Using a new gene-editing technique, a team of scientists from UT Southwestern Medical Center stopped progression of Duchenne muscular dystrophy (DMD) in young mice.
If efficiently and safely scaled up in DMD patients, this technique could lead to one of the first successful genome editing-based treatments for this fatal disease, researchers said.
DMD, the most common and severe form of muscular dystrophy among boys, is characterised by progressive muscle degeneration and weakness. It is caused by mutations in the X-linked DMD gene that encodes the protein dystrophin. The disease affects one in 3,500 to 5,000 boys, according to the Centers for Disease Control and Prevention and other estimates and often leads to premature death by the early 30s.
Although the genetic cause of DMD has been known for nearly 30 years, no effective treatments exist. The disease breaks down muscle fibres and replaces them with fibrous or fatty tissue, causing the muscle to gradually weaken. This condition often results in heart muscle disease, or cardiomyopathy, the leading cause of death in these patients.
In the study published in Science, UTSW researchers used a gene-editing approach to permanently correct the DMD mutation that causes the disease in young mice.
“This is different from other therapeutic approaches, because it eliminates the cause of the disease,” said senior author Dr. Eric Olson, Chairman of Molecular Biology, and Co-Director of the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center at UT Southwestern.
In 2014, Dr. Olson’s team first used this technique — called CRISPR/Cas9-mediated genome editing — to correct the mutation in the germ line of mice and prevent muscular dystrophy. This paved the way for novel genome editing-based therapeutics in DMD. It also raised several challenges for clinical applications of gene editing. Since germ line editing is not feasible in humans, strategies would need to be developed to deliver gene-editing components to postnatal tissues.
To test this out, researchers delivered gene-editing components to the mice via adeno-associated virus 9 (AAV9). DMD mice treated with this technique produced dystrophin protein and progressively showed improved structure and function of skeletal muscle and heart.
“AAV9 can efficiently infect humans in a tissue-specific manner, but it does not cause human disease or toxicity. It’s a molecular missile for gene therapy,” said Dr. Leonela Amoasii, a postdoctoral researcher in the Olson lab and co-lead author of the study with Dr. Chengzu Long, Instructor of Molecular Biology.
“The CRISPR/Cas9 system is an adaptive immune system of single-celled organisms against invading virus. Ironically, this system was hijacked, we packaged it into a nonpathogenic virus, and corrected a genetic mutation in an animal model,” added Dr. Long.
The CRISPR genome-editing technology, which was developed by a researcher at University of California at Berkeley, was picked as the “Breakthrough of the Year” scientific development by Science.
“This study represents a very important translational application of genome editing of DMD mutations in young mice. It’s a solid step toward a practical cure for DMD,” said Dr. Rhonda Bassel-Duby, Professor of Molecular Biology and Co-Principal Investigator of a genomic editing project with Dr. Olson at the Wellstone Center.
“Importantly, in principle, the same strategy can be applied to numerous types of mutations within the human DMD patients,” added Dr. Olson, who also serves as Director of the Hamon Center for Regenerative Science and Medicine, and holds the Annie and Willie Nelson Professorship in Stem Cell Research, the Pogue Distinguished Chair in Research on Cardiac Birth Defects, and the Robert A. Welch Distinguished Chair in Science.
Now, the research team is working to apply this gene-editing technique to cells from DMD patients and in larger preclinical animal models.
This marks the first major finding of the UTSW Wellstone Center, which was recently established with $7.8 million in funding from the National Institutes of Health. UTSW is one of six Wellstone Centers across the country, which work to translate scientific findings and technological developments into novel treatments for muscular dystrophy, and to promote basic, translational, and clinical research. UT Southwestern’s Wellstone Center focuses on Duchenne muscular dystrophy.
“The recent groundbreaking discoveries from the Olson laboratory using genome editing to correct the genetic mutation that causes DMD have accelerated the race to find a cure for this deadly disease,” said Dr. Pradeep Mammen, Associate Professor of Internal Medicine and Co-Director of the UTSW Wellstone Center. “The challenge now lies before Wellstone Center researchers to translate these discoveries in the mouse model of DMD into a therapy for patients with DMD.”
Duke University Research.
Researchers have used CRISPR to treat an adult mouse model of Duchenne muscular dystrophy. This marks the first time that CRISPR has successfully treated a genetic disease inside a fully developed living mammal with a strategy that has the potential to be translated to human therapy.
Researchers from Duke University had previously used CRISPR to correct genetic mutations in cultured cells from Duchenne patients, and other labs had corrected genes in single-cell embryos in a laboratory environment. But the latter approach is currently unethical to attempt in humans, and the former faces many obstacles in delivering treated cells back to muscle tissues.
Another approach, which involves taking CRISPR directly to the affected tissues through gene therapy techniques, also faces challenges, particularly with delivery. In the new study, Duke University researchers overcame several of these obstacles by using a non-pathogenic carrier called adeno-associated virus, or AAV, to deliver the gene-editing system.
