Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy. The main characteristics of DM1 are myotonia, progressive muscle weakness and wasting, and extensive systemic symptoms. DM1 belongs to a larger group of microsatellite disorders, which is associated with expansions of simple repetitive elements within specific genes. This autosomal dominant disease is caused by the expansion of CTG repeats in the 3'-UTR of the DMPK gene and its pathogenesis is mediated, at least in part, by a toxic RNA gain-of-function mechanism. Molecular hallmarks of DM1 cells expressing mutant DMPK transcripts are nuclear RNA foci. Their presence harms host cells resulting in a broad spectrum of abnormalities. Now, DM1 has become the paradigm for the RNA toxicity model of the disease pathogenesis.
CRISPR-Cas9 is an increasingly used technique for correcting genetic (DNA) defects that lead to various diseases. A few years ago, researchers at the University of California San Diego School of Medicine redirected the technique to instead modify RNA in a method they called RNA-targeting CRISPR-Cas9 (RCas9). In a new study, the researchers confirmed that a dose of RCas9 gene therapy can degrade toxic RNA and almost completely reverse the symptoms of a mouse model of myotonic dystrophy. The related research results were recently published online in the journal Nature Biomedical Engineering with the title of "The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1 ".
Usually, researchers can use CRISPR/Cas9 technology to quickly delete, insert, or alter a portion of a DNA sequence. Guided by gRNA, Cas9 targets specific sites in the DNA and forms a double-stranded break (DSB). During DSB repair, one or more genetic changes are introduced through the non-homologous end joining (NHEJ) or homology directed repair (HDR) pathway. RCas9 works similarly, but Cas9 is guided to an RNA molecule instead of DNA.
Researchers previously demonstrated that RCas9 efficiently and specifically eliminates repetitive RNAs—including the CUG repeats that cause DM1—in patient-derived cellular models. Motivated by the potential of a gene-therapy-based approach to promoting targeted, long-term treatments for genetic disease, here they further evaluated the potential clinical utility of RCas9 in a predictive animal model of DM1.
The researchers utilized a mouse model that expresses 250 CUG repeats driven by the human skeletal actin ACTA1 promoter (HSALR model) as an established preclinical model of DM1. They evaluated an AAV-packaged RCas9 system targeting CUG repeats in this model by either intramuscular or systemic administration in a manner that simulates potential treatments for adult-onset disease. Rcas9 reduced the abnormal repetitive RNA by more than 50%, which changed according to the tissue. And after treatment, the dystonic mice became almost the same as healthy mice.
Figure 1. Treatment scheme for dual-vector administration of RNA-targeting Cas9 and sgRNA. (Batra R, et al. 2020)
At first, the researchers were concerned that Rcas9 protein from bacteria might cause an immune response in mice and be quickly eliminated. So they tried to suppress the immune system briefly during the treatment. Their study shows that transient immunosuppression typically used in conjunction with AAV administration is sufficient to promote sustained transgene expression in mice without overt adverse immunological effects. There is also growing evidence that the expression of therapeutic transgenes at specific times in development or specific tissues can be sufficient to induce tolerance to the transgene. These results reveal a second route to sustained RCas9 expression in large tissue volumes in mice.
Generally, the study indicates the potential of RCas9 and potentially other RNA-targeting CRISPR-based approaches to address disease. However, it remains to be seen whether rcas9 based therapies will work in humans, or whether they will cause harmful side effects, such as an undesirable immune response. Advancement of this approach to patients will rely on future studies in large animals to further assess the safety, dose levels and immunosuppression regimens that are required for safe and long-term treatments.