CRISPR-Cas9 correction of OPA1 c.1334G>A: p.R445H restores mitochondrial homeostasis in dominant optic atrophy patient-derived iPSCs

Abstract

Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy in the United Kingdom. DOA has an insidious onset in early childhood, typically presenting with bilateral, central visual loss caused by the preferential loss of retinal ganglion cells. 60%–70% of genetically confirmed DOA cases are associated with variants in OPA1 , a ubiquitously expressed GTPase that regulates mitochondrial homeostasis through coordination of inner membrane fusion, maintenance of cristae structure, and regulation of bioenergetic output. Whether genetic correction of OPA1 pathogenic variants can alleviate disease-associated phenotypes remains unknown. Here, we demonstrate generation of patient-derived OPA1 c.1334G>A: p.R445H mutant induced pluripotent stem cells (iPSCs), followed by correction of OPA1 through CRISPR-Cas9-guided homology-directed repair (HDR) and evaluate the effect of OPA1 correction on mitochondrial homeostasis. CRISPR-Cas9 gene editing demonstrated an efficient method of OPA1 correction, with successful gene correction in 57% of isolated iPSCs. Correction of OPA1 restored mitochondrial homeostasis, re-establishing the mitochondrial network and basal respiration and ATP production levels. In addition, correction of OPA1 re-established the levels of wild-type (WT) mitochondrial DNA (mtDNA) and reduced susceptibility to apoptotic stimuli. These data demonstrate that nuclear gene correction can restore mitochondrial homeostasis and improve mtDNA integrity in DOA patient-derived cells carrying an OPA1 variant.

CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient

“…As we wanted to achieve a permanent correction of the GPR143 intron 7 mutation, we explored the more recent option of genome editing by using the CRISPR-Cas system to promote HDR, a strategy that has previously been proven feasible by other groups. 41 , 44 , 55 For its delivery, we opted for an RNP formulation instead of a plasmid-based approach. RNPs are less toxic, ready to act, and not constantly produced in the host cells.…”

Role of Oxidative Stress in Ocular Diseases Associated with Retinal Ganglion Cells Degeneration

“…In this section, we briefly highlight some of the iPSC-derived RGC disease models that have been developed using 2D and 3D culture approaches. Several teams have now made patient derived and CRISPR/Cas edited iPSC lines for the investigation of glaucoma [ 199 , 209 , 210 ], LHON [ 211 , 212 , 213 , 214 ], and DOA [ 215 , 216 , 217 , 218 ].…”

“…Several teams have also begun trying to model DOA from patients with mutations in the OPA1 gene [ 215 , 216 , 217 , 218 ]. Chen et al found that OPA1 mutation increased necrosis and apoptosis in the patient iPSCs and could not efficiently differentiate them into RGCs using their initial methodology.…”

“…Further, they generated an isogenic control through CRISPR/Cas9-mediated HDR. Correction of OPA1 led to restoration of mitochondrial homeostasis, including mtDNA stability and cellular bioenergetics [ 218 ]. Together, these initial studies set a foundation for more in-depth analysis in OPA1 patient iPSC-derived RGCs.…”

Functional genomics and the future of iPSCs in disease modeling

“…As the mutations are all engineered into the same isogenic cell line background, the effects observed are due to the individual mutations and not confounded by factors such as genetic background and sex ( Anastasaki et al., 2020 ). As an alternative approach, patient hiPSC lines can be compared with gene-corrected isogenic control cell lines ( Figure 2 ) ( Sladen et al., 2021 ).

Figure 2 Generation of isogenic pairs of cell lines that differ by a single genetic modification Isogenic control cell lines can be created from healthy pluripotent stem cells to model the effect of a specific patient population.

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