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

Abstract

Purpose: 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.

Methods: 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.

Results: 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 improved; in others, it was unchanged or marginally reduced. Retinal-specific promoters were also active in both cell lines, with the 661W cells showing a pattern consistent with the in vivo activity of the respective promoters tested. The prediction based on in vitro data that AAV2 sextuple Y-F mutants would show higher transduction efficiency in RPE relative to AAV2 triple Y-F capsid mutants was validated by evaluating the transduction characteristics of the two mutant vectors in mouse retina.

Conclusions: Our results suggest that this rapid and quantifiable cell-based assay using two biologically relevant ocular cell lines will prove useful in screening and optimizing AAV vectors for application in retina-targeted gene therapies.

Figure 1

Transduction efficiency of unmodified scAAV vectors in ARPE19 and 661W cells. Cells were infected with scAAV1, 2, 5, 8, and 9 at a multiplicity of infection (MOI) of 10,000. mCherry expression is shown in arbitrary units calculated by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. Each value represents the average of three samples (eight pooled wells of a 24-well plate/sample), based on 10,000 counted cells.

Figure 2

Transduction efficiency of unmodified scAAV2 and its single/multiple Y-F capsid mutant vectors. Cells were infected with unmodified scAAV2, scAAV2 single mutant (Y444F), double mutant (Y444F+Y730F), triple mutant (Y444F+Y730F+Y500F), quadruple mutant (Y444F+Y730F+Y500F+Y272F), pentuple mutant (Y444F+Y730F+Y500F+Y272F+Y704F), and sextuple mutant (Y444F+Y730F+Y500F+Y272F+Y704F+Y252F) at a multiplicity of infection (MOI) of 10,000. mCherry expression is shown in arbitrary units calculated by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. Each value represents the average of three samples (eight pooled wells of a 24-well plate/sample), based on 10,000 counted cells.

Figure 3

In vivo transduction pattern of AAV2 triple and sextuple Y-F capsid mutants. Green fluorescent protein (GFP) expression in retinal sections of adult mice at one month following subretinal delivery of (A) AAV2 triple mutant (Y444F+Y730F+Y500F) or (B) sextuple mutant (Y444F+Y730F+Y500F+Y272F+Y704F+Y252F) and (C) a comparison of the relative GFP fluorescence intensity in the retinal pigment epithelium (RPE) layer of retinas treated with the triple and sextuple AAV2 vectors, respectively. Values indicate percent GFP intensity relative to treatment with AAV2 triple mutant vector. All pictures were evaluated using Image J software. The data are shown as the mean±SEM; n=3 for each group (p<0.0001). PR represents photoreceptor layer.

Figure 4

scAAV-mediated mCherry expression in human retinal pigment epithelial (ARPE19) and 661W cone photoreceptor cells, at three days post infection. The top row consists of representative images of mCherry expression in ARPE19 cells infected with (A) scAAV1, (B) scAAV2, and (C) scAAV2 sextuple mutants at a multiplicity of infection (MOI) of 10,000. The bottom row consists of representative images of mCherry expression in 661W cells infected with (D) scAAV8, (E) scAAV2, and (F) scAAV2 sextuple mutants at an MOI of 10,000. All 10× images were taken with identical exposure times (800 ms). The scale bar in A=200 µm.

Figure 5

Transduction efficiency of unmodified scAAV1, scAAV8, and their respective single Y-F capsid mutant vectors. Transduction efficiency of scAAV1 and scAAV1 Y731F (A), scAAV8, scAAV8 Y733F, and scAAV8 Y447F (B) in ARPE19 and 661W cells measured by mCherry-mediated and fluorescence-activated cell sorting (FACS) analysis. Data represents infections at a multiplicity of infection (MOI) of 10,000. mCherry expression is shown in arbitrary units calculated by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. Each value represents the average of three samples (eight pooled wells of a 24-well plate/sample), based on 10,000 counted cells.

Figure 6

Human retinal pigment epithelial cell line (ARPE19) cells remain viable upon lengthy post infection incubation. mCherry expression in ARPE19 cells infected with scAAV2-smCBA-mCherry at an MOI of 10,000 (A) 3 days, (B) 7 days, and (C) 14 days post infection. All 10× images were taken at identical exposure settings (800 ms). The scale bar in A=200 µm.

Figure 7

Retina-specific promoters are capable of transducing both human retinal pigment epithelial cell line (ARPE19) and 661W cells. Representative images of green fluorescent protein (GFP) expression in ARPE19 cells at 3 days post infection with AAV2 (A) sc-mCARpro-GFP, (B) sc-MOPS500-eGFP, (C) sc-VMD2-GFP, and (D) sc-smCBA-GFP. Representative images of GFP expression in 661W cells at 3 days post infection with AAV2 (E) sc-mCARpro-GFP, (F) sc-MOPS500-eGFP, (G) sc-VMD2-GFP, and (H) sc-smCBA-GFP. All infections were done at a multiplicity of infection (MOI) of 10,000. 10× images A-C and E-G were taken at an ET of 800 ms, whereas images D and H were taken at an ET of 300 ms. The scale bar in A=200 µm.