ARTICLE IN BRIEF
At a plenary at the AAN Annual Meeting, Charles Gersbach, PhD, of Duke University, discusses advances in editing of the dystrophin gene in Duchenne muscular dystrophy using CRISPR/Cas9, removing its defects, and allowing it to create a nearly full-length functional protein.
Duchenne muscular dystrophy (DMD) has seen a surge of developments in gene-based therapies in recent years, most recently with the approval of two antisense treatments, eteplirsen and nusinersen, which prevent expression of mutation-containing exons. But an even more remarkable approach is in the works: editing of the dystrophin gene using CRISPR/Cas9, removing its defects, and allowing it to create a nearly full-length functional protein.
The technique has been successful in mice, and research is underway that will attempt to translate it to humans. In a plenary lecture here at the AAN Annual Meeting in April, Charles Gersbach, PhD, professor of biomedical engineering and pediatrics at Duke University in Durham, NC, reported on progress and advances from using this approach.
The genome revolution, from the publishing of the human genome sequence in 2001 to the subsequent detailed annotation of gene function and disease association throughout the next decade, “was intended to have a dramatic effect on medicine,” Dr. Gersbach said. “I would argue that one of the obstacles that has held back the translation of this information has been an inability to go in and precisely manipulate the genome,” either to test hypotheses about disease causation “or to reprogram the genome in order to treat the disease state.”
The development of CRISPR/Cas9 technology is poised to change that, he said. Cas9 is a DNA-cleaving enzyme that is directed to its target by a short complementary RNA sequence, called a guide RNA. In the bacteria from which the system is derived, these are copies of genetic material from viral invaders (clustered regularly interspaced short palindromic repeats), used as templates for warding off future infection.
In the lab, with an RNA sequence chosen to match a region of interest in the target gene, the system allows the researcher to create a break in the DNA with surgical precision, “and then guide how the genome repairs that break, and in the process incorporate specific sequences into, or remove them from, the genome.” Most relevant for Duchenne muscular dystrophy, he said, is the ability to create two breaks that flank the mutation, cutting out the intervening region and creating a shorter but more functional gene after repair.
“CRISPR/Cas9 has revolutionized the field by developing a technology that is fast, easy, cheap, and can be used very easily by any lab,” Dr. Gersbach said. The technique is being developed for treatment of many genetic diseases, including hemophilia and some cancers, as well as HIV and other blood-borne diseases where the affected tissue is readily accessible.
“Neuromuscular disease is more of a challenge,” he said, because of the need to deliver treatment to muscles throughout the body.
But the fact that the full-length gene is too large for conventional gene therapy vectors makes gene editing an attractive strategy nonetheless. In addition, because the muscle cell is multinucleated, “this gives us multiple shots on goal” from a single treatment.
The most common types of dystrophin mutations are amenable to gene editing correction, Dr. Gersbach said. “For example, deletion of exons 45 to 50 causes exon 51 to be out of frame, and creates a stop codon. If you remove exon 51, you can restore the reading frame, and restore the expression of a protein that is largely functional, simply missing an internal part. This leads to a mild phenotype.”
Exon 51 skipping is the strategy for both eteplirsen and nusinersen. But that strategy requires constant re-treatment, while a single CRISPR/Cas9 treatment has the potential to be permanent, he said.
In 2015, Dr. Gersbach showed in Nature Communications that by using guide RNAs to remove exons 45 to 55 in patient myoblasts in vitro, “we get a targeted deletion that removes exon 51 from the genome,” a strategy potentially applicable to more than 60 percent of patients.
To translate this into human patients, Dr. Gersbach is developing adeno-associated virus gene therapy to deliver the genes for the CRISPR/Cas9 system. In 2016, he reported in Science this can be done in mice through a single injection, with restoration of dystrophin production to 70 percent of muscle fibers, and an increase in muscle strength, with no obvious adverse effects after one year of observation. In his lecture, he reported further success with a new model, with restoration of dystrophin in cardiac muscle from a tail vein injection.
More work remains, he stressed, including further validation of safety and improvements in delivery efficiency, as well as scaling up to larger animal models.
“For the majority of mutations, the editing should enable the production of fairly large and highly functional dystrophin proteins,” commented Jeffrey S. Chamberlain, PhD, professor of neurology, medicine, and biochemistry at the University of Washington School of Medicine, and director of the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center of Seattle. Dr. Chamberlain's own work has shown that muscle-specific gene editing led to widespread dystrophin expression and increased force production in mice.
“This is an advantage compared to the best current alternative, which is delivering a microdystrophin gene using an AAV vector,” a strategy that is set to move into clinical trials in the near future.
Microdystrophin encodes only 30 percent of the dystrophin gene, removing much of the gene's repetitive middle region. In the DMD dog model, the gene restores function and prolongs survival, “but if you can use gene editing to make a larger protein, in theory that should be more functional.” On the other hand, he pointed out, microdystrophin is potentially beneficial to every DMD patient, while the diversity of dystrophin mutations means that a variety of guide RNAs will be needed to treat everyone.
Off-target effects may be another important difference between the two approaches. “The microdystrophin therapies don't seem to have any off-target effects,” because the gene doesn't disrupt the genome. “Gene editing does, and though the guide RNAs target the enzyme to the dystrophin gene, the specificity is not 100 percent,” Dr. Chamberlain said. “You get enormous enrichment when the system is properly designed, but you are always going to get some off-target effects. And any time you start editing the genome, you have a risk of disrupting an important cellular process,” potentially leading to tumors. “But so far, studies in mice have gone quite well, and overall I think this is an encouraging approach.”
“Ultimately, I think what is going to be needed is some type of self-inactivating vector, which would shut itself down after editing its target gene,” Dr. Chamberlain said. “The alternative is that the editing system remains active, potentially for a lifetime.”
“One qualification for both approaches is that a lot will depend on when the treatment is given,” Dr. Chamberlain said. “If the muscle has gone through many cycles of regeneration already, it is a little unclear how much reversal you are going to get in terms of strength,” at least for skeletal muscles.
“The reversibility of the disease is going to decline,” he continued, but because the heart doesn't go through the same cycles of fibrosis and regeneration, it is potentially more correctable at later stages than skeletal muscle. “This is all somewhat theoretical until we get into clinical trials,” and begin to see what the data show, he said.