Research ArticleNeuroscienceTherapeutics Open Access | 10.1172/JCI164792

Susumu Ikenoshita,1,2 Kazuya Matsuo,1 Yasushi Yabuki,1,3 Kosuke Kawakubo,1,3 Sefan Asamitsu,1 Karin Hori,1 Shingo Usuki,4 Yuki Hirose,5 Toshikazu Bando,5 Kimi Araki,6,7 Mitsuharu Ueda,2 Hiroshi Sugiyama,5,8 and Norifumi Shioda1,3

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

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1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Matsuo, K. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Yabuki, Y. in: JCI | PubMed | Google Scholar

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Kawakubo, K. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Asamitsu, S. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Hori, K. in: JCI | PubMed | Google Scholar

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Usuki, S. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Hirose, Y. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Bando, T. in: JCI | PubMed | Google Scholar

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Araki, K. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Ueda, M. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Sugiyama, H. in: JCI | PubMed | Google Scholar |

1Department of Genomic Neurology, Institute of Molecular Embryology and Genetics (IMEG),

2Department of Neurology, Graduate School of Medical Sciences,

3Graduate School of Pharmaceutical Sciences, and

4Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, Japan.

5Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

6Institute of Resource Development and Analysis and

7Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan.

8Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Kyoto, Japan.

Address correspondence to: Norifumi Shioda, Genomic Neurology, IMEG, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Phone: 81.96.373.6633; Email: shioda@kumamoto-u.ac.jp.

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Find articles by Shioda, N. in: JCI | PubMed | Google Scholar |

Authorship note: SI and KM are co–first authors and contributed equally to this work.

Published September 14, 2023 - More info

Published in Volume 133, Issue 22 on November 15, 2023
J Clin Invest. 2023;133(22):e164792. https://doi.org/10.1172/JCI164792.
© 2023 Ikenoshita et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published September 14, 2023 - Version history
Received: August 24, 2022; Accepted: September 12, 2023 View PDF 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.

Introduction

Short tandem repeats (STRs), also known as microsatellites, are polymorphic repeat sequences with 1–6 bp motifs scattered throughout the human genome (1). STRs are highly unstable in a repeat length–dependent manner, and the expansion of repeat length across generations results in diseases that primarily affect the central nervous system (2, 3). In particular, the expansion of CAG and CTG (CWG) triplet repeats cause many neurological diseases. These repeats can be classified into the following 2 types according to their genomic location: (a) CAG repeat expansion in coding regions; for example, in Huntington’s disease (HD); spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, and 17; spinal and bulbar muscular atrophy; and dentatorubral pallidoluysian atrophy and (b) CTG repeat expansion in noncoding regions, especially the 3′-UTRs; for example, in myotonic dystrophy type 1 (DM1) and SCA8 (2–5). While CAG repeat diseases in coding regions typically change the repeat tract size by 10 or fewer units per generation, CTG repeat diseases in noncoding regions increase by 100 to 10,000 units per generation (6, 7).

The mechanisms by which expanded CAG repeats in coding genes contribute to disease pathogenesis have been extensively discussed at DNA, RNA, and polyglutamine (polyQ) levels. Translated polyQ tracts form amyloid cores, initiating protein misfolding and aggregation that ultimately leads to neurodegeneration (8, 9). As causative genes with CAG repeat expansion have no sequence homology or functional similarity (2), expanded polyQ tracts are implicated as causal factors in CAG repeat diseases of coding regions. At the RNA level, the interruption of penultimate CAA within the glutamine-encoding sequence is closely linked to the timing of HD onset, and the mutation with loss of the CAA codon accelerates the onset, regardless of the polyQ tract length (10–12). In addition, the CAA interruption thermodynamically destabilizes the hairpin-structured RNA transcripts from the CAG tract in SCA1 and SCA2 (13), suggesting a link between RNA secondary structures and the pathogenesis of polyQ diseases. At the DNA level, some genes involved in DNA maintenance, such as MLH1 and PMS2, are implicated as rate determinants for the onset of HD by modifying the somatic expansion of CAG repeat DNA (11, 14).

CTG repeat expansion diseases in noncoding regions are mainly driven by RNA toxicity (5). DM1 is caused by a CTG repeat expansion in the 3′-UTR of DMPK and is the most common neuromuscular disorder (15–17). DM1 (OMIM #160900) is characterized by myotonia, muscle weakness, and cognitive dysfunction. CUG RNA transcribed from the expanded CTG repeats adopts a highly stable mismatched hairpin structure that forms nuclear RNA foci (18–20). Although the toxic mechanism of nuclear RNA foci remains unclear, CUG RNA–binding proteins such as the muscleblind-like (MBNL) family are sequestered, and CUG-binding protein 1 (CUGBP1) is upregulated by nuclear RNA foci, triggering aberrant alternative splicing of specific pre-mRNAs (21, 22).

