Pentamidine reverses the splicing defects associated with myotonic dystrophy

Warf, M. Bryan; Nakamori, Mikihito; Matthys, Catherine M.; Thornton, Charles A.; Berglund, J. Andrew

doi:10.1073/pnas.0903234106

Cited by 235 publications

(317 citation statements)

References 49 publications

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“…A potential alternative approach is to use combined administration of CAG morpholinos with gapmers targeting different sequences within the DMPK mRNA to avoid binding competition. In addition, several reports have identified small molecules and peptides with the ability to disrupt RNA foci formation (31,32). This opens up the possibility of combining ASOs with other nonantisense strategies for DM1 therapy.…”

Section: Discussionmentioning

confidence: 99%

RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1

Lee

Bennett

Cooper

2012

Proc. Natl. Acad. Sci. U.S.A.

143

134

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Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease caused by abnormal transcripts containing expanded CUG repeats. The CUG transcripts aggregate in the nucleus to form RNA foci and lead to nuclear depletion of Muscleblind-like 1 (MBNL1) and stabilized expression of CUGBP Elav like family 1 (CELF1), both of which are splicing regulatory proteins. The imbalance of these proteins results in misregulation of alternative splicing and neuromuscular abnormalities. Here, we report the use of antisense oligonucleotides (ASOs) as a therapeutic approach to target the pathogenic RNA in DM1. We designed chimeric ASOs, termed gapmers, containing modified nucleic acid residues to induce RNase H-mediated degradation of CUG-repeat transcripts. The gapmers selectively knockdown expanded CUG transcripts and are sufficient to disrupt RNA foci both in cell culture and mouse models for DM1. Furthermore, combination of gapmers with morpholino ASOs that help release binding of MBNL1 to the toxic RNA can potentially enhance the knockdown effect. Additional optimization will be required for systemic delivery; however, our study provides an alternative strategy for the use of ASOs in DM1 therapy.microsatellite expansion | muscular dystrophy | phosphorothioate | gapmer

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Section: Discussionmentioning

confidence: 99%

RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1

Lee

Bennett

Cooper

2012

Proc. Natl. Acad. Sci. U.S.A.

143

134

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“…Significantly, CUG focus formation requires the binding of the alternative splice factor, muscleblind 1 (MBNL1) to the expanded CUG repeat expansions (11,12). The importance of the aberrant MBNL1-CUG interaction in DM1 is underscored by the use of pentamidine and morpholino antisense oligonucleotides that dislodge MBNL1 from expanded CUG RNA, restore free MBNL1 levels, and rescue splice defects in DM1 mouse models (13,14). Consistent with these observations, inactivation of Mbnl1 in mice recapitulates a large fraction of the splice defects and several key features of DM1 pathology (15,16).…”

Section: Myotonic Dystrophy I (Dm1)mentioning

confidence: 99%

Expanded CUG Repeats Dysregulate RNA Splicing by Altering the Stoichiometry of the Muscleblind 1 Complex

Paul

Dansithong

Jog

et al. 2011

Journal of Biological Chemistry

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To understand the role of the splice regulator muscleblind 1 (MBNL1) in the development of RNA splice defects in myotonic dystrophy I (DM1), we purified RNA-independent MBNL1 complexes from normal human myoblasts and examined the behavior of these complexes in DM1 myoblasts. Antibodies recognizing MBNL1 variants (MBNL1 CUG ), which can sequester in the toxic CUG RNA foci that develop in DM1 nuclei, were used to purify MBNL1 CUG complexes from normal myoblasts. In normal myoblasts, MBNL1 CUG bind 10 proteins involved in remodeling ribonucleoprotein complexes including hnRNP H, H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9. Of these proteins, only MBNL1 CUG colocalizes extensively with DM1 CUG foci (>80% of foci) with its partners being present in <10% of foci. Importantly, the stoichiometry of MBNL1 CUG complexes is altered in DM1 myoblasts, demonstrating an increase in the steady state levels of nine of its partner proteins. These changes are recapitulated by the expression of expanded CUG repeat RNA in Cos7 cells. Altered stoichiometry of MBNL1 CUG complexes results from aberrant protein synthesis or stability and is unlinked to PKC␣ function. Modeling these changes in normal myoblasts demonstrates that increased levels of hnRNP H, H2, H3, F, and DDX5 independently dysregulate splicing in overlapping RNA subsets. Thus expression of expanded CUG repeats alters the stoichiometry of MBNL1 CUG complexes to allow both the reinforcement and expansion of RNA processing defects. Myotonic dystrophy I (DM1)4 is a multi-system disorder resulting from the expansion of a CTG repeat sequence in the 3Ј-untranslated region of DMPK, located on chromosome 19 (1-4). A key molecular consequence of expanded CUG repeat expression is the abnormal splicing of physiologically important RNAs. Specifically, abnormal splicing of the chloride channel (ClC-1) and the insulin receptor (IR) RNAs have been implicated in the development of myotonia and insulin resistance, respectively, in DM1 patients (5-8). In DM1 cells, expression of expanded CUG repeats results in the formation of aberrant nuclear CUG RNA aggregates or foci (9). The significance of nuclear CUG aggregate formation in the development of aberrant splicing and DM1 pathology is reflected in a recent study, which demonstrates that antisense RNA-mediated silencing of the mutant DMPK RNA and the consequent decrease of CUG foci allow the correction of splice defects in DM1 animal models (10). Significantly, CUG focus formation requires the binding of the alternative splice factor, muscleblind 1 (MBNL1) to the expanded CUG repeat expansions (11, 12). The importance of the aberrant MBNL1-CUG interaction in DM1 is underscored by the use of pentamidine and morpholino antisense oligonucleotides that dislodge MBNL1 from expanded CUG RNA, restore free MBNL1 levels, and rescue splice defects in DM1 mouse models (13,14). Consistent with these observations, inactivation of Mbnl1 in mice recapitulates a large fraction of the splice defects and several key features of DM1 pathology (15,16). Thus...

