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Updated: Nov 19, 2025
Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells
Published on: December 9, 2022
Gene correction of the CLN3 c.175G>A variant in patient-derived induced pluripotent stem cells prevents pathological
Xiao Zhang1,2, Dan Zhang1,2, Jennifer A Thompson3
1Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, WA, Australia.
Background:
Mutations in CLN3 cause Batten disease, however non-syndromic CLN3 disease, characterized by retinal-specific degeneration, has been also described. Here, we characterized an induced pluripotent stem cell (iPSC)-derived disease model derived from a patient with non-syndromic CLN3-associated retinopathy.
Methods:
Patient-iPSC, carrying the 1 kb-deletion and c.175G>A variants in CLN3, coisogenic iPSC, in which the c.175G>A variant was corrected, and control iPSC were differentiated into neural retinal organoids (NRO) and cardiomyocytes. CLN3 transcripts were analyzed by Sanger sequencing. Gene expression was characterized by qPCR and western blotting. NRO were characterized by immunostaining and electron microscopy.
Results:
Novel CLN3 transcripts were detected in adult human retina and control-NRO. The major transcript detected in patient-NRO displayed skipping of exons 2 and 4-9. Accumulation of subunit-C of mitochondrial ATPase (SCMAS) protein was demonstrated in patient-derived cells. Photoreceptor progenitor cells in patient-NRO displayed accumulation of peroxisomes and vacuolization of inner segments. Correction of the c.175G>A variant restored CLN3 mRNA and protein expression and prevented SCMAS and inner segment vacuolization.
Conclusion:
Our results demonstrate the expression of novel CLN3 transcripts in human retinal tissues. The c.175G>A variant alters splicing of the CLN3 pre-mRNA, leading to features consistent with CLN3 deficiency, which were prevented by gene correction.
Insights
Non-syndromic CLN3 disease causes retinal degeneration due to altered CLN3 gene splicing. Gene correction restored CLN3 expression and prevented disease features in patient-derived retinal organoids.
Area of Science:
- Genetics
- Cell Biology
- Ophthalmology
Background:
- Mutations in the CLN3 gene cause Batten disease.
- Non-syndromic CLN3 disease presents with retinal degeneration.
- This study focuses on a patient with non-syndromic CLN3-associated retinopathy.
Purpose of the Study:
- To characterize an induced pluripotent stem cell (iPSC)-derived disease model of non-syndromic CLN3 retinopathy.
- To investigate the molecular mechanisms underlying CLN3 deficiency in the retina.
- To assess the efficacy of gene correction in a cellular model.
Main Methods:
- Generated patient-iPSC and isogenic control iPSC lines.
- Differentiated iPSCs into neural retinal organoids (NRO) and cardiomyocytes.
- Analyzed CLN3 transcripts, gene expression (qPCR, Western blotting), and NRO morphology (immunostaining, electron microscopy).
Main Results:
- Identified novel CLN3 transcripts in human retina and NRO.
- Patient-derived NRO showed aberrant CLN3 splicing (exon skipping).
- Accumulation of mitochondrial ATPase subunit C (SCMAS) and photoreceptor defects (peroxisome accumulation, vacuolization) were observed in patient-NRO; gene correction reversed these phenotypes.
Conclusions:
- The c.175G>A variant in CLN3 alters pre-mRNA splicing, leading to retinal degeneration.
- This iPSC-derived NRO model recapitulates key features of non-syndromic CLN3 retinopathy.
- Gene correction is a viable strategy to restore CLN3 function and prevent disease pathology.
