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Paul Edward Sladen, Katarina Jovanovic, Mònica Aguilà, Patrick Yu-Wai-Man, Michael E Cheetham; Modelling Autosomal Dominant Optic Atrophy using Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2864.
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© ARVO (1962-2015); The Authors (2016-present)
Autosomal Dominant Optic Atrophy (DOA) is the most prevalent inherited optic neuropathy. DOA is characterised by the preferential loss of retinal ganglion cells (RGCs) and progressive blindness. 60-70% of DOA patients harbour mutations in the OPA1 gene, encoding a mitochondrial membrane protein that regulates mitochondrial network stability and biogenesis. Currently, there are no therapeutic options and the mechanisms causing RGC loss are poorly understood. Thus we set out to create a biobank of OPA1-mutant induced pluripotent stem cells (iPSCs) to model DOA via in vitro RGC generation.
Six patient derived OPA1 mutant cell lines were reprogrammed to iPSCs using non-integrating vectors expressing 4 Yamanaka factors. iPSCs were characterised using immunohistochemistry (IHC) and quantitative PCR (qPCR). In parallel, OPA1 exon 2 was targeted in a control cells by simultaneous reprogramming and CRISPR/cas9 genome editing to generate a haploinsufficient OPA1 isogenic cell line. OPA1 mutations were determined by Sanger sequencing and protein expression was confirmed by Western blot. OPA1-mutant iPSC were differentiated towards RGCs and analysed by IHC and qPCR.
CRISPR-cas9 targeting of OPA1 induced InDel mutations in 7/52 of isolated clones, an editing efficiency of 13%. All 7 clones had heterozygous frameshift mutations, no homozygous knock-out clones were identified. Western blot analysis of CRISPR-edited OPA1+/- iPSC showed OPA1 levels of 52 ± 5.9% vs controls. Sequencing analysis confirmed patient-derived iPSCs maintained patient specific mutations. IHC confirmed iPSCs expressed embryonic stem cell markers, Oct4, Tra-160 and SSEA4. qPCR validated the trilineage differentiation potential of all generated iPSCs with notable upregulation of genes associated to RGC differentiation, including Pax6 and Brn3A. IHC analysis demonstrated the feasibility of generating RGCs from all derived OPA1-mutant cell lines, showing expression of RGC specific markers Brn3 and Atoh7, and neuronal markers Pax6 and BIII tubulin.
We have generated an OPA1 mutant iPSC biobank encompassing the clinical disease spectrum. Additionally, an isogenic OPA1 haploinsufficiency model was created using CRISPR-cas9 gene editing. We have demonstrated the feasibility of in vitro RGC differentiation through iPSC technologies, providing an exciting model for studying OPA1-mediated disease pathways and targeted therapeutics.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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