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“…This demonstrates that the suppression of CUG-dependent phenotypes and recovery of Mbnl1 activity can be achieved without directly competing with Muscleblind binding to CUG repeats but by reducing the fraction of double-stranded CUG RNA from the cellular pool of toxic RNA, hence preventing sequestration of the protein, which requires CUG RNA hairpins. This is a unique mechanism that contrasts with that of other recently characterized molecules that target the binding of MBNL1 to toxic RNAs (17)(18)(19)(20)(21)(22)33). Disrupting Muscleblind binding to RNA could affect its function as a splicing factor in non-CUG-expressing cells.…”

Section: Abp1 Reversed Missplicing and Muscle Histopathology In A Dm1mentioning

confidence: 62%

“…They demonstrated that expanded CTG-induced effects could be reverted if CTG-repeat transgene expression was interrupted in a DM1 mouse model. Several other groups developed synthetic molecules and (CAG)n oligonucleotides that disrupted MBNL1 interaction with expanded CUG repeats (17)(18)(19)(20)(21)(22). However, those approaches may not address all the pathological consequences of expanded CUG RNAs.…”

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confidence: 99%

In vivo discovery of a peptide that prevents CUG–RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models

Garcia-Lopez

Llamusí

Orzáez

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Myotonic dystrophy type 1 (DM1) is caused by the expansion of noncoding CTG repeats in the dystrophia myotonica-protein kinase gene. Mutant transcripts form CUG hairpins that sequester RNAbinding factors into nuclear foci, including Muscleblind-like-1 protein (MBNL1), which regulate alternative splicing and gene expression. To identify molecules that target toxic CUG transcripts in vivo, we performed a positional scanning combinatorial peptide library screen using a Drosophila model of DM1. The screen identified a D-amino acid hexapeptide (ABP1) that reduced CUG foci formation and suppressed CUG-induced lethality and muscle degeneration when administered orally. Transgenic expression of natural, L-amino acid ABP1 analogues reduced CUG-induced toxicity in fly eyes and muscles. Furthermore, ABP1 reversed muscle histopathology and splicing misregulation of MBNL1 targets in DM1 model mice. In vitro, ABP1 bound to CUG hairpins and induced a switch to a single-stranded conformation. Our findings demonstrate that ABP1 shows antimyotonic dystrophy activity by targeting the core of CUG toxicity.disease model | drug discovery | non-coding RNA disease | medicinal chemistry | RNA secondary structure M yotonic dystrophy type 1 (DM1, OMIM #160900) is an autosomal dominant disease caused by the expansion of a CTG trinucleotide repeat in the 3′ untranslated region (UTR) of the dystrophia myotonica-protein kinase (DMPK) gene. Characteristic symptoms include myotonia, progressive muscle wasting, and cardiac conduction defects, among other systemic manifestations. The molecular mechanisms underlying DM1 pathogenesis are complex and affect a large number of cellular processes (1). However, most data suggest that the main triggering event is a toxic gain-of-function of the expanded CUG RNA. CUG-repeat expansions form double-stranded hairpins that are retained as inclusions within the nucleus. These hairpins recruit a number of transcription and splicing factors, including Muscleblind-like 1 (MBNL1) (2-6). Sequestration of MBNL1 originates a loss of function, which plays a key role in the development of DM1 symptoms. Mbnl1 knockout mice reproduced typical features of DM1, and overexpression of Mbnl1 in a mouse model that expressed CTG repeats reversed these phenotypes (7,8). CTGrepeat expression in mice caused misregulation of at least 156 alternative splicing events. Of these, 128 also occurred in Mbnl1 knockout animals (9-12). The splicing factor CUG-binding protein 1 (CUGBP1) is another key component in the development of DM1 phenotypes. CUGBP1 antagonizes MBNL1 activity in the regulated use of alternative exons in a number of transcripts and is abnormally upregulated in patients with DM1, further contributing to splicing misregulation (13-15).Mahadevan et al. provided the first in vivo proof-of-principle for a therapeutic strategy based on ablating toxic RNA molecules in DM1 (16). They demonstrated that expanded CTG-induced effects could be reverted if CTG-repeat transgene expression was interrupted in a DM1 mouse model. Several ...

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Pentamidine reverses the splicing defects associated with myotonic dystrophy

Cited by 235 publications

References 49 publications

RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1

RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1

Expanded CUG Repeats Dysregulate RNA Splicing by Altering the Stoichiometry of the Muscleblind 1 Complex

In vivo discovery of a peptide that prevents CUG–RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models

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