Personalized Pathogenicity Assessment of RPE65 Gene Mutations Using Patient-Specific hiPSC-Derived Retinal Pigment...

Area of Science:

• Ophthalmology and Genetics
• Molecular Biology and Cell Biology
• Gene Therapy and Regenerative Medicine

Background:

• Leber's congenital amaurosis (LCA) is a severe childhood retinal dystrophy linked to over 115 missense variants in the RPE65 gene.
• The RPE65 isomerohydrolase is crucial for the visual cycle, and its dysfunction leads to retinal degeneration.
• Variant-specific pathogenic mechanisms in RPE65-associated LCA remain largely uncharacterized due to genetic heterogeneity.

Purpose of the Study:

• To dissect the mechanistic and functional basis of mutated RPE65 protein in retinal degeneration.
• To evaluate gene therapy-mediated restoration of RPE65 function using patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE).
• To investigate the impact of compound heterozygous RPE65 variants (c.200T > G, c.430T > C) on RPE cell function.

Main Methods:

• Overexpression of wild-type and mutant RPE65 in HEK293T cells to assess protein stability and degradation pathways.
• Establishment and characterization of patient-specific iPSC-RPE (iRPE) for enzymatic activity analysis.
• Adeno-associated viral (AAV) vector-mediated delivery of RPE65 into patient iRPE to validate gene therapy efficacy.

Main Results:

• Both RPE65 variants significantly destabilize the RPE65 protein via the autophagosome-lysosome pathway, impairing isomerohydrolase activity.
• Patient-specific iRPE cells exhibited normal morphology and core physiological features but reduced endogenous RPE65 protein and enzymatic function.
• Exogenous RPE65 supplementation using AAV vectors effectively restored deficient isomerohydrolase activity in the patient-derived iRPE model.

Conclusions:

• The study elucidates variant-specific pathogenesis in RPE65-associated LCA, highlighting protein destabilization and functional impairment.
• Gene augmentation therapy using AAV vectors shows promise for restoring RPE65 function.
• Preclinical evidence supports gene replacement therapy for LCA patients with these specific RPE65 variants.