April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Quantifying Transduction Efficiencies Of Unmodified And Tyrosine Capsid Mutant AAV Vectors In A Cone Photoreceptor Cell Line
Author Affiliations & Notes
  • Renee C. Ryals
    Ophthalmology, University of Florida, Gainesville, Florida
  • Sanford L. Boye
    Ophthalmology, University of Florida, Gainesville, Florida
  • William W. Hauswirth
    Ophthalmology, University of Florida, Gainesville, Florida
  • Shannon E. Boye
    Ophthalmology, University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  Renee C. Ryals, None; Sanford L. Boye, None; William W. Hauswirth, W.W.H. and the University of Florida have a financial interest in the use of AAV therapies, and own equity in a company (AGTC Inc.) that might, in the future, commercialize some aspects of this work. (F); Shannon E. Boye, None
  • Footnotes
    Support  NIH grants EY13729, EY11123, EY08571
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 5474. doi:
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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)

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Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: photoreceptors • gene transfer/gene therapy • flow cytometry 

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