Glaucoma gene therapy on positive trajectory using CRISPR-Cas9

Results from a series of preclinical studies are providing proof of principle that gene targeting using CRISPR-Cas9 genome-editing technology can prevent or treat glaucoma associated with mutations in the myocilin (MYOC) gene.

Seattle-Results from a series of preclinical studies are providing proof of principle that gene targeting using CRISPR-Cas9 genome-editing technology can prevent or treat glaucoma associated with mutations in the myocilin (MYOC) gene, said Val Sheffield, MD, PhD.

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Speaking at ARVO 2016, Dr. Sheffield, professor of ophthalmology and visual sciences, University of Iowa Carver College of Medicine Iowa City, reviewed findings from a research project that has its roots more than 25 years ago.

It began with determination that mutations in myocilin coded for by the MYOC gene were the cause for an autosomal dominant early onset form of open-angle glaucoma. Next it was determined that the pathophysiology involved protein misfolding leading to endoplasmic reticulum stress in the trabecular meshwork and subsequently elevated IOP.

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Discussing the research focusing on gene therapy using the CRISPR-Cas9 system, Dr. Sheffield said the strategy is to create insertions or deletions resulting in frameshift mutations leading to stop mutations.

The first studies were done in vitro using mouse trabecular meshwork cells and human primary trabecular meshwork cells. In the next phase, the research advanced into in vivo studies, first using the ashMYOCY437H transgenic animal.

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Treatment of 1-month old mice, which had not yet developed elevated IOP, using Ad-5-CRISPR-Cas9 virus with guide RNAs targeting exon 1 of the MYOC gene was shown to relieve endoplasmic reticulum stress in the trabecular meshwork cells and mitigate elevations in IOP that occurred in animals treated with CRISPR-Cas9 with control guide RNA.

Targeting MYOC

In a rescue model using older animals that had already developed elevated IOP, treatment with CRISPR-Cas9 with the MYOC-targeting guide RNA resulted in decrease in IOP and increase in outflow facility.

A similar experiment was conducted with success in a mouse model of glucocorticoid-induced glaucoma for which the pathophysiology also seems to involve endoplasmic reticulum stress. Next, the researchers moved onto human cadaver eyes with positive results.

“We have successfully targeted MYOC using the CRISPR-Cas9 system that rescues the endoplasmic reticulum stress in cultured human trabecular meshwork cells, lowers the IOP, and improves outflow facility in the transgenic MYOC mouse model,” Dr. Sheffield said. “We think this approach also applies to glaucomas not associated with myocilin and that other CRISPR-Cas9 targets for glaucoma exist.”

He noted that gene therapy research starts and ends with patients, and now, it is ramping up toward the latter phase.

“We begin by identifying the gene and from there determine the molecular mechanisms so that we can understand the pathophysiology,” Dr. Sheffield said. “Then we move onto animal models with the ultimate hope of being able to translate the research to patients.”