Using CRISPR-Cas9 to identify new gene drug targets

CRISPR-Cas9 can be used as a tool to find genes that make a disease better or worse, providing targets for new treatments.

A new study led by Dr Louis Kunkel and research fellow Dr Angela Lek at Boston Children’s Hospital, US, used CRISPR-Cas9 to better understand facioscapulohumeral muscular dystrophy (FSHD) and explore potential treatments. 

In FSHD, the gene DUX4, normally active mainly during foetal development, is inappropriately switched on. This causes the toxic DUX4 protein to be produced in muscle cells when it should not, leading to cell death and muscle weakness.

Kunkel, Lek and colleagues wondered if other genes could be targeted to prevent or compensate for this problem. They decided to use CRISPR-Cas9 to systematically mutate every gene in the genome. Their goal was to find genes that, when knocked out, enable human muscle cells to survive even when the DUX4 protein is being made.

“We essentially utilised the CRISPR screen technique as a shortcut to illuminate ‘druggable’ pathways for FSHD,” explained Lek, the paper’s first author.

The screening process yielded about a half-dozen strong hits. Among them were several genes that play a role in the cellular response to low-oxygen conditions, or hypoxia. That, it turns out, is the main driver of cell death caused by DUX4. When the team exposed muscle cells to compounds known to inhibit this hypoxia response, the cells stayed alive.

“Our results show that knockout of key genes involved in hypoxia signalling can desensitise cells to toxicity from DUX4 and prevent them from dying,” said Kunkel.

Going a step further, the team created muscle cell lines from actual patients with FSHD. When treated with the same compounds, these cells showed fewer of the known biomarkers of the disease.

Finally, the researchers created two live zebrafish models of FSHD. When they exposed the fish to compounds that inhibit hypoxia signalling, the fish showed improvements in muscle structure and function and more swimming activity.

Kunkel and Lek have filed a patent application covering their discoveries. Lek, now at the Yale School of Medicine, US, is moving the drug experiments into mouse models of FSHD, while Kunkel plans further zebrafish studies at Boston Children’s Hospital.

“The most encouraging finding about this study is that we discovered that there are US Food and Drug Administration (FDA)-approved drugs that can overcome DUX4’s toxic effect,” said Lek. “We now have a collection of drugs to test and figure out which is most suitable for long-term dosing in patients with FSHD.”

Kunkel believes the process used in this study could be applied to many other diseases: “Our approach could provide an accelerated path to understanding complex genetic diseases, discovering therapeutic targets and testing potential treatments.”