CRISPR/Cas9 Tackles Duchenne Muscular Dystrophy in Mice

Ricki Lewis, PhD

The gene editing technique CRISPR/Cas9 is breathing new life into the effort to deliver dystrophin protein to the muscles of boys with Duchenne muscular dystrophy (DMD). Three papers in Science detail the “myoediting” approach in mice, offering hope at the end of a year that saw serious setbacks in a clinical trial for another approach to treat the disease.

The Case of the Giant Gene

The challenges of treating DMD are more formidable than those for most single-gene disorders. When the gene and its encoded protein were discovered and described in 1986 and 1987, just when discussions about clinical trials for the first gene therapies were heating up, researchers knew they were dealing with a beast.1,2 The gene that is deleted in whole in DMD and in part in the milder Becker form is gigantic at 2.4 million bases long, and the corresponding protein too huge to stuff into safe, small vectors like adeno-associated virus (AAV).
Dystrophin protein conjures up curious extremes. It serves as a dynamic linchpin in the cell membranes of muscle cells, where it is the major component of a complex of glycoproteins that connect the extracellular matrix outside the cell with the cytoskeleton within. Comprising a scant 0.002 percent of the muscle proteins – barely a speck compared to the pervasive sliding filaments of actin and myosin and the troponins that dot them – dystrophin is nevertheless crucial in a human body that is about 40 percent skeletal muscle.
The dystrophin gene is on the X chromosome, so girls inheriting mutations are protected by their second X. Boys without dystrophin have DMD, and live with progressive muscle degeneration, typically losing the battle in their late twenties when the heart and diaphragm fail.

 Strategy 1: Gene Therapy

 Animal models of DMD – mice  and dogs – provided a robust body of preclinical work in support of gene therapy clinical trials.3,4 But the giant gene was a poor fit for the diminutive AAV, once adenovirus entered the dark ages following the death of Jesse Gelsinger in 1999. Much of the 1990s and early 2000s were spent cutting the gene into “minigenes” in the 3.5 kilobase (35,000 base) range.5
Clinical trials began. Six of 9 boys given one or two injections of plasmids bearing full-length dystrophin genes into arm muscles showed a weak response in a phase 1 clinical trial, with results published in 2004. A trial of a minigene that began in 2006, with results published in 2010, unexpectedly showed response of the immune system to even undetectable amounts of dystrophin.6 Still, a second phase 1 trial began in 2011 with a vector variant optimized for delivery into muscle.7
Despite disappointing results, gene therapy clinical trials for DMD continue, because the approach does have benefits. “The minigene approach can be applied to all DMD patients and is not mutation-specific. The minigene also does not require DNA repair mechanisms and does not introduce the foreign gene editing proteins into the body” necessary with CRISPR/Cas9 editing, Charles A. Gersbach, Ph.D., Associate Professor, Department of Biomedical Engineering Investigator, Center for Genomic and Computational Biology Director, Center for Biomolecular and Tissue Engineering at Duke University and corresponding author of the Nelson paper told RDR.
Encouraging preclinical results have kept gene therapy hopes afloat too. In October, for example, a “microdystrophin” gene delivered in AAV9 to three 2-month-old dogs led to dystrophin in all muscles, even the crucial cardiac muscle of the heart and skeletal muscle of the diaphragm, when the experiment stopped at 4 months.8

Strategy 2: Antisense Oligonucleotides

A second strategy to counter DMD is to mimic natural mutations in some patients with Becker MD – these variants cause the mRNA-making machinery to skip key parts of the DNA sequence that harbor premature stop signals when transcribing the gene. This “exon-skipping” restores the reading frame, enabling production of a smaller but functional version of dystrophin.
Antisense oligonucleotides are synthetic short sequences of RNA-like molecules that bind to and shield key sites in the DNA in ways that echo natural exon-skipping. In DMD, skipping exon 51 can help about 13% of patients. “Removing exons 45 through 55 could treat 62% of DMD patients,” said Dr. Gersbach.9
Two antisense oligos in clinical trials for DMD skip exon 51: drisapersen (Prosensa and Biomarin Pharmaceuticals) and eteplirsen (Sarepta Therapeutics). “The drugs must be delivered every week in large quantities, are complex and expensive to manufacture, and have some safety concerns,” said Dr. Gersbach. Uptake into muscle cells is poor and clearance from the body rapid.
But it was whole-body responses, in addition to biomarkers revealing no detectable dystrophin, that alerted an advisory committee at FDA evaluating phase 2 results for drisapersen. The key 6-minute walk test needed to have run for 48 weeks to evaluate such a chronic disease, rather than the cut off at 25 weeks, according to the committee. Perhaps most telling, though, the study was not deemed sufficiently blinded. “The primary review team is concerned that treatment allocation may have been substantially unmasked in the clinical trials because of a high incidence of outwardly obvious injection site reactions from drisapersen. The distance walked in 6 minutes is clearly related to effort, and might have been affected by patient and investigator expectation bias if treatment assignments could be deduced,” the report states. Adverse effects (on kidneys, clotting, blood vessels, and skin) noted in preclinical work were apparent in the clinical trial as well, causing the agency to table a decision for now.

