The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1

Abstract

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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Fig. 1: Treatment of adult skeletal muscle in the HSALR DM1 mouse model with RNA-targeting Cas9 eliminates CUG RNA foci.
Fig. 2: RNA-targeting Cas9 releases MBNL1 protein and reverses hallmarks of splicing dysfunction in HSALR DM1 adult muscle.
Fig. 3: RNA-targeting Cas9 promotes a global reversal of DM1-associated splicing dysfunction and increases the expression of genes associated with proper muscle function and mature muscle.
Fig. 4: Sustained expression of RNA-targeting Cas9 in WT adult muscle.
Fig. 5: Transient pharmacological immunosuppression promotes sustained expression of RNA-targeting Cas9 in adult muscle.
Fig. 6: Systemic treatment of HSALR DM1 mice with RNA-targeting Cas9 leads to sustained expression in various tissues, eliminates toxic RNA foci and reverses DM1-related mis-splicing.
Fig. 7: Systemic treatment of HSALR DM1 mouse model with RNA-targeting Cas9 reverses behavioural and electrophysiological features of the disease.

Data availability

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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Acknowledgements

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.

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Authors

Contributions

G.W.Y., R.B., D.A.N. and M.S.S. conceptualized and designed the study and wrote the paper. R.B., D.A.N., F.K. and H.L.G. performed tissue sectioning, staining and histopathology. R.B., D.M.R., S.M.B. and H.L.G. performed RNA extractions and qPCR. M.S.S. provided the HSALR mouse colony. R.B., J.D.T., C.A.N. and Ł.J.S. maintained the HSALR colony, performed adult injections and collected tissues. T.T. and M.M. performed P0 neonatal injections. S.A. and R.B. generated next-generation sequencing (NGS) libraries. R.B., P.L., F.K. and G.W.Y. performed NGS data analysis. A.M. packaged AAV and performed quality control of the virus. R.B., O.P. and M.M. performed electrophysiology. R.B. performed functional assays. R.B., D.A.N. and G.W.Y. analysed all data. G.W.Y. supervised the study.

Corresponding author

Correspondence to Gene W. Yeo.

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Competing interests

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 information

Supplementary Information

Supplementary figures, video captions and dataset captions.

Reporting Summary

Supplementary Video 1

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.

Supplementary Video 2

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.

Supplementary Dataset 1

Differential alternative-splicing events inferred by analysis of RNA-seq data.

Supplementary Dataset 2

Differential gene expression inferred by analysis of RNA-seq data.

Supplementary Dataset 3

GO analysis.

Supplementary Dataset 4

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

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