A CRISPR gene activation technique has been shown to prevent and reverse disease symptoms in a mouse model of muscular dystrophy. This no-cut CRISPR method increased the expression of the laminin-α1 gene, something that had been difficult to do previously, because of the gene’s large size. Not only were muscle wasting and paralysis symptoms prevented in the pre-symptomatic transgenic mice that were treated with this technique, but symptomatic mice were also helped.

The work was published in Nature in a paper titled, “A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene.”

Muscular dystrophies are a group of inherited muscle-wasting diseases. One subtype, congenital muscular dystrophy type 1A (MDC1A), is caused by mutations in the gene Lama2, which encodes laminin-α2. The Lama2 mutations can cause the loss of the peripheral nerves’ protective myelin coating and disrupt the stability of muscle fibers.

Rodent studies have previously shown that increasing expression of the related gene, Lama1 (which encodes laminin-α1), can alleviate symptoms in a muscular dystrophy mouse model, but the gene’s large size makes it technically challenging to achieve with standard gene therapy methods.

To address this issue, Ronald Cohn, MD, president and CEO of the Hospital for Sick Children in Toronto, Ontario, Canada, and Evgueni Ivakine, PhD, senior research associate at the Hospital for Sick Children, co-senior authors on the paper, developed a CRISPR-mediated gene activation system to increase the expression of the Lama1 in a mouse model of MDC1A.

The team modulated expression of Lama1 in the MDC1A mouse model using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators, and single-guide RNAs (gRNA) that target the Lama1 promoter. The gRNA directs the inactive Cas9 and the VP64 activators to the site of the Lama1 gene, resulting in the increase of Lama1 gene expression without genome editing.

When pre-symptomatic mice were treated with this approach, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis.

“Targeting disease modifier genes in this way could benefit a wider range of patients” noted Kate Adcock, PhD, director of research and innovation, Muscular Dystrophy UK, “as the technique doesn’t depend on an individual mutation.”

“The major advance of this approach is the potential to treat complex diseases caused by multiple mutations, maybe even affecting different genes,” noted Alena Pance, PhD, senior staff scientist at the Wellcome Trust Sanger Institute, UK. “Rather than attempting to repair all the defects,” Pance added, “if a modifier gene can be found as was shown in this study, its activation can overcome the pathology of the disease.”

In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, the team showed that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic transgenic mice with apparent hindlimb paralysis and muscle fibrosis.

“As this system does not lead to breaks in the DNA, it is deemed safer,” notes Pance. “However, the design of the guide RNAs to provide the specific location in the genome follows the same principles as those applied to genome editing, so the potential for off-target effects is still an important issue to consider.” Although the authors assessed this point by identifying potential binding sites, Pance notes that it is unclear what using a general gene activator, which has the potential to induce expression of any gene to which it is delivered, could do in an off target location.

“Finally” asserts Pance, “the long term effects of the therapy will also need to be evaluated.” She adds that “it will be important to understand the therapeutic timeline to establish any potential clinical protocol that could be applied to humans.”

CRISPR is a versatile tool, in part because it can be used to upregulate and downregulate the expression of key genes. The authors propose that in the future, combinatorial therapeutic approaches might help to treat this and other genetic disorders by “turning up” protective genes and “turning down” detrimental genes.