Dantrolene Enhances Antisense-Mediated Exon Skipping in Human and Mouse Models of Duchenne Muscular Dystrophy

Kendall, Genevieve C.; Mokhonova, Ekaterina; Moran, Miriana; Sejbuk, Natalia E.; Wang, Derek W.; Silva, Oscar; Wang, Richard T.; Martinez, Leonel; Lü, Qi; Damoiseaux, Robert; Spencer, Melissa J.; Nelson, Stanley F.; Miceli, M. Carrie

doi:10.1126/scitranslmed.3005054

Cited by 79 publications

(83 citation statements)

References 59 publications

(86 reference statements)

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“…We established inducible fibroblast-derived myogenic (iFDM) cell cultures from patient and normal NHDF fibroblast lines using methods described 26,27. Fibroblasts were transduced with a tamoxifen-inducible MyoD lentivirus (Lv-CMV-MyoD-ER(T),26 Addgene, Cambridge, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

Dominov

Uyan

Sapp

et al. 2014

Ann Clin Transl Neurol

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ObjectiveMutations in dysferlin (DYSF), a Ca2+-sensitive ferlin family protein important for membrane repair, vesicle trafficking, and T-tubule function, cause Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal myopathy. More than 330 pathogenic DYSF mutations have been identified within exons or near exon–intron junctions. In ~17% of patients who lack normal DYSF, only a single disease-causing mutation has been identified. We studied one family with one known mutant allele to identify both the second underlying genetic defect and potential therapeutic approaches.MethodsWe sequenced the full DYSF cDNA and investigated antisense oligonucleotides (AONs) as a tool to modify splicing of the mRNA transcripts in order to process out mutant sequences.ResultsWe identified a novel pseudoexon between exons 44 and 45, (pseudoexon 44.1, PE44.1), which inserts an additional 177 nucleotides into the mRNA and 59 amino acids within the conserved C2F domain of the DYSF protein. Two unrelated dysferlinopathy patients were also found to carry this mutation. Using AONs targeting PE44.1, we blocked the abnormal splicing event, yielding normal, full-length DYSF mRNA, and increased DYSF protein expression.InterpretationThis is the first report of a deep intronic mutation in DYSF that alters mRNA splicing to include a mutant peptide fragment within a key DYSF domain. We report that AON-mediated exon-skipping restores production of normal, full-length DYSF in patients’ cells in vitro, offering hope that this approach will be therapeutic in this genetic context, and providing a foundation for AON therapeutics targeting other pathogenic DYSF alleles.

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Section: Methodsmentioning

confidence: 99%

A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

Dominov

Uyan

Sapp

et al. 2014

Ann Clin Transl Neurol

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“…A recent report showed that the small molecule dantrolene could produce about a 2.5-fold enhancement of SSO-mediated exon skipping (Kendall et al, 2012). Dantrolene acts on the muscle cell ryanodine receptor, a calcium transporter, and is currently used clinically in treatment of malignant hyperthermia.…”

Section: Enhancing the Pharmacological Effects Of Oligonucleotides Usmentioning

confidence: 99%

Cellular Uptake and Intracellular Trafficking of Oligonucleotides: Implications for Oligonucleotide Pharmacology

Juliano

Xin

Carver

et al. 2014

Nucleic Acid Therapeutics

101

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One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed.

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“…In the heart specifically, proteolytic degradation by the UPS is highly regulated at multiple levels to affect gating, assembly, and/or activity (37). Heterogeneity of the proteasome subpopulations represents one important mechanism by which proteasome activity and substrate specificity may be modulated.…”

Section: Regulation Of the Cardiac Upsmentioning

confidence: 99%