A team of researchers at the University of California, San Diego (UCSD) School of Medicine have developed a new technique using CRISPR-Cas9 and have successfully reversed muscular dystrophy in mice.
The most common form of muscular dystrophy in adults is myotonic dystrophy type 1 (DM1). DM1 is caused by DMPK gene mutations that result in the production of RNA that is toxic to cells. In healthy people, the DMPK gene has between 3 and 37 repetitions of the CTG sequence, whereas patients with DM1 have hundreds to thousands of CTG repetitions, which results in the production of abnormally long RNA. The toxic RNA builds up and causes progressive muscle wasting and weakness and other debilitating symptoms.
CRISPR-Cas9 is being increasingly used by researchers to make edits to remove defective genes, correct mutations, and insert functional copies of genes. Rather than use CRISPR-Cas9 to target the DMPK gene directly, the UCSD researchers redirected the Cas9 enzyme to modify RNA in a technique they call RNA-targeting Cas9 (RCas9).
The researchers had previously used RCas9 in 2017 to eliminate around 95% of the abnormal RNA that caused type 1 and type 2 myotonic dystrophy and also eliminated abnormal RNA linked to Huntingdon’s disease and a type of ALS. The latest study takes the research a step further, with the researchers successfully reversing DM1 in a mouse model of the disease. Prior to the study, there was little evidence of the efficacy of CRISPR-mediated RNA targeting in vivo.
“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, explained the researchers.”
Since there was potential for the immune system of the mice to attack the RCas9 proteins, the researchers suppressed the immune system in the mice during the treatment and prevented an immune reaction and clearance.
The researchers reduced the abnormal RNA repeats by more than 50%, which was sufficient to reverse DM1 in mice to the point where the treated mice were indistinguishable from healthy mice. The researchers did not see signs of muscle damage and noted an increase in activity of genes involved in new muscle formation.
“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.” The researcher’s technique could potentially serve as an effective treatment for muscular dystrophy and other severe neuromuscular diseases.
You can read more about the study in the paper – The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1 – which was recently published in Nature Biomedical Engineering. DOI: 10.1038/s41551-020-00607-7