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.

Structural Studies of a Complex of a CAG/CTG Repeat Sequence-Specific Binding Molecule and A–A-Mismatch-Containing DNA

“…Therefore, based on the structure observed in this experiment, it will be helpful to consider the structural changes to DNA strands caused by PIP in the nucleus. Although the existence of this branching structure was hypothesized in a previous study, this was the first direct observation of such a structure. We believe by observing the dynamic interaction between PIP and DNA, we can better understand how it behaves inside cells and pave the way for future studies in this area.…”

“…Although the existence of this branching structure was hypothesized in a previous study, 18 this was the first direct observation of such a structure. We believe by observing the dynamic interaction between PIP and DNA, we can better understand how it behaves inside cells and pave the way for future studies in this area.…”

“…The cPIP used in this research exhibits a high binding affinity for not only DNA strands containing A–A mismatched base pairs but also DNA strands containing A–T matches and T–T mismatches. , In previous studies, the melting temperature ( T m ) was mainly used for interpretation, but it reveals only the thermodynamic stability of DNA . The dissociation equilibrium constant ( K d ) of the cPIP toward DNA strands containing A–A mismatches and T–T mismatches was not determined yet.…”

“…Using this sequence specificity, many studies have been conducted using various PIPs as drug candidates, , to visualize telomere, , and as epigenetic regulators. − A cyclic-PIP (cPIP) that strongly binds to a sequence containing CAG/CTG repeats (5′-WGCWGCW-3′, W: A or T) has been developed . Furthermore, biological experiments using this cPIP revealed that it specifically binds to the hairpin structure that contains the mismatched base pairs formed thereby suppressing the genes that cause the expanded-repeat-associated diseases . Although this cPIP displays a remarkable binding affinity to DNA duplexes containing canonical A–T Watson–Crick base pairs, it also exhibits a high affinity to those containing A–A or T–T mismatched base pairs, i.e., 5′-AGCAGCA-3′/3′-AGCAGCA-5′ or 5′-TGCTGCT-3′/3′-TGCTGCT-5′ .…”

“… 17 Furthermore, biological experiments using this cPIP revealed that it specifically binds to the hairpin structure that contains the mismatched base pairs formed thereby suppressing the genes that cause the expanded-repeat-associated diseases. 18 Although this cPIP displays a remarkable binding affinity to DNA duplexes containing canonical A–T Watson–Crick base pairs, it also exhibits a high affinity to those containing A–A or T–T mismatched base pairs, i.e., 5′-AGCAGCA-3′/3′-AGCAGCA-5′ or 5′-TGCTGCT-3′/3′-TGCTGCT-5′. 17 It has been suggested that such A–A and T–T mismatches exist in hairpin structures, which can be formed by CAG/CTG repeat sequences and are believed to be involved in the elongation of repeated sequences.…”