In addition to the pathogenic mechanisms of polyQ toxicity and RNA toxicity, the CWG repeats expansion may also induce cell death indirectly by repeat-associated non-AUG (RAN) translation into toxic polypeptides (23, 24). RAN translation was first reported in the noncoding CTG repeat diseases DM1 and SCA8 (23) and has also been found in some coding CAG repeat diseases, including HD (24, 25).

CWG repeat diseases are thought to be caused by highly complex intracellular mechanisms, and no effective treatment has been developed to date. Antisense oligonucleotides (ASOs) that eliminate pathogenic repeat RNAs have been developed. However, a series of clinical trials using ASOs have been terminated (26–29). To address this issue, we focused on the transcription inhibition of CWG repeat DNA as a therapeutic target. Here, we assessed the potential of a CWG triplet repeat DNA-targeting compound cyclic pyrrole–imidazole polyamide (CWG-cPIP) to inhibit expanded CWG repeat–derived mRNA transcription in DM1- and HD-derived human cells, as well as to control neuronal dysfunction in adeno-associated virus–mediated (AAV-mediated) CWG repeat–expressing mice and a genetic mouse model of HD.

Results

CWG-cPIP binds preferentially to repeat-expanded CWG DNA. PIPs are composed of amide-linked N-methyl pyrrole (Py) and N-methyl imidazole (Im) residues. PIPs can be optimized and synthesized to target DNA sequences and bind noncovalently to DNA minor grooves in a sequence-specific manner. Im/Py pairs recognize G/C base pairs, whereas Py/Py, β-alanine, and γ-turn pairs recognize A/T and T/A bp (30, 31). We have previously developed many types of PIPs with sequence specificity, including anticancer agents (32), DNA fluorescence probes (33), and gene regulators (34, 35). In addition, we recently found that cyclic-type PIPs (cPIPs) with 2 γ-turn units showed higher DNA sequence selectivity and binding affinity than did traditional hairpin-type PIPs (hPIPs) (36). On the basis of these chemical discoveries, we developed a CWG-cPIP for CWG repeat diseases (Figure 1A) (37).

Figure 1

Transcriptional inhibition of CWG repeat DNA by CWG-cPIP. (A) Chemical structure of CWG-cPIP and CWG-hPIP; a schematic illustration of DNA sequence recognition of CWG-cPIP (bottom left); and molecular models of CWG-cPIP/double-stranded CWG-DNA complex by computer-assisted molecular simulation. (B) Nucleic acid sequences used for the Tm assay and quantification of ΔTm. The number on the x axis corresponds to the nucleic acid sequence on the left legend. *P < 0.05 and **P < 0.01, by 2-sided, unpaired Student’s t test. n = 2 [1. d(CAG/CTG); 3. AT rich; 5. d(CAG)10; 7. d(CGG)10; 9. r(CUG)10; 10. r(CAG)10]; n = 3 [2. d(CCG/CGG); 4. GC rich; 6. d(CTG)10; 8. d(CCG)10]. (C) Schematic representation of the in vitro transcription arrest assay. (D) Representative urea polyacrylamide gel electrophoresis for the in vitro transcription arrest assay (left). CWG-cPIP concentrations were 1.25, 2.5, and 3.75 μM. The arrow and bracket represent transcribed full-length RNAs and arrested-form RNAs, respectively. Graph on the right shows quantification of the arrested RNAs. **P < 0.01, by 2-way ANOVA with Bonferroni’s multiple-comparison test. n = 3 each. L, ladder; nt, nucleotide. Data represent the mean ± SEM. Statistical data are provided in Supplemental Data File 6.

