Abstract
Purpose :
The use of optogenetic gene therapy for the treatment of inherited retinal diseases including retinitis pigmentosa (RP) has become a significant therapeutic option for late-stage disease. Several ongoing clinical trials use optogenetic tools for vision restoration in patients affected with late-stage RP. Pre-clinical models remain limited and development of human retinal organoids has potential to aid translational testing of optogenetic vectors. Herein, we aim to evaluate the transduction efficiency of optogenetic tools in human-derived retinal organoids and assess their impact on organoid viability.
Methods :
Human-derived retinal organoids were differentiated to 180 days and treated with human rhodopsin optogene driven by ubiquitous chicken beta actin (CAG) promoter and packaged into AAV vectors (AAV2.2 and AAV2.5) at 2 different doses, 1E+10 (n = 5) and 1E+11 (n = 5). Organoids were kept in culture media and live cell imaging was performed weekly using an EVOS Cell Imaging System (Thermo Fisher Scientific). 3D live organoid imagining was done with a spinning disk confocal microscope (Olympus). An adenosine triphosphate (ATP) assay was performed using the CellTiter-Glo 3D Cell Viability kit (Promega, Madison, WI).
Results :
The live cell assay demonstrated a steady increase in rhodopsin expression for up to four weeks of assessment (Figure. 1). The expression was dose dependent with higher AAV dose (1E+11) leading to higher levels of transduction efficiency compared to the lower dose (1E+10). The transduction efficiency also depended on the capsid, with AAV2.5 showing overall more expression compared to AAV2.2 at the same dose. According to the ATP assay, transgene expression in the organoids had no significant effect on the viability of retinal organoids (97%) compared to untreated organoids (100%) (P = 0.8699, n = 5).
Conclusions :
Using the human retinal organoid model, we were able to assess preliminary expression patterns and impact on the cell viability of optogenetic tools. The efficiency of organoid transduction varies based on the AAV capsid and vector dose. Notably, expression of human rhodopsin in retinal organoids does not affect the viability of retinal organoids, indicating safety of this optogenetic tool for further translational studies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.