Abstract
Purpose :
Autosomal dominant optic atrophy (DOA) is the commonest inherited optic neuropathy characterised by selective degeneration of the retinal ganglion cells (RGCs). OPA1 is the commonest causative gene and over 450 variants have been identified. Splice site variants make up a large proportion of pathological variants, but little is known about the tissue-specific differences in splicing. OPA1 has 8 alternatively splice isoforms that have distinct tissue-dependent patterns, but a lack of readily accessible human RGCs has limited further investigation. This study sought to investigate the effect of an OPA1 splice site variant (c.2356-1G>T) in RGCs derived from induced pluripotent stem cells (iPSC-RGCs).
Methods :
Fibroblasts with the c.2356-1G>T OPA1 variant were obtained from a DOA patient and reprogrammed to iPSCs. The variant was corrected using CRISPR-Cas9 gene editing to produce an isogenic control, c.2356-1G>WT. iPSCs were used to generate RGCs using a 2D 42-day differentiation protocol. OPA1 expression was characterised using qPCR and western blotting. Long-read sequencing using Oxford Nanopore Technologies sequencing was used to characterise splicing in the c.2356-1G>T and c.2356-1G>WT lines. Mitochondrial bioenergetics was assessed using the Seahorse XFe96 platform.
Results :
Analyses of c.2356-1G>T fibroblast RNA by RT-PCR revealed a 21-base pair skipped in-frame OPA1 transcript that could have dominant-negative effects, with no evidence of additional splicing outcomes in the presence of NMD inhibition. By contrast, analyses of OPA1 transcripts obtained from c.2356-1G>T and c.2356-1G>WT iPSC-RGCs confirmed the 21-base pair skip, but also identified several different splice outcomes at low-frequency. Correct splicing was restored through CRISPR correction. Correction also led to an increase in OPA1 protein expression and improved mitochondrial bioenergetics.
Conclusions :
iPSC-RGCs recapitulate RGC-specific OPA1 splicing patterns, providing a useful in vitro model for investigating RNA processing and associated mechanisms that could contribute to the selective vulnerability of RGCs in DOA. iPSC-RGCs established from a DOA patient demonstrate varied OPA1 splicing patterns highlighting the importance of isogenic controls. OPA1 splice variants can have multiple splicing outcomes that can manifest with both haploinsufficiency and dominant-negative disease mechanisms.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.