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Jie Cheng, Cynthia A Berlinicke, Valentin Sluch, Xitiz Chamling, Donald J Zack; Modeling of autosomal dominant optic atrophy with human embryonic stem cell derived retinal ganglion cells by CRISPR-Cas9 mediated editing of OPA1. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1363.
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© ARVO (1962-2015); The Authors (2016-present)
Autosomal dominant optic atrophy (DOA) is the most commonly diagnosed inherited optic neuropathy, and there are currently no treatments for this condition. Mutations in the OPA1 gene account for 50-60% of all DOA cases. OPA1 haploinsufficiency was proposed to cause mitochondrial dysfunction, leading to retinal ganglion cell (RGC) injury and death. To generate a human model to study both the disease mechanism as well as for use in drug discovery, we set out to make human embryonic stem cell (hESC)-derived RGCs harboring OPA1 mutations.
CRISPR guide RNAs (gRNA) targeted to the first exon of human OPA1 together with S. pyogenes Cas9 were transfected into an H9-hESC line carrying a BRN3B-tdTomato reporter (H9-Brx). CRISPR-Cas9 induced indels by non-homologous end joining were identified through two rounds of colony selection, followed by PCR-amplification of genomic DNA and sequencing. Alteration in OPA1 protein expression levels were assessed by Western blotting. CRISPR-edited hESC lines and congenic H9-Brx line were differentiated into RGCs using a newly developed large-scale RGC differentiation protocol. Differentiated RGCs were evaluated by quantitative morphological analysis, immunocytochemistry and qPCR.
Two gRNAs were used to generate OPA1 mutations in H9-Brx. Sequencing of genomic DNA revealed that mutation rate was 52% (16/31) for gRNA1 and 29% (9/31) for gRNA2. Western blotting of three clones with a frameshift mutation demonstrated a 54.6 ± 2.9 % reduction of OPA1 protein. These mutant clones were differentiated to RGCs using a combination of small molecules. At day 35, differentiated RGCs were immuno-purified. When compared to RGCs derived from the parental H9-Brx, they exhibited similar morphological properties, such as average neurite counts per cell and average neurite length; they stained positive for neuron specific beta-3-tubulin; and they expressed similar level of RGC markers BRN3B, RBPMS and SPP-1 RNA.
In this study, we have generated several independent lines of hESCs with OPA1 frameshift mutations. We demonstrated that these hESCs can be differentiated into RGCs. Functional evaluation of mutant cell lines is under way. The large-scale production of OPA1 mutant RGCs paves the way for biochemical analysis as well as drug discovery efforts for DOA and other RGC-related neurodegenerative diseases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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