Abstract
Purpose :
The majority of dominant optic atrophy (DOA) is caused by mutations in the OPA1 gene that encodes a mitochondrial targeted protein playing important roles in mitochondrial dynamics, membrane integrity, cristae structure, and cellular respiration. Despite its expression in most somatic tissue types, OPA1 mutations most prominently affect retinal ganglion cells (RGCs). The purpose of the study is to use pluripotent stem cell (PSC)-derived retinal organoids to overcome the scarcity of human RGCs and establish DOA disease models in vitro.
Methods :
The peripheral blood cells of DOA patients with distinct OPA1 mutations were reprogrammed to obtain iPSC lines. CRISPR-Cas mediated homologous directed repair (HDR) was subsequently performed to correct the mutation of a DOA patient’s iPSCs to establish an isogenic control iPSC line. In addition, CRISPR-Cas9-mediated gene editing was used to create homozygous and heterozygous OPA1 mutant ESCs from a control line with a normal OPA1 gene. The OPA1 mutant and control PSC lines, and 3D retinal organoids derived from these PSCs were characterized for protein expression by Western blot, mitochondrial morphology by super resolution imaging, cellular respiration by Seahorse assay, and neuronal properties by whole cell patch clamp recording.
Results :
We have established isogenic OPA1 mutant and control PSC lines. The OPA1 mutant PSCs show reduced OPA1 protein expression, altered mitochondrial morphology, and reduced cell respiration and mitochondrial ATP production rates compared with their isogenic controls. All heterozygous OPA1 mutant PSC lines can give rise to retinal organoids and produce RGCs. Retinal organoids show different OPA1 protein isomer ratios compared with PSCs. Similar reduced cellular respiration and ATP production rates have been observed in OPA1 mutant retinal organoids compared with their corresponding isogenic controls. Electrophysiological analysis of OPA1 mutant and control PSC-derived neurons has detected altered physiological properties between isogenic pairs.
Conclusions :
The isogenic OPA1 mutant and control PSC lines provide useful tools for establishing OPA1-DOA disease models using 3D-retinal organoid-derived RGCs and induced neurons. The identified OPA1 mutant phenotypes serve as the basis for using PSC-based DOA disease models in vitro to develop therapies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.