When it comes to Duchenne muscular dystrophy (DMD), there is rarely good news to share. It is an inherited disorder caused by the mutation of a gene that makes dystrophin, a protein on the X chromosome needed for healthy muscle development. Without it, victims suffer progressively worsening muscle failure beginning in early childhood. By the time they reach puberty, 90% of DMD victims cannot walk. By their mid-20s, most are dead – victims of an incurable disorder that finally shuts down their ability to breathe or their hearts to beat.1
Thanks to something called X-linked recessive inheritance, every boy inherits an X chromosome from his mother and a Y chromosome (which makes him male) from his father. Girls inherit two X chromosomes, one from each parent.2
Both boys and girls have a 50/50 chance of inheriting the protein disorder if their mother has the dystrophin mutation. Most girls never develop DMD, but they are carriers of the disorder and may likely suffer from cardiomyopathy, or weakening of the heart muscle, later in life. Boys with the flawed gene lack the protection of that second X chromosome and constitute the vast majority of DMD sufferers. By one estimate, 1 in every 2,400 boys worldwide is born with DMD. By the age of five, most are already suffering from the relentlessly progressive muscle-wasting disease. There are no cures, no treatments, and no survivors.3
Of Mice and Men
Don’t tell that to a team of researchers at The University of Texas Southwestern Medical Center in Dallas. They recently developed a new plasmid-based technique using CRISPR/Cas9-mediated genome editing to eliminate defective dystrophin mutations from DNA in mice, which develop similar DMD symptoms to humans. CRISPR (clustered regularly interspaced short palindromic repeat) Cas (CRISPR-associated) systems are a revolutionary approach to alter an organism's complete set of DNA, including all of its genes. It does so by targeting and cutting the DNA chain at a specific site to remove the mutated gene. The double-strand break in the DNA is then repaired by one of two pathways. The first pathway is known as Non-Homologous End-Joining (NHEJ), meaning the broken ends are tied off in the cell. The second pathway is called Homology Directed Repair (HDR). It is based on a DNA template that is injected into the cells of mice zygotes. Zygotes are the earliest developmental stages of fertilized eggs. The treated mice zygotes were then implanted into surrogate mothers. Now carrying the correct DNA template, it knows exactly which DNA sequence to use to repair the cut DNA. The result is an error-free restoration of the DNA strand minus the mutated gene.4
Why is this so important? Unlike other therapies meant to supplant or bypass the defective gene, the CRISPR/Cas9-mediated genome editing approach created by the UT Southwestern Medical Center team eliminates the dystrophin mutation defect completely. It is not simply a work-around; it is a cure… at least in mice. "Even in mice with only a small percentage of corrected cells, we saw widespread and progressive improvement of the condition over time, likely reflecting a survival advantage of the corrected cells and their contribution to regenerating muscle," Dr. Eric Olson, Director of the Hamon Center for Regenerative Science and Medicine at UT Southwestern and Chairman of Molecular Biology, said in a UT press release.5, 6
Scaling Up a Possible Cure in Humans
Dr. Olson is the first to point out that his team’s genomic editing approach is currently not a fix for DMD in people. Technical challenges remain before that becomes a reality. Most important is how to scale up the CRISPR treatment for humans. It is no longer a question of how, but by how much, another team member says, adding that the UT team is already working on a more clinically possible method to correct mutations in adult tissue.
Dr. Olson notes that the current CRISPR technique could well lead to new treatments for DMD sufferers. “…in the future we might be able to use this technique therapeutically… to directly target and correct the mutation in muscle stem cells and muscle fibers,” he says.7
In addition, UT Southwestern Medical Center researchers made another important discovery while testing their CRISPR technique on mice. Correction of only 17% of the mutant protein was enough to allow dystrophin expression in a majority of muscle fibers in the mice. That suggested a “selective advantage” of the corrected skeletal muscle cells, as shown with subsequent Western blot analysis testing that verified that MDM was effectively eradicated in the mice.8 The team also performed a series of cellular and muscle tissue analyses to verify their findings. These included capturing a large number of critically important specimen images using PerkinElmer’s OpenLab™ 4.0 acquisition and control software. OpenLab software was also used to magnify and color the slides to create image overlays.9
Building On a Strong Relationship
The UT Southwestern Medical Center is one of the foremost academic and research centers in the U.S. In addition to the OpenLab software, PerkinElmer supports the Center with a number of analytical and imaging instruments, including an Operetta® high-content microplate imaging solution, a Vectra® automated quantitative pathology imaging system, a LabChip® GXII Touch for genomics instrument, as well as EnVision® plate readers for research and assay development applications.
Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA, Science, published online August 14, 2014.
X-Linked Recessive Inheritance – Traditional Patterns of Inheritance 3 (Fact Sheet 10), New South Wales (Australia) Government Ministry of Health – Centre for Genetics Education.
Duchenne Muscular Dystrophy Research Fund (DMDfund).
CRISPR gene therapy prevents muscular dystrophy, leahcanscience.com.
New gene editing method shows promising results for correcting muscular dystrophy, Newsroom, The University of Texas Southwestern.
Supplementary Materials for Prevention of muscular dystrophy in mice by CRISPR/Cas9 – mediated editing of germline DNA, Science.