Myotonic dystrophy type I (DM1) is the most common type of adult-onset muscular dystrophy. DM1 is caused by mutations in the DMPK gene. A normal DMPK gene has 3 to 37 repetitions of the CTG sequence, while in DM1, there are hundreds to thousands of repetitions of this sequence. When a DMPK gene with too many CTG repeats is transcribed, the resulting RNA is too long. This abnormally long RNA is toxic to cells, and those affected experience progressive muscle wasting and weakness.

CRISPR-Cas9 is a technique increasingly used in efforts to correct the genetic defects that cause a variety of diseases. Now a research team from the University of California, San Diego (UCSD), School of Medicine, reports they redirected the technique to modify RNA in a method they call RNA-targeting Cas9 (RCas9), to eliminate the toxic RNA and almost fully reverse symptoms in a mouse model of myotonic dystrophy.

Their findings, “The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1,” was published in Nature Biomedical Engineering and led by Gene Yeo, PhD, professor of cellular and molecular medicine at UCSD School of Medicine.

Myotonic dystrophy is part of a group of inherited disorders called muscular dystrophies. There are two major types of myotonic dystrophy: type 1 and type 2. The muscle weakness associated with type 1 particularly affects muscles farthest from the center of the body, such as those of the lower legs, hands, neck, and face. Muscle weakness in type 2 primarily involves muscles close to the center of the body, such as those of the neck, shoulders, elbows, and hips. The two types of myotonic dystrophy are caused by mutations in different genes.

“Many other severe neuromuscular diseases, such as Huntington’s and ALS, are also caused by similar RNA buildup,” explained Yeo. “There are no cures for these diseases.” Yeo led the study with collaborators at Locanabio and the University of Florida.

CRISPR-Cas9 works by directing Cas9 to cut a specific target gene, allowing researchers to inactivate or replace the gene. However, the Cas9 in the RCas9 method is guided to an RNA molecule instead of DNA. In a previous study, Yeo and his team established RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags. In a 2017 study, in lab models and patient-derived cells, the researchers used RCas9 to eliminate 95% of the abnormal RNA linked to myotonic dystrophy type 1 and type 2, one type of ALS and Huntington’s disease.

In the current study, the method goes further, by reversing myotonic dystrophy type 1 in a mouse model of the disease. “Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking,” noted the researchers. “Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia.”

The researchers packaged RCas9 in a non-infectious virus. They then gave the mice a single dose of the therapy or a placebo. RCas9 reduced the abnormal RNA repeats by more than 50%, varying a bit depending on the tissue, and the treated myotonic dystrophy mice became indistinguishable from healthy mice.

Green muscle fibers with RCas9 (the therapeutic candidate for myotonic dystrophy) have eliminated their toxic RNA (red), whereas fibers lacking RCas9 (dark) have persisting toxic RNA (red). Source: UC San Diego Health Sciences

To prevent the potential of the RCas9 proteins, developing an immune reaction in the mice, the researchers tried suppressing the mice’s immune systems briefly during treatment. As a result, they were surprised to see that they successfully prevented immune reaction and clearance. The researchers did not see signs of muscle damage, but found an increase in the activity of genes involved in new muscle formation.

Yeo believes the findings will open a new avenue of understanding and lead the way for treating other genetic diseases. “This opens up the floodgates to start testing RNA-targeting CRISPR-Cas9 as a potential approach to treat other human genetic diseases—there are at least 20 caused by buildup of repetitive RNAs,” Yeo added.

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