To investigate the selectivity and binding affinity of CWG-cPIP to the target DNA sequence, we conducted a melting temperature (Tm) assay, wherein ΔTm was measured for several sequences (ΔTm = Tm [DNA or RNA + PIP] – Tm [DNA or RNA]). CWG-cPIP bound to double-stranded CWG DNA but not to AT-rich or GC-rich double-stranded DNA with high specificity (Figure 1B and Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI164792DS1). To further investigate the binding properties of CWG-cPIP, we performed a Tm assay using 5′-(CAG)10-3′ and 5′-(CTG)10-3′ repeat DNAs containing 3 A/A and T/T mismatched pairs, respectively. CWG-cPIP also showed a high binding affinity for these CWG-mismatched repeat DNAs Figure 1B) (37). Furthermore, we confirmed that CWG-cPIP does not bind to CWG repeat RNA. Importantly, CWG-cPIP showed a significantly higher binding affinity than did a traditional CWG-hPIP for CWG repeat DNA in both double-stranded and mismatched structures (38). Unexpectedly, both CWG-cPIP and CWG-hPIP showed high affinity for the 5′-(CCG)10-3′ repeat DNA (Figure 1B and Supplemental Table 1). To elucidate the underlying cause of this phenomenon, we performed molecular modeling studies of CWG-cPIP binding to repeat DNA (Supplemental Figure 1). We found that CCG repeat DNA interacted with CWG-cPIP at the same proximal distance as CWG repeat DNA, suggesting that high affinity for CCG repeat DNA is a common characteristic of PIPs targeting CWG repeat DNA.

PIPs are known to stably interfere with transcription elongation by RNA polymerase II (pol II) for more than 20 hours in vitro (39). During transcription elongation, pol II recognizes PIPs bound to DNA through its own Switch 1 region and is arrested 2–5 bp upstream of the site (39). To investigate the inhibitory effect of CWG-cPIP on transcription elongation by pol II, we performed an in vitro transcription arrest assay using CTG repeat DNA containing the normal range (CTG)10 or the pathogenic range (CTG)73, which produces 321 nt RNA under the T7 promoter (Figure 1C). As the concentration of CWG-cPIP increased, the amount of transcribed full-length RNA decreased (arrow, 321 nucleotide), and multiple arrested RNAs accumulated (bracket) (Figure 1D). Quantitative evaluation showed that CWG-cPIP treatment produced significantly more arrested products from (CTG)73 DNA than from (CTG)10 DNA (Figure 1D). These results suggested that CWG-cPIP inhibited pol II transcription elongation by preferentially targeting repeat-expanded DNA rather than normal repeat DNA.

CWG-cPIP inhibits the production of pathogenic CUG RNA in DM1 and polyQ in HD cells. We examined whether CWG-cPIP is effective in cells with pathogenic CWG repeat DNA sequences. First, we investigated the cytotoxicity of CWG-cPIP in intact Neuro-2a cells using a cell viability assay. We found that CWG-cPIP, even at a concentration of 30 μM for 48 hours, had no significant impact on cell viability (Figure 2A). To investigate cell membrane permeability and intracellular residence duration of CWG-cPIP, we synthesized FITC-labeled CWG-cPIP (Supplemental Figure 2) and administered it to intact Neuro-2a cells. We observed FITC-labeled CWG-cPIP (1 μM) in cell nuclei using confocal microscopy for more than 3 days without drug delivery systems (DDSs) such as liposomes (Figure 2B). To assess the off-target effects of CWG-cPIP on gene expression, we performed RNA-Seq analysis of RNAs extracted from the control fibroblasts along with spike-in control RNAs (40) 7 days after treatment with CWG-cPIP (1 μM). Based on a cutoff of an adjusted P value of less than 0.05 and a |log2 fold change| of greater than 0.5, we observed no changes in gene expression levels following the treatment, suggesting that CWG-cPIP had no significant effect on global transcription (Supplemental Figure 3A and Supplemental Data File 1).

Figure 2

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

Next, we examined the effect of CWG-cPIP on the production of pathogenic CUG RNA in Neuro-2a cells transfected with a plasmid expressing (CUG)10, (CUG)180, or (CUG)700 repeats in the 3′-UTR of HaloTag mRNA. To normalize transfection efficiency, we used a dual-promoter vector expressing 2 different genes: HaloTag with CTG repeats and Egfp as an internal reference (Figure 2C). We observed that HaloTag-(CUG)700 mRNA expression was significantly decreased 12 hours after CWG-cPIP treatment at concentrations as low as 0.1 μM and over 50% at 1 μM compared with that after vehicle treatment. In contrast, HaloTag-(CUG)10 mRNA expression was suppressed by only 20%, even at the highest concentration of 1 μM CWG-cPIP, compared with expression levels after vehicle treatment. Thus, treatment with CWG-cPIP considerably suppressed the expression of HaloTag-CUG mRNA with expanded repeats (Figure 2C). Compared with expression levels after vehicle treatment, treatment with 3 μM CWG-hPIP suppressed HaloTag-(CUG)700 mRNA expression by approximately 15% (Supplemental Figure 4A). In primary mouse cortical neurons transfected with a plasmid expressing (CUG)10 or (CUG)700 repeats in the 3′-UTR of Egfp mRNA, treatment with CWG-cPIP (1 μM for 14 days) considerably suppressed the expression of EGFP-(CUG)700 mRNA but not EGFP-(CUG)10 mRNA (Supplemental Figure 4B).

