June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Use of iPSC-derived retinal ganglion cells to investigate cell specific OPA1 RNA processing
Author Affiliations & Notes
  • Joshua Harvey
    Institute of Ophthalmology, University College London, London, London, United Kingdom
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
  • Neringa Jurkute
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Gavin Arno
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Patrick Yu-Wai-Man
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
    University of Cambridge Department of Clinical Neurosciences, Cambridge, Cambridgeshire, United Kingdom
  • Michael E Cheetham
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Joshua Harvey None; Neringa Jurkute None; Gavin Arno None; Patrick Yu-Wai-Man Transine Therapeutics, Code C (Consultant/Contractor), Chiesi GmbH, Code C (Consultant/Contractor), GenSight Biologics, Code C (Consultant/Contractor), Stoke Therapeutics, Code C (Consultant/Contractor); Michael Cheetham None
  • Footnotes
    Support  Moorfields Eye Charity GR001138
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1896. doi:
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      Joshua Harvey, Neringa Jurkute, Gavin Arno, Patrick Yu-Wai-Man, Michael E Cheetham; Use of iPSC-derived retinal ganglion cells to investigate cell specific OPA1 RNA processing. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1896.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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