CRISPR-mediated RNA targeting to treat Myotonic Dystrophy

RNA-targeting CRISPR

Date: 16th September 2020

Many severe neuromuscular diseases are caused by a build-up of toxic repetitive RNA.  Now scientists have used CRISPR-mediated RNA targeting to eliminate this RNA, thereby reversing the disease phenotypes in mouse models of myotonic dystrophy type 1.

Many eukaryotic genomes contain small repetitive tracts, called microsatellites, that serve regulatory functions.  However, errors during replication or DNA repair can cause these repetitive elements to enlarge leading to an accumulation of RNA that is toxic to the cell.  This results in cellular damage and diseases such as myotonic dystrophy type 1 (DM1) – the most common adult onset muscular dystrophy, Huntington’s disease and familial amyotrophic lateral sclerosis (ALS) to name but a few.

Now scientists from the University of California San Diego, US, led by Gene Yeo, have packaged RNA-targeting CRISPR–Cas9 (RCas9) into adeno-associated virus (AAV) vectors and shown that intramuscular or systemic injections of the therapy into adult and neonatal mouse models of DM1 can target and eliminate the toxic repetitive elements leading to an overall improvement in muscle health.

Yeo and his team had previously generated a RNA-specific CRISPR system by fusing a RNA endonuclease to nuclease-null (or dead) Cas9  (Rcas9).  This RCas9 could reverse dysfunctional RNA splicing in myotonic dystrophy type 1 patient cells which cause CUG repeats.  Now the team wanted to build on this system and assess its potential as a RNA-targeting gene therapy for DM1.

RCas9 as a potential gene therapy

To start the researchers packaged the system into two AAV vectors; one containing the RCas9 and the other a promoter-driven single-guide RNA (sgRNA).  Upon intramuscular injection into adult DM1 modelled mice the RCas9 eliminated CUG repeat foci and reversed the hallmark splicing defects in adult DM1 skeletal muscle 4 weeks post injection.  Furthermore, sequestration of MBNL family proteins by the CUG-repeat RNA foci, which is an important molecular pathology feature of DM1, was ameliorated in the treated mice.

Encouraged by the efficient reversal of characteristic molecular and cellular manifestations of DM1 the team went on to show that RCas9 was able to reverse transcriptome-wide DM1-associated splicing defects in adult skeletal muscle and increased the expression of genes associated with proper muscle function and mature muscle.

Induction of the immune response

Perhaps not surprisingly, the team also found that immune response-linked genes were upregulated in the RCas9 treated mice, as Cas9 proteins are potentially immunogenic, this phenomenon was explored in further depth. Whilst there was an increase in antigen presentation and T-cell response pathways there was no substantial alterations in muscle morphology or signs of immune-response-linked muscle damage suggesting this response was not deleterious to the muscle tissue.

However, whilst there appeared to be little muscle damage induced by the immune response, the adaptive immune response could potentially target the gene therapy over time, rendering it ineffective.  To address this the team pharmacologically suppressed the immune system of the mice for two weeks post treatment.  They found that transient immunosuppression could increase transduction and was able to prevent a low-level cytotoxic immune response to treated cells suggesting this may be an important strategy for increasing efficiency of the system.

gene therapy

Systemic administration of RCas9

Finally, the team assessed systemic administration of the RCas9 therapy in neonatal and adult DM1 modelled mice.  This led to sustained expression of dCas9 in in disease-relevant tissues, such as the lower leg muscle, quadriceps, diaphragm and heart, and eliminated toxic RNA foci and reversed DM1-related mis-splicing.  The team saw an efficient and sustained reversal of physiological manifestations of the disease, with behavioural and electrophysiological features of the disease being reversed.

Conclusions and future applications

The data presented here demonstrated that RCas9 treatment together with transient immunosuppression promoted sustained and specific therapeutic activity through both local and systemic administration in adult and neonatal mice.  The therapy reversed many physiological manifestations of DM1, and importantly there were no significant adverse responses to the treatment.

The team are now looking to expand this pre-clinical trial, hoping to advance the development of the program to the benefit thousands of DM1 patients.  Indeed, Yeo is placed well to accelerate this potential therapy into the clinic.   He is co-founder of Locanabio, a leader in RNA-targeted gene therapy, and the work was partially funded by the company, their aim to bring RNA-targeting CRISPR-Cas9 to clinical trials and beyond.

Whilst several clinical trials are already underway to assess the efficacy and safety of CRISPR therapies most involve in vitro editing of the cells which are then transplanted back into the patient.  However, in March this year Allergan and Editas Medicine were the first to attempt in vivo CRISPR therapy in patients to treat Leber congenital amaurosis 10, an eye disorder.  It will be the first trial to shed light on the safety of in vivo CRISPR editing and will be an important milestone for the CRISPR field and for RCas9 therapy of DM1.

In contrast, as a concept several clinical trials have supported the use of AAV delivery for genetic muscular diseases indicating they are potentially safe for systemic gene therapies.  However, yet to be assessed is the safety of RNA-targeting CRISPR therapies in humans.  The first step is likely to involve studies in larger animal models to assess this more thoroughly and to identify proper AAV dosing, administration methods and specific immunosuppression regimens.

One likely adaptation to the RCas9 therapy described here is the recent emergence of smaller RNA-specific Cas proteins.  These could be packaged together with a sgRNA into one AAV vector decreasing AAV dosage and therapeutic cost.

We have seen here the potential limitation of gene therapies that invoke an immune response. Whilst suppressing the immune system with drugs moderately enhanced the expression of RCas9 there may be more efficient ways of circumventing the immune system. We recently reported that scientists have modulated the immune system using a CRISPR-based synthetic repressor which subsequently enhanced AAV-delivered gene therapy whilst others are trying to immunosilence Cas9 bringing it into stealth mode.

However RCas9 proceeds, this work highlights the exciting potential of this CRISPR system as a viable treatment for the many crippling diseases that involve the accumulation of RNA repeats. We hope that by treating the underlying biology and cause of the disease rather than treating the symptoms this can improve patient outcomes and health.


For more information please see the press release from the University of California San Diego or Locanabio, or visit them on our synthetic biology map.

Batra, R., D. A. Nelles, D. M. Roth, F. Krach, C. A. Nutter, T. Tadokoro, J. D. Thomas, Ł. J. Sznajder, S. M. Blue, H. L. Gutierrez, P. Liu, S. Aigner, O. Platoshyn, A. Miyanohara, M. Marsala, M. S. Swanson and G. W. Yeo (2020). “The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1.” Nature Biomedical Engineering.