified scAAV1, scAAV2, scAAV5 and scAAV8 vectors was detectable by FACS in 661W cells with scAAV1 and scAAV2 being the most efficient. scAAV5 and scAAV8 showed moderate efficiency while scAAV9 performed poorly. With some exceptions, the Y-F capsid mutations generally increased the efficiency of scAAV vector transduction, with increasing number of mutated residues improving efficiency. Results for single scAAV1 and scAAV8 capsid mutants were mixed. In some cases efficiency improved whereas in others it was unchanged or marginally reduced. Retinal specific promoters were also active and showed a pattern consistent with the in-vivo activity of the respective promoters tested.
Our results suggest that this rapid and quantifiable cell-based assay using a biologically relevant ocular cell line will prove useful in optimizing AAV vector constructs and qualifying clinical-grade vector for the treatment of retinal disease.
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