Quantifying transduction efficiencies of unmodified and tyrosine capsid mutant AAV vectors in vitro using two ocular cell lines

• Published in: Molecular vision 2011-04, Vol.17, p.1090-1102
• Main Authors: Ryals, Renee C, Boye, Sanford L, Dinculescu, Astra, Hauswirth, William W, Boye, Shannon E
• Format: Article
• Language: English
• Subjects: Adeno-associated virus; Animals; Antibodies; Capsid - chemistry; Capsid - metabolism; Cell Line; Dependovirus - genetics; Dependovirus - metabolism; Epithelial Cells - cytology; Epithelial Cells - metabolism; Flow Cytometry; Gene Transfer Techniques; Genetic Therapy - methods; Genetic Vectors - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; High-Throughput Screening Assays; Humans; Mice; Phenylalanine - genetics; Phenylalanine - metabolism; Photoreceptor Cells - cytology; Photoreceptor Cells - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Retina - cytology; Retina - metabolism; Transformation, Genetic; Transgenes; Tyrosine - genetics; Tyrosine - metabolism; Vision, Ocular
• ISSN: 1090-0535; 1090-0535

With the increasing number of retinal gene-based therapies and therapeutic constructs, in vitro bioassays characterizing vector transduction efficiency and quality are becoming increasingly important. Currently, in vitro assays quantifying vector transduction efficiency are performed predominantly for non-ocular tissues. A human retinal pigment epithelial cell line (ARPE19) and a mouse cone photoreceptor cell line, 661W, have been well characterized and are used for many retinal metabolism and biologic pathway studies. The purpose of this study is to quantify transduction efficiencies of a variety of self-complementary (sc) adeno-associated virus (AAV) vectors in these biologically relevant ocular cell lines using high-throughput fluorescence-activated cell sorting (FACS) analysis. ARPE19 and 661W cells were infected with sc-smCBA-mCherry packaged in unmodified AAV capsids or capsids containing single/multiple tyrosine-phenylalanine (Y-F) mutations at multiplicity of infections (MOIs) ranging from 100 to 10,000. Three 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. The scAAV vectors containing cone-specific (sc-mCARpro-green fluorescent protein [GFP]), rod-specific (sc-MOPS500-eGFP), retinal pigment epithelium (RPE)-specific (sc-VMD2-GFP), or ubiquitous (sc-smCBA-GFP) promoters were used to infect both cell lines at an MOI of 10,000. Three days post infection, cells were immunostained with an antibody raised against GFP and imaged. Finally, based on our in vitro results, we tested a prediction of transduction efficiency in vivo. Expression from unmodified scAAV1, scAAV2, scAAV5, and scAAV8 vectors was detectable by FACS in both ARPE19 and 661W cells, with scAAV1 and scAAV2 being the most efficient in both cell lines. scAAV5 showed moderate efficiency in both ARPE19 and 661W cells. scAAV8 was moderately efficient in 661W cells and was by comparison less so in ARPE19 cells; however, transduction was still apparent. scAAV9 performed poorly in both cell types. With some exceptions, the Y-F capsid mutations generally increased the efficiency of scAAV vector transduction, with the increasing number of mutated residues improving efficiency. Results for single scAAV1 and scAAV8 capsid mutants were mixed. In some cases, efficiency im