Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip.

Mutations in the tripeptidyl peptidase 1 (TPP1) gene lead to neuronal ceroid lipofuscinosis type 2 (CLN2), characterized by lysosomal accumulation of lipofuscins predominantly in the brain and retina. The ocular phenotype is characterized by outer retinal degeneration that leads to vision loss. Leveraging human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs), retinal pigmented epithelial cells, and the retina-on-chip system, we establish an in vitro CLN2 model that recreates the principal histological hallmarks, namely the accumulation of subunit C of mitochondrial ATP synthase (SCMAS) and lipids mainly in the outer retina. Furthermore, single-cell RNA sequencing reveals a dysregulation of translational and mitochondrial function in CLN2 cones. Finally, adeno-associated virus (AAV)-mediated TPP1 gene therapy can restore TPP1 expression and decrease and even prevent SCMAS accumulations. Our study uses an innovative human-relevant microphysiological retinal disease models, uncovers previously uncharacterized mechanisms of CLN2 pathophysiology, and demonstrates the potential of AAV9.hCLN2 gene therapy for CLN2 disease, potentially treating patient blindness.