25 May Using CRISPR to Identify Novel Therapeutic Targets in FSHD

Written by Justin Cohen, PhD

CRISPR-Cas9, originally discovered in prokaryotic organisms to be part of their immune system, has rapidly developed into a gene editing tool far more accurate than its predecessors. This technology is thus generating a lot of buzz due to its potential to correct genetic diseases. However, what if it could be used to identify factors that can lessen disease severity? These targets could be the source of novel treatments. A recent study out in Science Translational Medicine led by Angela Lek PhD and Louis Kunkel PhD explored this in Facioscapulohumeral Muscular Dystrophy (FSHD), a type of muscular dystrophy for which there is no treatment or cure.  

     FSHD patients typically experience skeletal muscle weakness in their face, shoulders and upper arms (hence the name) before progression to other muscles. While symptoms typically appear during the second decade of life, there is a wide range of disease severity, ranging from  being wheelchair-bound by their teens to others who can reach their 60s and barely need a cane.  

     The genetics of FSHD is highly complex and only beginning to be understood. FSHD can ultimately be traced to misexpression of double homeobox 4 (DUX4), a protein that is normally only active in embryonic development. In FSHD, the gene for this protein is erroneously “turned on”, causing toxicity in muscle. How DUX4 leads to muscle weakness is still unknown due to the many different proteins it can affect. However, if the right protein is blocked, muscle damage can be prevented, even in the presence of DUX4.        

    Lek et al., used CRISPR-Cas9 gene editing technology to identify these targets, taking advantage of an FSHD cell culture model in which DUX4 can be “turned on”, killing most cells within 48 hours. By using CRISPR-Cas9 to systematically “turn off” each gene in the human genome, they identified which genes prevent DUX4-mediated cell death when switched off. They identified several related clusters, one of which is associated with the cellular compensation for low oxygen conditions, or hypoxia. This result has major implications for FSHD, as it suggests that DUX4 could be driving cell death through hypoxia signaling and that targeting this response could be therapeutic. 

     The team supports this possibility in follow-up experiments using compounds which impair this cellular response. In the presence of these compounds, muscle cells stayed alive when DUX4 was “turned on”. Confirming this further, the authors treated muscle cells taken from FSHD patient biopsies with these compounds and found a decrease in FSHD “biomarkers”, known indicators of the disease.    

      Lastly, these compounds were tested in a zebrafish model of FSHD. Zebrafish are a common model for muscle diseases as their translucent skin allows easy visualization of their muscle structure. These compounds reduced cell death, improved muscle fiber structure and increased swimming activity in these zebrafish. 

       These results suggest that targeting the cellular response to low oxygen conditions could be a therapeutic approach for FSHD. Importantly, there are numerous proteins that modulate this signaling, meaning there may be drugs that are already approved by the Food and Drug Administration (FDA) for use in other diseases which target aspects of this response. If so, time in the clinical trial pipeline can be reduced as the safety profile has already been tested. The team is now exploring if any FDA-approved compounds suitable for long-term use are effective in their models and plan to begin mouse studies on the most promising candidates. If effective, this approach can be expanded to other diseases, broadening the capability of CRISPR-Cas9 technology.