Exon Skipping Therapy Using Phosphorodiamidate Morpholino Oligomers in the mdx52 Mouse Model of Duchenne Muscular Dystrophy

Miyatake, Shouta; Mizobe, Yoshitaka; Takizawa, Hotake; Hara, Yuko; Yokota, Toshifumi; Takeda, Shin’ichi; Aoki, Yosuke

doi:10.1007/978-1-4939-7374-3_9

Cited by 15 publications

(7 citation statements)

References 29 publications

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“…Importantly, the mdx52 model also allows for the testing of patient mutation-relevant exon skipping strategies in vivo ( e.g. targeting exon 51 or exon 53) ( 33 – 35 ).…”

mentioning

confidence: 99%

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

Tle.

Johansson

Hanson

et al. 2020

Molecular & Cellular Proteomics

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The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. While a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n=3. High-resolution isoelectric focusing liquid chromatography-tandem mass spectrometry (HiRIEF-LC-MS/MS) was used to quantify the expression of 4,974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to wild-type (C57BL/6) controls at each age. Furthermore, 1,795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function which have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators which together are indicative of crosstalk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Up-regulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was up-regulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.

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“…Importantly, the mdx52 model also allows for the testing of patient mutation-relevant exon skipping strategies in vivo ( e.g. targeting exon 51 or exon 53) ( 33 – 35 ).…”

mentioning

confidence: 99%

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

Tle.

Johansson

Hanson

et al. 2020

Molecular & Cellular Proteomics

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“…In saying that, it is important to note that in vitro studies and in vivo animal models do not always translate into successful treatments for patients. This is mainly due to sequence differences in the cases of in vivo models, with a prime example being a mouse model of Duchenne muscular dystrophy, whereby subtle changes in sequences can drastically affect AO efficiency [46].…”

Section: Discussionmentioning

confidence: 99%

Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing

McIntosh¹,

Aung-Htut²,

Fletcher³

et al. 2019

IJMS

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Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

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“…Muscle lysates from mice were prepared according to our protocol 30 . In brief, 20 × 12 μm muscle cryosections were homogenized in 100 μL of radioimmunoprecipitation assay (RIPA) buffer with Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland), heated at 95°C for 5 min, and centrifuged at 16,500 × g for 15 min.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we investigated the relationship between cell-surface SR-As and endocytic uptake of PMOs in dystrophic skeletal muscle, using the H2K- mdx 52 myogenic cell line and mdx 52 mice, a murine exon 52-deleted model, 30 and also measured the zeta potential of this PMO and its effect on the binding to SR-A1.…”

Section: Introductionmentioning

confidence: 99%

Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle

Miyatake

Mizobe

Tsoumpra

et al. 2019

Molecular Therapy - Nucleic Acids

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Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) is a promising treatment strategy for Duchenne muscular dystrophy (DMD). The most significant limitation of these clinically used compounds is their lack of delivery systems that target muscles; thus, cell-penetrating peptides are being developed to enhance uptake into muscles. Recently, we reported that uptake of peptide-conjugated PMOs into myofibers was mediated by scavenger receptor class A (SR-A), which binds negatively charged ligands. However, the mechanism by which the naked PMOs are taken up into fibers is poorly understood. In this study, we found that PMO uptake and exon-skipping efficiency were promoted in dystrophin-deficient myotubes via endocytosis through a caveolin-dependent pathway. Interestingly, SR-A1 was upregulated and localized in juxtaposition with caveolin-3 in these myotubes and promoted PMO-induced exon skipping. SR-A1 was also upregulated in the skeletal muscle of mdx52 mice and mediated PMO uptake. In addition, PMOs with neutral backbones had negative zeta potentials owing to their nucleobase compositions and interacted with SR-A1. In conclusion, PMOs with negative zeta potential were taken up into dystrophin-deficient skeletal muscle by upregulated SR-A1. Therefore, the development of a drug delivery system targeting SR-A1 could lead to highly efficient exon-skipping therapies for DMD.

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Exon Skipping Therapy Using Phosphorodiamidate Morpholino Oligomers in the mdx52 Mouse Model of Duchenne Muscular Dystrophy

Cited by 15 publications

References 29 publications

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing

Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle

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