Next, we performed FISH with a Cy5-labeled (CAG)10 repeat probe to detect CUG RNA foci in mouse primary neurons transfected with a plasmid expressing (CUG)10 or (CUG)700 repeats in the 3′-UTR of Egfp mRNA. EGFP-(CUG)10 mRNA–expressing neurons had no detectable CUG RNA foci, whereas EGFP-(CUG)700 mRNA–expressing neurons remarkably accumulated nuclear CUG RNA foci. The number of nuclear CUG RNA foci was significantly decreased following CWG-cPIP treatment at 1 μM for 14 days (Figure 2D). We examined the inhibitory effect of CWG-cPIP on endogenous CTG repeat–derived CUG RNA foci using DM1 patient–derived fibroblasts and induced neurons (iNeurons). Treatment with 1 μM CWG-cPIP for 3 days significantly reduced the number of nuclear CUG RNA foci in DM1 fibroblasts and iNeurons (Figure 2E and Supplemental Figure 4C).

We further examined whether CWG-cPIP inhibits pathogenic mRNA derived from coding gene expansion. Neuro-2a cells were transfected with a plasmid expressing HaloTag mRNA tagged with a (CAG)23 or (CAG)74 repeat sequence within a part of exon 1 of the HTT gene (Supplemental Figure 4D) and treated with CWG-cPIP for 12 hours. CWG-cPIP effectively suppressed HaloTag-(CAG)74 mRNA expression but not HaloTag-(CAG)23 mRNA expression at a lower concentration (Supplemental Figure 4D).

We also assessed whether treatment with CWG-cPIP suppresses polyQ inclusion body formation in Neuro-2a cells transfected with a plasmid expressing Egfp tagged with a (CAG)23 or (CAG)74 repeat sequence within exon 1 of the HTT gene, termed EGFP-Q23 and EGFP-Q74, respectively. We observed EGFP-positive aggregates of various sizes in the nuclei and cytoplasm of EGFP-Q74–expressing cells but not in EGFP-Q23–expressing cells, and EGFP-positive aggregates were significantly reduced by CWG-cPIP treatment (Figure 2F). The levels of polyQ-expanded huntingtin (HTT) protein detected by an anti-polyQ tract antibody (clone 1C2) markedly decreased following CWG-cPIP treatment in HD patient–derived fibroblasts compared with their levels in vehicle-treated fibroblasts. Importantly, there were no changes in normal HTT protein levels in HD fibroblasts following CWG-cPIP treatment (Figure 2G).

Treatment with CWG-cPIP ameliorates cognitive deficit in AAV-mediated CWG repeat–expressing mice. We assessed the potential of CWG-cPIP in inhibiting the production of pathogenic CUG RNA foci and polyQ in vivo and restoring CWG repeat disease–mediated changes at the behavioral, physiological, and molecular levels. Intravenously administered PIPs could not be detected in the mouse brain by PET imaging (41), suggesting that there was little brain translocation of PIPs following peripheral administration. Thus, we administered CWG-cPIP intracerebrally to investigate its effect on brain function in mouse models of CWG repeat diseases.

First, FITC-labeled CWG-cPIP (Supplemental Figure 2) was injected bilaterally into the mouse hippocampus, and its tissue distribution and retention for up to 7 days were assessed by histological analysis. FITC-labeled 83 μg/kg CWG-cPIP (1.5 nmol) was rapidly delivered to the cell nuclei of the hippocampus without any DDS and retained for at least 7 days. Moreover, we observed no cell death in the CWG-cPIP–injected hippocampus, as determined by cleaved caspase-3 immunoreactivity (Supplemental Figure 5).

The off-target effects of CWG-cPIP in vivo were investigated in the hippocampi 21 days after the treatment (83 μg/kg), and differentially expressed genes were detected only in 0.74% (Supplemental Figure 3B and Supplemental Data File 2). Among these genes, only Inhbe contained a (CTG)16 repeat, which is predominantly expressed in the liver (42).

