Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1-9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

Abstract

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Figure 1

Schematic of rAAV tropism testing for in vitro and ex vivo-isolated cells. Human in vitro cellular models (including induced pluripotent stem cells (iPSC), iPSC-derived retinal pigment epithelium (iPSC-RPE), and iPSC-derived human cortical neurons) and ex vivo-isolated rat cortical neurons were generated and cultured. Before transduction testing, cells were collected and seeded at equal numbers in each well of two Matrigel-coated 96-well plates. Eleven AAV serotypes at different multiplicity of infection (MOI) were introduced to the cells in triplicate. GFP expression was measured by scanning the plate in Typhoon scanner followed by an analysis with protein array analyzer on ImageJ. The AAV-eGFP transgene expression was normalized with background and used as data to compare the critical effect of cell types and AAV serotypes on transgene expression.

Figure 2

iPSC and iPSC-RPE tropism of 11 rAAV serotypes. (a) Representative phase image showing morphology of IPSC cultures and flow cytometry analysis of pluripotency surface marker expression (SSEA3 and SSEA4) of iPS cells. (b) The onset of AAV-eGFP expression in iPSCs at a dosage of 1E6 vg/cell for all rAAV at 48, 72, and 96 h posttransduction. There is no significant difference between any two time points (48, 72, and 96 h) after correction for multiple comparisons using Tukey method. (c) Heat map showing relative AAV-GFP expression per cell across all rAAV, dosages, and time in iPSCs. The scale bar shows the intensity of AAV-eGFP expression presented as an arbitrary relative fluorescence unit (A.U.) per cell. (d) Retinal pigmented epithelium (RPE) showing “cobblestone” appearance in phase (10x magnification), and immunofluorescent labeling with expression of ZO-1 (red) and MITF (green), and merged images showing uniform RPE monolayer. Images were captured at 10x magnification. (e) AAV-eGFP expression in RPEs at a dosage of 1E6 vg/cell for all rAAV at 48, 72, and 96 h postinfection. Analysis of variance for repeated measures with post hoc pairwise comparisons between time points were performed with statistical significance indicated with pvalue < 0.05^∗, <0.01^∗∗, and <0.001^∗∗∗ after correction for multiple comparisons using Tukey method. (f) Heat map showing relative AAV-eGFP expression per cell across all rAAV, dosages, and time in RPE cells.

Figure 3

rAAV tropism of in vitro-derived cortical neurons compared to ex vivo-isolated rat cortical neurons. (a) iPSC-derived cortical neurons show dorsal forebrain identity at 82 days in vitro (DIV). The expression of pyramidal neuron markers, Tuj1 (green)/Satb2 (red) or Tuj1 (green)/CTIP2 (red), is shown. Images were captured at 10x magnification. (b) The onset of GFP expression in iPSC-derived cortical neurons (82-86 DIV) at a dosage of 1E6 vg/cell for all rAAV at 48, 72, and 96 h postinfection. Analysis of variance with post hoc pairwise comparisons between time points were performed with statistical significance indicated with pvalue < 0.05^∗, <0.01^∗∗, and <0.001^∗∗∗ after correction for multiple comparisons using Tukey method. (c) The relative AAV-eGFP per cells was observed to be lowest in iPSC-cortical neurons compared to all cells analyzed. The heat map showing relative AAV-GFP expression per cell across all rAAV, dosages, and time in iPSC-derived cortical neurons. The scale bar shows the intensity of AAV-eGFP expression presented as an arbitrary relative fluorescence unit (R.F.U.) per cell. (d) Ex vivo cortical rat neurons were isolated at embryonic day 18. Similar to iPSC-cortical neurons expression, rat cortical neurons express Tuj1 (green)/Satb2 (red) or Tuj1 (green)/CTIP2 (red). Images were captured at 10x magnification. (e) AAV-eGFP expression in rat cortical neurons at a dosage of 1E6 vg/cell for all rAAV at 48, 72, and 96 h. Analysis of variance with post hoc pairwise comparisons between time points were performed with statistical significance indicated with pvalue < 0.05^∗, <0.01^∗∗, and <0.001^∗∗∗ after correction for multiple comparisons using Tukey method. (f) Rat cortical neurons show the highest AAV-GFP expression among cell types, and heat map shows the relative AAV-GFP expression per cell across all rAAV, dosages, and time in rat cortical cells.

Figure 4

Overview of AAV-eGFP transgene expression across four cell types. The illustration shows cell type and serotype comparison between induced pluripotent stem cells (iPSC), iPSC-derived retinal pigmented epithelium (RPE), iPSC-derived cortical neurons, and ex vivo-isolated rat cortical neurons. The size of the bubble is proportional to the level of GFP expression measure within and across cell type.