X-linked retinoschisis Advancing disease modeling with retinal organoids

X-linked retinoschisis (XLRS) is an inherited retinal disease caused by mutations in the RS1 gene, primarily affecting the macula of young males and leading to progressive loss of central vision. Despite successful preclinical studies in animal models, no treatment is currently available. This thesis explores the potential of patient-derived retinal organoids (ROs) as an alternative model to study XLRS. First, a systematic review of 25 preclinical gene therapy studies, including a meta-analysis of 19 rodent studies, showed that AAV-mediated RS1 augmentation improved retinal function, while highlighting that careful study design and reporting are essential to overcome barriers to clinical translation. Next, we reviewed the signaling pathways targeted by supplements in published RO protocols and experimentally compared the most common ones, identifying the Wnt activator CHIR99021 as the factor that significantly improved RO yield. We showed that embryoid body formation from single cells or cell clumps affects early differentiation but has minimal impact on final RO architecture, cellular composition and photoreceptor maturation. Using induced pluripotent stem cells from patients carrying two Dutch and Belgian founder variants (p.E72K and a deletion in exon 3), we generated XLRS ROs. Control ROs faithfully recapitulated the temporal and spatial expression of RS1, whereas patient-derived ROs showed reduced RS1 and disrupted photoreceptor development. However, the characteristic schisis phenotype found in patients was not consistently observed in patient-derived retinal organoids. Overall, ROs replicate intracellular XLRS pathomechanisms, but modeling tissue splitting will require integrating vasculature, microglia and interactions with the retinal pigment epithelium.