Thirty five years ago the human gene that causes Duchenne Muscular Dystrophy (DMD) was discovered. It was hailed as a light bulb moment in understanding the disease mechanism. However, there was little practical progress towards new treatments or therapies over the next two decades. Now, the rapid emergence of CRISPR gene editing technique is proving a game changer in the revised battleplan against the distressing condition.
Image: Dystrophin muscle protein domain (N-terminal actin binding domain). Defects cause Duchenne muscular dystrophy (DMD). Cartoon & stick representation with backbone gradient coloring. Credit: StudioMolekuul
Dystrophin, the rod-shaped protein which connects the cytoskeleton of a muscle fibre to the surrounding cell matrix through the cell membrane, lies at the heart of DMD enigma.
We now know DMD arises from a recessive, fatal, X-linked gene occurring at a frequency of about 1 in 3,500 new-born boys. Becker MD is a milder allelic form of the same condition.
Several CRISPR techniques have been applied to correct some of the thousands of documented mutations in the dystrophin gene at the core of the fatal disease that leads to progressive muscle weakness and wasting.
While most attempts have relied on the generation of DNA double-strand breaks (DSBs) by the more conventional Cas9 nuclease activity, only a few studies have taken advantage of more forensic method of base editing and prime editing. Since these relatively new methods only introduce single-strand breaks and make minimal change to the genome, they can serve as potentially safe and efficient alternatives to genetic correction of DMD disease-causing mutations. It helps that the DMD gene encoding the dystrophin protein is one of the longest human genes covering 2.3 megabases. It means we may be close to a breakthrough.
A new CRISPR gene-editing strategy for DMD therapy
A research group under Eric Olson at the University of Texas Southwestern Medical Centre in Dallas, followed this approach in developing a new CRISPR gene-editing strategy for DMD therapy. Their work was published in Science Advances last month (April).
Olson’s group focused one of the most common DMD causing mutations. It centred on deletion of exon 51 in DMD which causes disruption to the dystrophin reading frame and generates a premature ‘stop’ codon in exon 52. The researchers say they have managed to restore the reading frame with both the base editing and prime editing by the introduction of exon “skipping” and reframing.
In both cases the strategy restored in vitro dystrophin expression in human cardiomyocytes, the cells responsibly for normal heart contraction function and it is being seen as potentially a major breakthrough.
The team reportedly tested the efficacy of the single-swap gene-editing strategy in a human disease model. Firstly, an sgRNA with 38 per cent on-target editing at the SDS of exon 50 was selected using human HEK293T cells. Then human ?Ex51 iPSCs were nucleofected* with this sgRNA and the base-editing machinery. Single clones of edited cells – 88 per cent of which contained the on-target edit - were isolated and differentiated into cardiomyocytes.
DNA sequence analyses of these edited iPSC-derived cardiomyocytes revealed successful splicing of exon 49 to 52, indicating that the skipping of exon 50 had taken place. Just as in the mouse experiment, edited human cardiomyocytes showed restoration of dystrophin protein expression as revealed by Western blot analysis and immunocytochemistry.
Single-swap gene-editing strategy
So the single-swap gene-editing strategy for exon 50 skipping appears to work in both human and murine cells. Eric Olson added, the strategy also exerts significant functional, disease-alleviating changes in mouse muscles.
“That's an important point because, in this disease, the muscle tissue is destroyed and replaced by fibrotic scar and fat infiltration, so normal muscle structure is preserved after editing,” he says in the paper.
“Our next step will be to demonstrate if this technology also works for other mutations in DMD. The longer term goal is to move this work to the clinic, but we don’t have a specific timeline established yet. We have to ensure safety and continue to optimise delivery methods,” says Olson.
He concludes: “This new research adds to the toolbox of genome-editing technologies that we can deploy to correct genetic defects. It may depend on the type of mutation as to which one of those technologies will be the most effective.
“We are more and more optimistic and hopeful about our path toward a new treatment for DMD.” His hope is that going forward methods of delivery to edit the genome in DMD patients will be improved.
At last, more than three decades after the gene was identified there are exciting times for MDM research and fresh hope for sufferers and their families.
Read the original paper, 'Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing' in Science Advances
* Nucleofection is a method of delivering nucleic acid to the cells by creating transient small pores in the cell membrane by applying an electric pulse. It was invented by Amaxa.
Electroporation and NucleofectorTM Technology, Lonza.com
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Author: Dermot Martin is a specialist writer and reporter on science, technology and medical research