A new method of using CRISPR-Cas9 has been developed by scientists at the University of Texas Southwestern Medical Center that can potentially be used to treat Duchenne muscular dystrophy.
Duchenne muscular dystrophy affects approximately one in 5000 males. The disease is caused by X-linked dystrophin gene mutations. The DMD gene is one of the largest in the human genome – containing around 2.6 million base pairs – and the gene has a complex structure. These factors contribute to the high rate of spontaneous mutations. So far, around 3,000 different mutations have been identified that can cause the disease.
Several research teams have turned to the gene editing tool CRISPR to try to correct genetic mutations that cause Duchenne muscular dystrophy. Dr. Eric Olson, director of UT Southwestern’s Hamon Center for Regenerative Science and Medicine, and his team have previously successfully corrected point mutations in mouse models of the disease using CRISPR. However, in humans, only 5%-9% of DMD patients have point mutations. Most DMD patients have deletions in one or more exons.
The high number of potential mutations that cause the disease means conventional molecular therapies need to be developed to treat each single mutation, whereas Dr. Olson and his team have conducted proof-of-concept studies that suggest CRISPR-Cas9 could easily be used to correct the majority, if not all of mutations that cause the disease.
For the study the researchers obtained cardiomyocytes (heart muscle cells) from patients diagnosed with DMD and determined that making a single cut in strategic locations using CRISPR, it was possible to restore dystrophin levels to near normal levels.
“Not only did we find a practical way of treating many mutations, we have developed a less disruptive method that skips over defective DNA instead of removing it. The genome is highly structured, and you don’t want to remove DNA that could potentially be important,” said co-author of the study, Rhonda Bassel-Duby, PhD.
The team used a dozen guide RNAs for the Cas9 enzyme to make the cuts and remove the mutations. Those gRNAs targeted hotspots on the dystrophin gene where up to 60% of the mutations occur.
“We screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting,” the researchers explained in the paper. “We performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes.”
The team found that they only needed to correct between 30% and 50% of myocytes to bring dystrophin levels up to near-normal levels.
Before this new technique can be used on patients, the U.S Food and Drug Administration must first approve the use of CRISPR on humans. So far, no trials using the gene editing tool have taken place in the United States. China has been using CRISPR for some time, but the regulatory environment in the US is far stricter, and new treatments take much longer to be approved. That said, the first trials using CRISPR are expected to be authorized later this year, with blood disorders the most likely candidates for this new treatment.
In the meantime, Dr. Olson and his team will be conducting further experiments to test and refining the technique to ensure off-target gene edits do not occur. Dr. Olson’s optimistic view is CRISPR could be used to treat Duchenne muscular dystrophy in as little as 5 years.
The study – Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing – was recently published in the journal Science Advances.