Finding new treatments for muscular dystrophy with CRISPR-Cas9

By Dr. Liji Thomas, MDMar 25 2020

A new study out of Boston's Children's Hospital has used the gene-editing tool CRISPR-Cas9 to explore the fatal genetic condition called facioscapulohumeral dystrophy (FSHD, one of the family of muscular dystrophies), and to test out the potential utility of various genes involved in this disorder. The research is published in the journal Science Translational Medicine.

3D Rendering Crispr DNA Editing. Image Credit: Nathan Devery / Shutterstock

What is CRISPR-Cas9?

CRISPR-Cas9 (short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) is an adaptation of a natural genome editing system in bacteria, where bits of DNA are removed from viral invaders by the bacterial host and converted into arrays of DNA called CRISPR arrays. The function of these arrays is to ensure bacterial recognition of the virus or closely related ones so that any return of the virus will prompt the immediate transcription of RNA fragments from the CRISPR arrays. The RNA is specifically directed against the viral DNA, as a result, which is then cut up using the enzyme Cas9 or something like it. This prevents the virus from doing any harm.

The CRISPR-Cas9 system has deservedly been in the spotlight for several years because of the ease, smoothness, and rapidity with which it introduces vital changes into the genome of an organism. As such, it is viewed as our best hope of inserting genes that can correct defective or missing genes responsible for various genetic diseases.

However, the CRISPR-Cas9 system has also proved to be a useful tool for the detection of genes that support other, more active genes that cause genetic diseases. By changing the way such ancillary genes regulate the main gene players, it could be possible to design new treatments for such diseases.

What is FSHD?

The condition called FSHD is an incurable condition in which there is a severe muscular weakness in the face, the shoulder blades, and the upper arms. The patient receives only supportive care at present.

The cause of FSHD is the unwanted switching on of the DUX4 gene, which is usually only active while the fetus is developing. This inappropriate activity of the gene causes the production of the DUX4 protein within the muscle cells after birth. This is toxic to the muscle cells, causing them to die and resulting in muscle weakness.

The study

The scientists wanted to see if the DUX4 gene activity could be compensated for or even avoided by using other gene targets. They aimed to empower the affected muscle cell despite the presence of the toxic protein.

To find out which gene they should use, they turned to CRISPR-Cas9. Their objective was simple: turn off every gene in the genome, one by one. At some point, they hoped they would find one or more genes that could be switched off permanently within the human muscle cell, enabling it to go on living in the presence of the DUX4 protein. In other words, says author Angela Lek, "We essentially utilized the CRISPR screen technique as a shortcut to illuminate 'druggable' pathways for FSHD."

The findings

The study threw up about six promising candidate genes via CRISPR-Cas9 screening. Some of these were genes that are activated under conditions of hypoxia or low oxygen levels. Further exploration showed that cell death caused by DUX4 is caused primarily by hypoxia. To combat this, the team took these muscle cells and exposed them to molecules that prevent this hypoxic response from occurring. The results were cheering: the cells remained alive.

In other words, researcher Louis Kunkel says, "Our results show that knockout of key genes involved in hypoxia signaling can desensitize cells to toxicity from DUX4, and prevent them from dying."

In the second stage, the researchers then cultured muscle cells from FSHD patients and treated them by exposing them to the same hypoxia-signal inhibiting compounds, the levels of known disease markers within the cells dropped significantly, which could mean reduced disease activity.

Finally, the investigators worked with two animal models of FSHD, in the form of zebrafish. When these fish were exposed to the same compounds, their muscle structure showed an improvement, as did muscle function. The fish began to swim more actively.

Implications and future directions

The researchers are excited about their discovery and have applied for a patent to protect it while they move on into other animal studies. Lek has since moved out of the team and is planning to conduct mouse experiments on the FSHD gene. Kunkel is continuing to work on zebrafish models.

The researchers point out, "The most encouraging finding of this study is that we discovered that there are FDA-approved drugs that can overcome DUX4's toxic effect." They plan to test the collection of approved drugs to find out which of them is best for the long-term treatment of this condition in humans.

A perhaps still more significant achievement is the use of a process that could, in practice, be used to help discover treatments for many other conditions, by contributing to their understanding, finding targets for therapy and testing promising candidates.