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Precise Targeted Cleavage of a r(CUG) Repeat Expansion in Cells by Using a Small-Molecule-Deglycobleomycin Conjugate.

ABSTRACT: RNA repeat expansions cause more than 30 neurological and neuromuscular diseases with no known cures. Since repeat expansions operate via diverse pathomechanisms, one potential therapeutic strategy is to rid them from disease-affected cells, using bifunctional small molecules that cleave the aberrant RNA. Such an approach has been previously implemented for the RNA repeat that causes myotonic dystrophy type 1 [DM1, r(CUG)^exp] with Cugamycin, which is a small molecule that selectively binds r(CUG)^exp conjugated to a bleomycin A5 cleaving module. Herein, we demonstrate that, by replacing bleomycin A5 with deglycobleomycin, an analogue in which the carbohydrate domain of bleomycin A5 is removed, the selectivity of the resulting small-molecule conjugate (DeglycoCugamycin) was enhanced, while maintaining potent and allele-selective cleavage of r(CUG)^exp and rescue of DM1-associated defects. In particular, DeglycoCugamycin did not induce the DNA damage that is observed with high concentrations (25 μM) of Cugamycin, while selectively cleaving the disease-causing allele and improving DM1 defects at 1 μM.

SUBMITTER: Angelbello AJ

PROVIDER: S-EPMC7360342 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model.

Project description:Myotonic dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by an expanded CTG repeat that is transcribed into r(CUG)exp The RNA repeat expansion sequesters regulatory proteins such as Muscleblind-like protein 1 (MBNL1), which causes pre-mRNA splicing defects. The disease-causing r(CUG)exp has been targeted by antisense oligonucleotides, CRISPR-based approaches, and RNA-targeting small molecules. Herein, we describe a designer small molecule, Cugamycin, that recognizes the structure of r(CUG)exp and cleaves it in both DM1 patient-derived myotubes and a DM1 mouse model, leaving short repeats of r(CUG) untouched. In contrast, oligonucleotides that recognize r(CUG) sequence rather than structure cleave both long and short r(CUG)-containing transcripts. Transcriptomic, histological, and phenotypic studies demonstrate that Cugamycin broadly and specifically relieves DM1-associated defects in vivo without detectable off-targets. Thus, small molecules that bind and cleave RNA have utility as lead chemical probes and medicines and can selectively target disease-causing RNA structures to broadly improve defects in preclinical animal models.

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Precise small-molecule recognition of a toxic CUG RNA repeat expansion.

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Precise small molecule cleavage of a r(CUG) repeat expansion in a myotonic dystrophy mouse model

Project description:Myotonic dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by an expanded CTG repeat that is transcribed into r(CUG)exp. The RNA repeat expansion sequesters regulatory proteins such as Muscleblind-like protein 1 (MBNL1), which causes pre-mRNA splicing defects. The disease-causing r(CUG)exp has been targeted by antisense oligonucleotides, CRISPR-based approaches, and RNA-targeting small molecules. Herein, we describe a designer small molecule, Cugamycin, that targets the structure of r(CUG)exp and cleaves it in cells and in vivo. Cugamycin selectively cleaves r(CUG)exp while leaving short repeats of r(CUG) untouched. In contrast, oligonucleotides that recognize r(CUG) sequence rather than structure cleave both long and short r(CUG)-containing transcripts. In the HSALR mouse model of DM1, Cugamycin selectively ablates r(CUG)exp in disease-affected muscle. Transcriptomic, histological, and phenotypic studies demonstrate that Cugamycin broadly and specifically relieves DM1-associated defects without detectable off-targets. Thus, small molecules that bind and cleave RNA may have utility as lead chemical probes and medicines.

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Precise small molecule cleavage of a r(CUG) repeat expansion in a myotonic dystrophy mouse model

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Massively Parallel Optimization of the Linker Domain in Small Molecule Dimers Targeting a Toxic r(CUG) Repeat Expansion.

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