The paper appears in Science.
“Recent discussion about using CRISPR to correct genetic mutations in human embryos has rightfully generated considerable concern regarding the ethical implications of such an approach,” said Gersbach, associate professor of biomedical engineering at Duke University. “But using CRISPR to correct genetic mutations in the affected tissues of sick patients is not under debate. These studies show a path where that’s possible, but there’s still a considerable amount of work to do.”
Duchenne muscular dystrophy is caused by problems with the body’s ability to produce dystrophin, a long protein chain that binds the interior of a muscle fiber to its surrounding support structure. Dystrophin is coded by a gene containing 79 protein-coding regions, called exons. If any one exon gets a debilitating mutation, the chain does not get built.
Without dystrophin providing support, muscle tends to shred and slowly deteriorate.
Duchenne affects one in 5,000 newborn males. Most patients are wheelchair-bound by age 10 and don’t live beyond their 20s or early 30s. The mutation is on the X chromosome so female children with two X chromosomes should have at least one functioning copy of the gene.
Gersbach has been working on potential genetic treatments for Duchenne with various gene-altering systems since starting his lab at Duke in 2009. His lab recently began focusing on CRISPR/Cas9 — a modified version of a bacterial defence system that targets and slices apart the DNA of familiar invading viruses.
While Gersbach has had success in cultured patient cells by using a jolt of electricity to punch holes in their membranes to deliver the CRISPR system, this strategy was not practical in a patient’s muscle tissues.
“A major hurdle for gene editing is delivery. We know what genes need to be fixed for certain diseases, but getting the gene editing tools where they need to go is a huge challenge,” said Chris Nelson, the fellow in Gersbach’s laboratory who led the work. “The best way we have to do it right now is to take advantage of viruses, because they have spent billions of years evolving to figure out how to get their own viral genes into cells.”
Nelson and Gersbach began working on packaging gene editing tools into AAV, the most popular virus for delivering genes today. They were assisted through collaborations with AAV experts Aravind Asokan, associate professor at the University of North Carolina, Chapel Hill School of Medicine and Dongsheng Duan at the University of Missouri School of Medicine. Duan also provided significant expertise from a long history of work on gene therapy for neuromuscular disorders.
To use viruses as delivery vehicles for gene therapy, researchers take all the harmful and replicative genes out of the virus and put in the therapeutic genes they want to deliver. While early virus types didn’t work well for various reasons, such as integrating into the genome and causing problems or triggering immune responses, AAV thus far has proven special. It’s a virus that many people are exposed to anyway and is non-pathogenic, but still exceptionally effective at getting into cells.
AAV is in use in many late-stage clinical trials in the United States, and has already been approved for use in one gene therapy drug in the European Union. There are also different versions of AAV that can preferentially go to different tissues, such as skeletal and cardiac muscle, so researchers can deliver them systemically.
But there’s always a catch, and for Gersbach it was a matter of size.
“AAV is a really small virus and CRISPR is relatively large,” said Gersbach. “It simply doesn’t fit well, so we still had a packaging problem.”
The solution came from Feng Zhang, an investigator at the Broad Institute of the Massachusetts Institute of Technology and Harvard. Earlier this year, Zhang described a CRISPR system from a different bacterium than the one commonly used.
In the natural bacterial immune system, CRISPR is the mug shot that helps identify the target DNA, and Cas9 is the blade that slices the strands. The large Cas9 protein typically used by researchers comes from the bacterial species Streptococcus pyogenes. After scouring the bacterial kingdom, Zhang discovered the much smaller Cas9 protein of Staphylococcus aureus.
Small enough to fit comfortably inside of AAV.
In the study, researchers worked with a mouse model that has a debilitating mutation on one of the exons of the dystrophin gene. They programmed the new CRISPR/Cas9 system to snip out the dysfunctional exon, leaving the body’s natural repair system to stitch the remaining gene back together to create a shortened but functional version of the gene.
Besides being much easier and more efficient than replacing the dysfunctional exon with a working copy, simply snipping out the weak link is a strategy that would be effective in a larger swath of the patient population.
Gersbach and his team first delivered the therapy directly to a leg muscle in an adult mouse, resulting in the restoration of functional dystrophin and an increase in muscle strength. They then injected the CRISPR/AAV combination into a mouse’s bloodstream to reach every muscle. The results showed some correction of muscles throughout the body, including in the heart, a major victory because heart failure is often the cause of death for Duchenne patients.
“There is still a significant amount of work to do to translate this to a human therapy and demonstrate safety,” said Gersbach. “But these results coming from our first experiments are very exciting. From here, we’ll be optimizing the delivery system, evaluating the approach in more severe models of DMD, and assessing efficiency and safety in larger animals with the eventual goal of getting into clinical trials.”
*CRISPR . Clustered regularly-interspaced short palindromic repeats