Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3′ untranslated region of DMPK. 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. 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 sustained reversal of DM1 phenotypes provides further support that RNA-targeting Cas9 is a viable strategy for treating DM1 and other MRE-associated diseases.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. Intramuscular RCas9 injection NGS data are available at the GEO repository (GSE152033) and can also be viewed using the UCSC genome browser (https://genome.ucsc.edu/s/ranjan99/RCas9_HSA_IM). The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request.
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This work was partially supported by the NIH (grant no. NS103172) to G.W.Y. and M.S.S. This work is also partially supported by the Muscular Dystrophy Association MVP (grant no. 575855) to R.B. and Locanabio.
G.W.Y. is a cofounder, member of the board of directors, equity holder and paid consultant of Locanabio. D.A.N. is a cofounder and an equity holder of Locanabio. R.B. is an equity holder and employee of Locanabio. M.S.S. is an equity holder of Locanabio and a Scientific Advisory Board member of Skyhawk Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California San Diego and the University of Florida, Gainesville in accordance with their conflict of interest policies. The other authors declare no other competing interests.
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Supplementary figures, video captions and dataset captions.
Representative video of hindlimb pull-test measurements showing myotonia and latency to muscle relaxation in P0 neonatal control-treated and P0 neonatal RCas9-CTG-treated HSALR mice.
Representative video of hindlimb pull-test measurements showing myotonia and latency to muscle relaxation in 8-week-old control-treated and 8-week-old RCas9-CTG-treated HSALR mice.
Differential alternative-splicing events inferred by analysis of RNA-seq data.
Differential gene expression inferred by analysis of RNA-seq data.
Differential gene expression inferred by analysis of RNA-seq data.
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Batra, R., Nelles, D.A., Roth, D.M. et al. The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1. Nat Biomed Eng (2020). https://doi.org/10.1038/s41551-020-00607-7