Strategy 3: “Myoediting” Using CRISPR/Cas9

The slow evolution of gene therapy for DMD, coupled with the recent FDA setback, could be reason for pessimism. But then along came CRISPR.
Three papers in Science this week (Nelson et al, Tabebordbar et al and Long et al)10-12  used what the Long paper calls “myoediting” to eliminate exon 23 from the mouse model, standing in for exon 51 skipping in humans. The experiments introduce the components of the gene editing machinery in AAV vectors directly into muscles of postnatal animals.
CRISPR/Cas9 works at the site of the targeted gene itself, rather than remaining aboard AAV as part of an episome outside the chromosomes, as is the case for the vector used in conventional gene therapy.
All 3 studies report the long-sought production of dystrophin in skeletal muscle, and even in the diaphragm and in the cardiac muscle of the heart. In addition, the protein remained for months and enabled muscles to resist force.
Amy Wagers, PhD’s, group from the Harvard Stem Cell Institute (Tabebordbar et al) report particularly exciting findings because their protocol targeted muscle satellite cells, which are stem cells. When activated by an injury, satellite cells supply the nuclei that become the headquarters of cells that rebuild the tissue.
“Gene editing in satellite cells creates a pool of regenerative cells that now harbor a therapeutically modified DMD gene, and participation of these edited cells in the repair process delivers that edited gene into the fibers, so that the modified gene product – a truncated dystrophin ­ continues to be expressed,” Dr. Wagers told RDR.
Even if gene editing could correct the much more abundant skeletal and cardiac muscle cells, if satellite cells were unedited, they would continue to provide nuclei bearing the original mutation, diluting the effects of manipulation. Plus, dystrophin, in addition to maintaining the structural integrity of contracting muscle cells, helps satellite cells directly in muscle repair. “Thus, editing the DMD gene in satellite cells provides the potential to improve two key aspects of DMD pathology: myofiber fragility and poor muscle repair potential,” Dr. Wagers said. With built-in perpetuation of the therapeutic edit via stem cells, one treatment could do the trick.
Tackling DMD with myoediting improves upon gene therapy and antisense oligos in several ways. “The dystrophin gene with mutations in the hotspot region around exons 45-50, along with the additional removal of exon 51, contains much more of the dystrophin gene than the minigene and thus also generates a protein with greater functionality that is more similar to the normal protein,” said Dr. Gersbach.
The response in terms of both gene expression and protein production is also superior to previous strategies, sufficient to potentially have clinical impact. Just restoring 3 to 15% of dystrophin levels in skeletal muscle, as seen in the gene-edited mice, could counter skeletal and cardiac muscle decline if extrapolated to humans, and a 30% restoration would completely ameliorate the pathology. “We know from animal studies and the exon skipping studies that low levels of dystrophin are sufficient to have a beneficial effect,” said Dr. Gersbach.
“Gene editing provides permanent correction of the mutation responsible for DMD. It does not require lifelong delivery,” pointed out Eric Olson, PhD, The Robert A. Welch Distinguished Chair in Science at the University of Texas Southwestern Medical Center and corresponding author on the Long et al paper.
And last but certainly not least, CRISPR/Cas9 makes possible the multiplexing that has eluded conventional gene therapy. The ability to target more than one part of the dystrophin gene could bring 83% of patients into the candidate pool.
Of course a long road stretches from promising results in mice to a boy who can walk farther in six minutes and live beyond young adulthood, especially when gene therapy and antisense oligos, which had a head start compared to CRISPR/Cas9, have yet to reach clinical practice. Finding a way to treat all muscle groups systemically is still a hurdle, as is designing the most meaningful clinical trials. “While there are obvious differences between an oligonucleotide drug and a gene editing therapy, there is much that we can learn from those trials and what the FDA would like to see in the development of a DMD drug. A tremendous amount of work remains to understand the safety of in vivo delivery of CRISPR/Cas9. But we envision that CRISPR may represent a possible single treatment therapy,” summed up Dr. Gersbach.
When might the first clinical trial using myoediting to treat DMD begin? “I think within a few years, as soon as the process can be scaled up and safety issues can be addressed,” said Dr. Olson.


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