To assess whether CWG-cPIP ameliorates brain dysfunction in CWG repeat diseases in vivo, we generated brain-specific and rapid-onset models through the following gene transfer into the bilateral CA1 region of the hippocampus using AAV serotype 9: insertion of (CTG)10 or (CTG)300 repeats into the 3′-UTR of Egfp mRNA (in the hippocampus of mice referred to herein as CUG10 and CUG300 mice), and EGFP-tagged (CAG)23 or (CAG)74 repeats within exon 1 of the HTT gene (in the hippocampus of mice referred to herein as Q23 and Q74 mice) (Figure 3A). CWG-cPIP did not affect the stability of recombinant AAV capsid proteins in vitro, nor did the AAV transduction efficiency when cotreated in HEK293 cells (Supplemental Figure 6). A mixture of CWG-cPIP (83 μg/kg) or vehicle and each AAV9 (1.0 × 1013 vector genomes/mL) was injected into the mouse hippocampus, and memory-related behaviors were evaluated in Y-maze, novel object recognition (NOR), and passive avoidance (PA) tests on days 21 to 27 after the injection. Hippocampal tissue was used for electrophysiology and immunohistochemistry on days 28 to 30 (Figure 3A).

Figure 3

Amelioration of cognitive deficits observed in AAV-mediated CWG repeat–expressing mice by CWG-cPIP treatment. (A) Schematic representation of viral constructs used in in vivo experiments and experimental schedules and representative confocal image of GFP expression in the hippocampus of CUG10 mice. Scale bar: 200 μm. (B and E) Spontaneous alternation behaviors and locomotor activities in the Y-maze test. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Bonferroni’s multiple-comparison test. CUG10 plus vehicle and CUG300 plus vehicle: n = 11 mice; CUG300 plus CWG-cPIP: n = 9 mice each (B); n = 10 mice each (E). (C and F) Discrimination indices for the NOR test sessions. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Bonferroni’s multiple-comparison test. CUG10 plus vehicle and CUG300 plus vehicle: n = 11 mice; CUG300 plus CWG-cPIP: n = 9 mice each (C); n = 10 mice each (F). (D and G) Latency to enter the dark compartment in the PA test sessions. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Bonferroni’s multiple-comparison test. CUG10 plus vehicle and CUG300 plus vehicle: n = 11 mice; CUG300 plus CWG-cPIP: n = 9 mice each (D); n = 10 mice each (G). Data represent the mean ± SEM. Statistical data are provided in Supplemental Data File 6.

In the Y-maze test, CUG300 and Q74 mice showed impaired memory-related behavior compared with CUG10 and Q23 mice. This was quantified by calculating the percentage of alternation behavior. The percentage of spontaneous alternation behavior significantly increased in CWG-cPIP–treated CUG300 and Q74 mice (Figure 3, B and E). CUG300 mice showed a characteristic behavior of dramatically increased locomotor activity, determined by the number of arm entries, and CWG-cPIP treatment did not improve hyperactivity (Figure 3B). In the NOR test, we observed no differences in the discrimination index using the same object for all mice during the training trials (Supplemental Figure 7A). After a 24-hour retention interval, CUG300 and Q74 mice had a significantly lower discrimination index for the novel object than did CUG10 and Q23 mice. The discrimination index for the novel object for CUG300 and Q74 mice treated with CWG-cPIP was significantly higher than that for the vehicle-treated mice (Figure 3, C and F). In the PA test, we observed no significant differences in latency to entering a dark room in the absence of a foot shock for all mice (Supplemental Figure 7B). However, latency to enter the dark compartment was markedly decreased 24 hours after foot shock for CUG300 and Q74 mice compared with CUG10 and Q23 mice. CWG-cPIP administration significantly restored the reduced latency time (Figure 3, D and G).

CWG-cPIP ameliorates neuronal dysfunction in AAV-mediated, CWG repeat–expressing mice. We next assessed the electrophysiology of hippocampal long-term potentiation (LTP), which is critical for learning and memory. Interestingly, we found that basal synaptic transmission in input-output relationships was impaired in CUG300 compared with CUG10 mice (Figure 4A). In addition, we observed a dramatic reduction in high-frequency stimulation–induced (HFS-induced) LTP in CUG300 mice compared with that in CUG10 mice, and the reduced basal synaptic transmission and LTP in CUG300 mice were significantly restored following CWG-cPIP treatment (Figure 4, A–C). In Q74 mice, HFS-induced LTP was significantly impaired compared with that in Q23 mice without changes in basal synaptic transmission, and CWG-cPIP treatment significantly restored the reduction in synaptic plasticity observed in Q74 mice (Figure 4, D–F).