Evaluation of retinal cells transduction with a high capacity viral vector using human iPSC- derived retinal organoids

Submission 82

PS2-65-Poster Presentation

Presented by: Luisina Cappellino

Luisina Cappellino ¹, Marcela Garita-Hernandez ¹, Hassan Boukhaddaoui ¹, Bertrand Beucher ^{2, 3}, Carla Jimenez-Medina ¹, Nejla Erkilic ¹, Eric Kremer ³, Vasiliki Kalatzis ¹

¹ Institute for Neurosciences of Montpellier, Inserm, University of Montpellier, Montpellier, France

² PVM, BioCampus, CNRS, Inserm, University of Montpellier, Montpellier, France

³ Institute of Molecular Genetics of Montpellier, CNRS, University of Montpellier, Montpellier, France

Objective

Inherited retinal dystrophies (IRDs) are a group of disorders caused by genetic variants in more than 300 genes. They affect the photoreceptors and retinal pigment epithelium (RPE) and provoke progressive visual impairment. Currently, there is an impasse in gene replacement therapy for IRDs due to mutations in large genes, such as MYO7A (6.7 kb), ABCA4 (6.8 kb) or USH2A (15.6 kb). These genes exceed the cargo capacity of AAVs (4.7 kb), the vector considered as gold standard for retinal gene therapy. We evaluated the retinal tropism of a high-capacity viral vector, using clinically relevant human retinal cell models: iPSCs-derived retinal organoids and RPE.

Methods

We characterized the temporo-spatial expression of the viral vector’s receptor on retinal organoids and assayed its expression on RPE by immunofluorescence (IF) studies. We tested transduction feasibility and dose-dependent effects on transduction efficacy using a reporter construct and subsequent IF analysis in both models. In the case of retinal organoids, we evaluated transduction efficacy at different time points during differentiation and carried out IF studies on sections and whole organoids to identify target cell types.

Results

Our data show that the receptor is expressed in both retinal models. In retinal organoids, it was detected from early retinal differentiation stages (day (D) 56). At immature stages (D100 and D150), the receptor was localized at the level of the outer limiting membrane (OLM). At maturity (D225), expression was detected in the outer nuclear layer. We developed a protocol for transduction of human retinal organoids, which allowed gene transfer at all time points tested during differentiation, despite the existence of the OLM. By IF studies, we observed that the vector can transduce mature photoreceptors and RPE in a dose-dependent manner.

Conclusion

The use of human iPSC-derived retinal organoids allowed us to identify a high-capacity viral vector capable of transducing human photoreceptors and RPE. These results have important potential in the development of novel gene therapy strategies for IRDs.