NEW YORK (GenomeWeb) – Researchers have used the CRISPR/Cas9 gene-editing tool in mice to disrupt a gene that causes glaucoma. As they also were able to target the gene in human eye cultures, the researchers said their study suggests CRISPR/Cas9 editing could work in the eye.
Researchers led by Val Sheffield at the University of Iowa used the gene-editing tool to disrupt the myocilin (MYOC) gene. Gain-of-function mutations in MYOC lead to an accumulation of the myocilin protein in cells, which triggers endoplasmic reticulum stress and increased intraocular pressure to cause both juvenile open-angle glaucoma and adult-onset primary open-angle glaucoma (POAG).
As the researchers reported in the early edition of the Proceedings of the National Academy of Sciences today, targeting this gene with CRISPR leads to a drop in ER stress and intraocular pressure in a mouse model of glaucoma.
"Our present study demonstrates the potential of CRISPR-mediated genome editing in human myocilin-associated POAG, as well as in other human disorders resulting from gain-of-function mutations," the authors wrote in their paper.
Sheffield and his colleagues generated eye tissue cell lines, one of which expressed wild-type MYOC and one of which expressed the Y437H mutation, which is one of the most common MYOC mutations linked to early-onset POAG. As they expected, the mutant cell lines had higher expression levels of myocilin and ER stress-related transcripts.
They also developed an adenovirus to contain and deliver the CRISPR machinery. In particular, they designed a guide RNA that targets a location near the first exon of MYOC to induce an early frameshift mutation and termination of the myocilin protein.
When they introduced this adenovirus complex into the cell line expressing the mutant MYOC, the cell line expressed lower levels of myocilin, as compared to a mock-treated mutant MYOC line. The treated mutant MYOC cell lines also expressed lower levels of proteins involved in the unfolded protein response cascade. The researchers estimated that they had between 60 percent and 70 percent transduction efficiency and additional assays confirmed that the gRNA targeted the MYOC gene.
They then injected this CRISPR-containing adenovirus into the eyes of mouse models of POAG. When they treated young mice, less than a month old, before they experienced increased intraocular pressure, they found that editing the gene could stave off glaucoma. Meanwhile, in mice older than nine months, treatment with the CRISPR construct led to lower intraocular pressure levels. This suggested to the researchers that they could prevent the development of glaucoma in young mice and treat it in older mice using their CRISPR-based approach.
Sheffield and his colleagues examined tissue sections from the treated mouse eyes to confirm that their CRISPR construct was targeting MYOC and again found a transduction efficiency of between 60 percent and 70 percent
They also tested their CRISPR system on cultured human tissue cells, including from trabecular meshwork eye tissue, and ex vivo perfusion-cultured eyes. Treatment with their construct led to reduced levels of myocilin mRNA and reduced secretion of myocilin protein into the medium, suggesting that the MYOC gene was disrupted.
"Our construct also works well in cultured human TM cells and skin fibroblasts in vitro, as well as in human ex vivo perfusion-cultured eyes, suggesting a feasible translational application of this technology to humans," Sheffield and his colleagues wrote.
They added that not only could CRISPR-based gene editing could be used to treat this form of glaucoma, it could also be applied to other human conditions caused by gain-of-function mutations.