A cyclic pyrrole-imidazole polyamide reduces pathogenic RNA in CAG/CTG triplet repeat neurological disease models
Susumu Ikenoshita, … , Hiroshi Sugiyama, Norifumi Shioda
Susumu Ikenoshita, … , Hiroshi Sugiyama, Norifumi Shioda
Published September 14, 2023
Citation Information: J Clin Invest. 2023;133(22):e164792. https://doi.org/10.1172/JCI164792.
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Research Article Neuroscience

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

Abstract

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.

Authors

Susumu Ikenoshita, Kazuya Matsuo, Yasushi Yabuki, Kosuke Kawakubo, Sefan Asamitsu, Karin Hori, Shingo Usuki, Yuki Hirose, Toshikazu Bando, Kimi Araki, Mitsuharu Ueda, Hiroshi Sugiyama, Norifumi Shioda

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Figure 2

Attenuation of pathogenic CUG RNA foci and polyQ aggregates in DM1 and HD cell models by CWG-cPIP treatment.

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Attenuation of pathogenic CUG RNA foci and polyQ aggregates in DM1 and H...
(A) Cell viability assay in Neuro-2a cells treated with CWG-cPIP at concentrations of 0.1, 0.3, 1, 3, 10, and 30 μM. Statistics were performed by 1-way ANOVA with Bonferroni’s multiple-comparison test. n = 6 each. (B) Chemical structure of FITC-labeled CWG-cPIP and representative confocal images of FITC-labeled CWG-cPIP. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (C) Schematic representation of constructs used for RT-qPCR in cellulo and quantification of HaloTag mRNA levels. **P < 0.01, by 1-way ANOVA with Bonferroni’s multiple-comparison test. n = 8 each. #Rep., CUG repeat lengths. (D) Representative confocal images of CUG-RNA foci (white) in mouse primary neurons (scale bars: 5 μm) and quantification of CUG-RNA foci (right). **P < 0.01, by 2-sided, unpaired Student’s t test. CUG700 plus vehicle: n = 49 cells; CUG700 plus CWG-cPIP: n = 36 cells. (E) Representative confocal images of CUG-RNA foci (white) in DM1 patient–derived iNeurons (scale bars: 5 μm) and quantification of CUG-RNA foci. **P < 0.01, by 2-sided, unpaired Student’s t test. Vehicle: n = 61 cells; CWG-cPIP: n = 49 cells. (F) Schematic representation of constructs containing Egfp tagged with CAG repeat sequences in a coding region and representative confocal images of GFP-positive aggregates in Neuro-2a cells. Scale bars: 10 μm. Graph shows quantification of GFP-positive aggregates. **P < 0.01, by 1-way ANOVA with Bonferroni’s multiple-comparison test. n = 6 wells each. (G) Representative blots of lysates from HD patient–derived fibroblasts probed with 1C2 and HTT antibodies. Arrow indicates HTT products corresponding to the normal allele. Graphs show quantification of 1C2 and HTT. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Bonferroni’s multiple-comparison test. n = 5 experiments each. Data represent the mean ± SEM. Statistical data are provided in Supplemental Data File 6. Veh., vehicle treatment.