Abstract
Purpose :
ADOA is the most common inherited optic neuropathy, resulting in severe and progressive visual failure due to loss of retinal ganglion cells (RGCs). Most patients harbor loss-of-function mutations in the OPA1 gene that lead to haploinsufficiency. Reduced OPA1 protein levels result in impaired mitochondrial function in RGCs leading to cell death. Currently, there is no treatment for patients with ADOA. TANGO ASOs reduce inclusion of a non-productive, alternatively spliced exon in OPA1, and leverage the wild-type (WT) allele to increase productive OPA1 mRNA and protein. To test TANGO ASOs on OPA1 expression and function in RGCs in a human ADOA model, we generated OPA1 haploinsufficient iPSCs for differentiating into retinal neurospheres.
Methods :
OPA1 mutations were introduced in a WT human iPSC line using CRISPR-Cas9. Clones were assessed for karyotype, pluripotency, heterozygous mutation status, and OPA1 expression. Mutant and isogenic control WT retinal neurospheres were generated over 45 days of differentiation (PMID: 25640818). RGCs were assessed by immunofluorescent microscopy and single-cell RNA sequencing. Relative levels of OPA1 were assessed by qPCR, quantitative western, in situ hybridization, and immunohistochemistry. Reactive oxygen species (ROS) and viability of RGCs were assessed by flow cytometry. We evaluated neurospheres treated with TANGO ASOs by gymnotic delivery.
Results :
Haploinsufficient OPA1 iPSC clones were generated with normal karyotype and pluripotency. Both OPA1 mutant and WT control neurospheres were generated with approximately 10-20% RGCs. OPA1 mutant cells had similar viability and increased cellular ROS levels compared to controls. ASO treatment of neurospheres increased OPA1 mRNA expression and OPA1 protein while reducing non-productive OPA1 mRNA.
Conclusions :
iPSC-derived RGC-containing neurospheres provide a useful in vitro model to evaluate TANGO ASOs. TANGO ASOs were able to partially correct RNA and protein deficiencies in OPA1 mutant retinal neurospheres. Our results support that TANGO ASOs can potentially be used to treat ADOA caused by OPA1 haploinsufficiency.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.