CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

Agtmaal, Ellen L. van; André, Laurène M.; Willemse, Marieke; Cumming, Sarah A.; Kessel, Ingeborg D G van; Broek, W.J.A.A. van den; Gourdon, Geneviève; Furling, Denis; Mouly, Vincent; Monckton, Darren G.; Wansink, Derick G.; Wieringa, Bé

doi:10.1016/j.ymthe.2016.10.014

Cited by 110 publications

(137 citation statements)

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“…Technologies that engage the DM1-linked repeat expansion on both the DNA and RNA levels have been investigated. Although excision of these large tracts in DNA followed by repair in their absence is possible (9), this genome engineering approach has not yet been realized with high efficiency. ASOs provide means to engage pathogenic RNAs directly but must be continuously re-administered for life and have experienced poor biodistribution in muscle in the clinic.…”

Section: Main Textmentioning

confidence: 99%

Reversal of molecular pathology by RNA-targeting Cas9 in a myotonic dystrophy mouse model

Batra

Nelles

Krach

et al. 2017

Preprint

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Abstract:The dominantly inherited, multi-systemic disease myotonic dystrophy type I (DM1) is caused by triplet repeat CTG expansions in the DMPK gene and is the most common form of adult-onset muscular dystrophy. Elimination of the toxic, repetitive CUG RNA constitutes a therapeutic for this disease. We report an RNA-targeting Cas9 (RCas9) system that supports efficient reversal of DM1 phenotypes via delivery to adult poly(CUG) DM1 mouse muscle using adeno-associated virus (AAV). We observe elimination of CUG RNA, restoration of CUG foci-associated Mbnl1 protein to wild-type subcellular localization, correction of DM1-type alternative splicing patterns in candidate genes including the voltage-gated chloride channel 1 (Clcn1) responsible for characteristic myotonia, recovery of Clcn1 staining, and reduction in centralized myonuclei. Our results establish RCas9 as a potential long-term in vivo therapeutic for DM1.One Sentence Summary: A repurposed CRISPR system termed RNA-targeting Cas9 reverses the molecular pathology associated with the most common type of adult onset muscular dystrophy in adult mouse muscle. Main text:Myotonic dystrophy type 1 is an autosomal inherited disorder characterized by CTG repeat expansions in the DMPK gene. The repetitive RNAs produced by this locus form nuclear RNA foci (1) that sequester RNA binding proteins such as MBNL1 (2, 3) and divert them from their homeostatic RNA processing activities (4, 5). Resulting loss of MBNL1 function is linked to hundreds of splicing defects that cause myotonia and progressive muscle degeneration (4,6,7). Since DMPK is widely expressed, DM1 pathology is multi-systemic leading to subcapsular cataracts, cognitive defects, and cardiac conduction defects that contribute to DM1-associated mortality (8). Currently available treatments for DM1 do not address the underlying etiology of this disease that affect more than 30,000 patients in the US alone and a recent clinical failure of a promising antisense oligonucleotide (ASO) targeting CUG repeat RNA (clinicaltrials.gov identifier: NCT02312011) highlights the need for new therapeutic modalities.Technologies that engage the DM1-linked repeat expansion on both the DNA and RNA levels have been investigated. Although excision of these large tracts in DNA followed by repair in their absence is possible (9), this genome engineering approach has not yet been realized with high efficiency. ASOs provide means to engage pathogenic RNAs directly but must be continuously re-administered for life and have experienced poor biodistribution in muscle in the clinic. RNA interference (RNAi) can be encoded in adenoassociated viral vectors to support long-term, targeted delivery to human tissue (10) but typically does not engage repetitive RNAs efficiently.By repurposing the CRISPR/Cas9 genome engineering system, we have recently demonstrated the ability of Cas9 to efficiently engage RNA (11) and eliminate microsatellite repeat expansion RNAs [Batra et al, 2017] in human cells. Utilizing a nuclease-null Cas9 (dCas9) fused to ...

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Section: Main Textmentioning

confidence: 99%

Reversal of molecular pathology by RNA-targeting Cas9 in a myotonic dystrophy mouse model

Batra

Nelles

Krach

et al. 2017

Preprint

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“…Reconstitution of these DNA-targeting systems in eukaryotic cells has supported powerful applications in genome editing and other DNA-level manipulations (Sheridan, 2017; Yu et al, 2017). A recent study demonstrated that MREs can be excised from DNA using genome engineering techniques (van Agtmaal et al, 2017), although simultaneous induction of a pair of double-stranded breaks flanking the MRE followed by DNA repair in the absence of the repeat has not been realized with sufficient efficiency. In contrast to RNA-directed systems, genome editing features the risk of permanent, off-target edits to DNA (Schaefer et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9

Batra

Nelles

Pirie

et al. 2017

Cell

226

200

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SUMMARY Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington’s disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of micro-satellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.

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“…Multiple groups have successfully edited genomes in cell and animal models of disease. Some have directly applied these approaches to microsatellite expansion diseases to remove the expanded repeat tract 40 or cause somatic instability leading to repeat contraction 41 . However, challenges remain to ensure both efficient and specific cleavage activity across multiple cells and tissues of the body.…”

Section: Discussionmentioning

confidence: 99%

Impeding transcription of expanded microsatellite repeats by deactivated Cas9

Pinto

Saxena

Oliveira

et al. 2017

Preprint

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SummaryTranscription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs' endothelial corneal dystrophy, and C9orf72-ALS/FTD.Eliminating or reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that a deactivated form of the Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction in the abundance of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/185496 doi: bioRxiv preprint first posted online Sep. 7, 2017; decreased myotonia. These observations suggest that transcription of microsatellite repeatcontaining RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

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CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

Cited by 110 publications

References 83 publications

Reversal of molecular pathology by RNA-targeting Cas9 in a myotonic dystrophy mouse model

Reversal of molecular pathology by RNA-targeting Cas9 in a myotonic dystrophy mouse model

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9

Impeding transcription of expanded microsatellite repeats by deactivated Cas9

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