Tropism of AAV Vectors in Photoreceptor-Like Cells of Human iPSC-Derived Retinal Organoids

Abstract

Purpose: To expand the use of human retinal organoids from induced pluripotent stem cells (iPSCs) as an in vitro model of the retina for assessing gene therapy treatments, it is essential to establish efficient transduction. To date, targeted transduction of the photoreceptor-like cells of retinal organoids with adeno-associated virus (AAV) vectors has had varied degrees of success, which we have looked to improve in this study.

Methods: Retinal organoids were differentiated from iPSCs of healthy donors and transduced with reporter AAV containing a CAG.GFP, CAG.RFP, GRK1.GFP, or EFS.GFP transgene. Capsid variants assessed were AAV5, AAV2 7m8, AAV2 quad mutant, AAV2 Y444F, and AAV8 Y733F. At 27 days post-transduction, retinal organoids were assessed for reporter expression and viability.

Results: The short intron-less elongation factor 1 alpha (EFS) promoter provided minimal reporter expression, whereas vectors containing the CAG promoter enabled transduction in 1% to 37% of cells depending on the AAV serotype; the AAV2 quad mutant (average 19.4%) and AAV2 7m8 (16.4%) outperformed AAV5 (12%) and AAV8 Y733F (2.1%). Reporter expression from rhodopsin kinase (GRK1) promoter transgenes occurred in ∼5% of cells regardless of the serotype. Positive co-localization with recoverin-expressing cells was achieved from all GRK1 vectors and the CAG AAV2 quad mutant variant. Treatment with the AAV vectors did not influence retinal organoid viability.

Conclusions: Reliable transduction of the photoreceptor-like cells of retinal organoids can be readily achieved. When using a CAG-driven transgene, transduction of a broad range of cell types is observed, and GRK1 transgenes provide a more restricted expression profile locating to the outer layer of photoreceptor-like cells of retinal organoids.

Translational relevance: This study expands the AAV capsid and transgene options for preclinical testing of gene therapy in iPSC-derived human retinal organoids.

Figure 1.

Live cell imaging of reporter gene expression up to 27 days post-transduction. Arrows indicate the areas where the onset of reporter gene expression first appeared. Each retinal organoid was treated with 1E+10 genome copies, except for GRK1.GFP AAV8 Y733F, for which 5E+10 genome copies were used. Background fluorescence of live retinal organoids was unavoidable, but reporter expression was evident as higher intensity areas above this background. Further examples of retinal organoids treated per condition are shown in Supplementary Figure S2.

Figure 2.

Reporter transgene expression in retinal organoids transduced with different vector types. (A) Z-stacks of retinal organoid sections were extracted using 40× magnification and analyzed for reporter-positive cells. Values were obtained from 27 to 35 individual images from at least three different retinal organoids per treatment group. One-way ANOVA with Tukey's multiple comparisons test revealed significant differences among groups. *P = 0.041, **P = 0.003, ****P < 0.0001. Error bars represent minimum/maximum. Example Z-stacks from which the data were extracted are provided in Figures 3 and 4. (B) Representative examples of whole retinal organoid sections taken with 10× magnification.

Figure 3.

Representative reporter gene expression profiles from CAG transgenes delivered in multiple AAV capsid types. For each sample type, example retinal organoid sections with each marker are shown in the upper panel, and only reporter and marker expression is shown in the lower panel, for which overlapping signals are highlighted in white. (A) AAV2 7m8; (B) AAV2 quad mutant; (C) AAV5; and (D) AAV8 Y733F. Each retinal organoid was treated with 1E+10 genome copies. Scale bars: 25 µm. Isolated green and red channels are shown in Supplementary Figure S5. GFAP, glial fibrillary acidic protein, a Müller glia cell marker; L/M, long-/medium-wavelength cone opsin, a cone photoreceptor marker; PKCα, protein kinase C alpha, a bipolar cell marker; REC, recoverin, a pan-photoreceptor marker; RHO, rhodopsin, a rod photoreceptor marker.

Figure 4.

Reporter gene expression profiles from GRK1 transgenes delivered in multiple AAV capsid types. For each sample type, example retinal organoid sections with each marker are shown in the upper panel, and only reporter and marker expression is shown in the lower panel, for which overlapping signals are highlighted in white. (A) GRK1.GFP AAV2 7m8; (B) GRK1.GFP AAV8 Y733F; and (C) GRK1.GFP AAV2 Y444F. Each retinal organoid was treated with 1E+10 genome copies, except in (B), where 5E+10 genome copies were used. Scale bars: 25 µm. Isolated green and red channels are shown in Supplementary Figure S6.

Figure 5.

AAV transduction did not influence retinal organoid viability. The average ATP levels from four individual untreated retinal organoids were used to determine the relative percentage ATP activity in each treatment group. (A) One-way ANOVA indicated no significant influence of AAV treatment on viability (F = 1.107, P = 0.3908), and Dunnett's multiple comparisons test revealed no significant differences in any treatment group compared to the untreated group. Error bars represent standard error of the mean. (B) ATP levels correlated with retinal organoid size (r = 0.8019, P < 0.0001, R² = 0.6431).