In the past several years, we have seen some major advancements in the study of Duchenne muscular dystrophy (DMD), a rare but devastating genetic disorder that causes muscle loss and physical impairments. Investigators using the gene-editing tool CRISPR have shown in previous studies, in rodent and canine models, that the repair or removal of key disease mutations is possible, as well as improvement of muscle function. Now, in a new study from researchers at the University of Missouri (MU) School of Medicine scientists have shown that employing CRISPR to edit mutations in muscle stem cells (MuSCs) may provide the means for lifelong correction of the genetic mutation responsible for the disorder.

Findings from the new study were published recently in Molecular Therapy through an article titled, “AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice.”

“Research has shown that CRISPR can be used to edit out the mutation that causes the early death of muscle cells in an animal model,” explained senior study investigator Dongsheng Duan, PhD, a professor in the department of molecular microbiology and immunology at the MU School of Medicine. “However, there is a major concern of relapse because these gene-edited muscle cells wear out over time. If we can correct the mutation in muscle stem cells, then cells regenerated from the edited stem cells will no longer carry the mutation. A one-time treatment of the muscle stem cells with CRISPR could result in continuous dystrophin expression in regenerated muscle cells.”

Children with DMD have a gene mutation that interrupts the production of a protein known as dystrophin. Without dystrophin, muscle cells become weaker and eventually die. Many children lose the ability to walk, and muscles essential for breathing and heart function ultimately stop working.

In the current study, researchers first delivered the gene-editing tools to normal mouse muscle through AAV9, a virus that was recently approved by the FDA to treat spinal muscular atrophy.

“CRISPR editing of MuSCs with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies,” the authors wrote. “However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice.”

Lead study investigator Michael Nance, an MD, PhD program student in Duan’s lab, added that “we transplanted AAV9 treated muscle into an immune-deficient mouse. The transplanted muscle died first then regenerated from its stem cells. If the stem cells were successfully edited, the regenerated muscle cells should also carry the edited gene.”

Amazingly, the researchers’ reasoning was correct, as they found abundant edited cells in the regenerated muscle. They then tested if muscle stem cells in a mouse model of DMD could be edited with CRISPR. Similar to what they found in normal muscle, the stem cells in the diseased muscle were also edited. Cells regenerated from these edited cells successfully produced dystrophin.

“This finding suggests that CRISPR gene editing may provide a method for lifelong correction of the genetic mutation in DMD and potentially other muscle diseases,” Duan concluded. “Our research shows that CRISPR can be used to effectively edit the stem cells responsible for muscle regeneration. The ability to treat the stem cells that are responsible for maintaining muscle growth may pave the way for a one-time treatment that can provide a source of gene-edited cells throughout a patient’s life.”

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