Résumé
IntroductionVariants in ~300 genes cause inherited retinal dystrophies (IRD). The majority areexpressed in photoreceptors and retinal pigment epithelium (RPE). Currently, there isan impasse in gene replacement therapy for IRDs due to mutations in genes such asABCA4 (6.8 kb) or USH2A (15.6 kb), which exceed the capacity of AAVs (4.7 kb). Weaddressed this issue by testing the retinal tropism of a high-capacity viral vector capableof accommodating cassettes up to 36 kb.ObjectivesThe main aim was to evaluate the feasibility of transducing clinically relevant humanretinal cell models (iPSC-derived retinal organoids and RPE) using this high-capacityvector. The secondary aim was to identify the transduced cell types in retinal organoids.MethodsFirst, we characterized the temporo-spatial expression of the viral vector’s receptor onretinal organoids and assayed its expression on RPE by immunofluorescence (IF)studies. Secondly, we tested transduction feasibility and dose-dependent effects ontransduction efficacy using a reporter construct (CAG-mCitrine) in both models.Additionally, in the case of retinal organoids, we evaluated transduction efficacy atdifferent time points during differentiation, and carried out IF studies on sections andwhole organoids to identify target cell types.ResultsOur data show that the receptor is expressed in both retinal models. In retinalorganoids, it was detected from early retinal differentiation stages (day (D) 56). Atimmature stages (D100 and D150), the receptor was localized at the level of the outerlimiting membrane (OLM). By contrast, at maturity (D225), it was detected beyond theOLM at the level of the outer segment-like structures. We developed a protocol fortransduction of human retinal organoids, which resulted in gene transfer at all timepoints tested during differentiation, despite the existence of the OLM. By IF studies, weobserved that the vector can transduce mature photoreceptors and RPE in a dosedependentmanner, with increased efficacy for the higher vector doses. Moreover,photoreceptors were preferentially transduced over Müller cells.ConclusionWe identified a high-capacity viral vector capable of transducing human photoreceptorsand RPE. These ground-breaking results have important potential in the developmentof novel gene therapy strategies for IRDs caused by mutations in large genes.