Impeding transcription of expanded microsatellite repeats by deactivated Cas9

Pinto, Belinda S.; Saxena, Tanvi; Oliveira, Rita C. M.; Méndez-Gómez, Héctor; Cleary, John D.; Denes, Lance T.; McConnell, Ona; Arboleda, J. D.; Xia, Guangbin; Swanson, Maurice S.; Wang, Eric T.

doi:10.1101/185496

Cited by 3 publications

(2 citation statements)

References 57 publications

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“…56 Finally, two other recent approaches exploited deactivated forms of Cas9, which do not cut DNA, to mediate the inhibition of DMPK gene transcription and degradation of toxic DMPK transcripts. 26,57 The relevance of these various approaches for clinical translation and therapeutic intervention in patients with DM1 remains to be assessed.…”

Section: Discussionmentioning

confidence: 99%

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice

et al. 2019

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Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3′ UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy.

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Section: Discussionmentioning

confidence: 99%

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice

et al. 2019

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“…However, when DNA breaks occur near repeat sequences, the repair machinery is activated, which leads to expansion growth and may cause further mutation of the repeats (56). Alternatively, CRISPR-based technologies capable of perturbing CWG repeat RNA have been extensively evaluated as potential therapeutics (57)(58)(59). However, because Cas proteins are bacterial in origin, they can be recognized as foreign by the immune system, and long-term expression may trigger an autoimmune response (60).…”

Section: Discussionmentioning

confidence: 99%

A cyclic pyrrole-imidazole polyamide reduces pathogenic RNA in CAG/CTG triplet repeat neurological disease models

Ikenoshita,

Matsuo,

Yabuki

et al. 2023

Journal of Clinical Investigation

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Expansion of CAG and CTG (CWG) triplet repeats causes several inherited neurological diseases. The CWG repeat diseases are thought to involve complex pathogenic mechanisms through expanded CWG repeat–derived RNAs in a noncoding region and polypeptides in a coding region, respectively. However, an effective therapeutic approach has not been established for the CWG repeat diseases. Here, we show that a CWG repeat DNA–targeting compound, cyclic pyrrole–imidazole polyamide (CWG-cPIP), suppressed the pathogenesis of coding and noncoding CWG repeat diseases. CWG-cPIP bound to the hairpin form of mismatched CWG DNA, interfering with transcription elongation by RNA polymerase through a preferential activity toward repeat-expanded DNA. We found that CWG-cPIP selectively inhibited pathogenic mRNA transcripts from expanded CWG repeats, reducing CUG RNA foci and polyglutamine accumulation in cells from patients with myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD). Treatment with CWG-cPIP ameliorated behavioral deficits in adeno-associated virus–mediated CWG repeat–expressing mice and in a genetic mouse model of HD, without cytotoxicity or off-target effects. Together, we present a candidate compound that targets expanded CWG repeat DNA independently of its genomic location and reduces both pathogenic RNA and protein levels. CWG-cPIP may be used for the treatment of CWG repeat diseases and improvement of clinical outcomes.

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Disease Mechanisms and Therapeutic Approaches in C9orf72 ALS-FTD

Mayl

Lee

2021

Biomedicines

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A hexanucleotide repeat expansion mutation in the first intron of C9orf72 is the most common known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Since the discovery in 2011, numerous pathogenic mechanisms, including both loss and gain of function, have been proposed. The body of work overall suggests that toxic gain of function arising from bidirectionally transcribed repeat RNA is likely to be the primary driver of disease. In this review, we outline the key pathogenic mechanisms that have been proposed to date and discuss some of the novel therapeutic approaches currently in development.

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Impeding transcription of expanded microsatellite repeats by deactivated Cas9

Cited by 3 publications

References 57 publications

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice

A cyclic pyrrole-imidazole polyamide reduces pathogenic RNA in CAG/CTG triplet repeat neurological disease models

Disease Mechanisms and Therapeutic Approaches in C9orf72 ALS-FTD

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