<i>In vivo</i> comparison of 2′‐<i>O</i>‐methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping

Heemskerk, Hans; Winter, C.L. de; Kimpe, Sjef J. De; Kuik-Romeijn, Petra van; Heuvelmans, Niki; Platenburg, Gerard; Ommen, Gert‐Jan B. van; Deutekom, J.C.T. van; Aartsma‐Rus, Annemieke

doi:10.1002/jgm.1288

Cited by 162 publications

(135 citation statements)

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“…6 These novel therapeutic options for DMD represent the first step toward a possible cure for this devastating disorder. However, several major obstacles, namely optimization of nontoxic effective doses, improvement of the delivery system, distribution to all affected tissues, achievement of a sustainable therapeutic effect and development of an administration strategy suitable for lifelong treatment, [7][8][9][10] still remain to be overcome.…”

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confidence: 99%

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

et al. 2009

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Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropilacrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as a-, b-, g-and d-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials. Gene Therapy ( Keywords: dystrophin; antisense; nanoparticles; arrector pili; smooth muscle Duchenne muscular dystrophy (DMD) is an inherited X-linked degenerative muscular disorder predominantly caused by frame-disrupting mutations arising from gross rearrangements in the dystrophin gene. 1 DMD patients are affected by both severe skeletal muscle wasting and dilated cardiomyopathy. The milder allelic form of the disease, Becker muscular dystrophy, is due to in-frame mutations that preserve a shortened but functional protein.Recently, novel therapeutic approaches for DMD have emerged, some of which have thus far only been tested in animal models. Others, however, such as drugs which induce stop-codon reversion 2 (PTC124) and antisense oligoribonucleotide (AON)-mediated exon skipping, [3][4][5] are already undergoing clinical trials. 6 These novel therapeutic options for DMD represent the first step toward a possible cure for this devastating disorder. However, several major obstacles, namely optimization of nontoxic effective doses, improvement of the delivery system, distribution to all affected tissues, achievement of a sustainable therapeutic effect and development of an administration strategy suitable for lifelong treatment, 7-10 still remain to be overcome.We previously showed that inert PMMA T1 na...

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Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

et al. 2009

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“…5 Briefly, 7 mdx mice were used that were injected in the gastrocnemius muscle on 2 consecutive days with 2.9 nmol of either morpholino or 2-OMePS AON (approximately 20 mg) at 4-5 weeks of age. They were killed by cervical dislocation 10 days after the last injection.…”

Section: Methodsmentioning

confidence: 99%

“…As anticipated, there appears to be a correlation between exon-skipping percentages and dystrophin restoration in that AONs that induce high levels of exon skipping also lead to higher dystrophin levels than AONs that are less efficient. 5 Thus, when optimizing AONs, assessment of exon skipping percentages is a relatively straightforward way to compare the efficiencies of different AONs. However, different ways to quantify exon-skipping levels are used by different groups, making it difficult to compare results between labs.…”

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confidence: 99%

“…[5][6][7][8][9][10][11] As dystrophin expression is rather low, cDNA synthesis followed by two rounds of PCR amplification (primary and nested PCR) is the method used most to detect and quantify the exon-skipping percentage, but protocols greatly differ and the total number of amplification cycles can vary from B50 to 70. 5,[7][8][9][10][11] Only one group has recently used a single round amplification 12 to assess exon 23 skipping in the mdx mouse, which is a dystrophin-negative mouse model with a point mutation in the in-frame exon 23 and which has been instrumental to optimize the exon-skipping approach in vivo. Although RT-PCR-based methods are inexpensive and allow high throughput, there is a possibility that the shorter skipped fragment is amplified more efficiently than the larger unskipped fragment, thus leading to an overestimation of exon-skipping percentages.…”

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confidence: 99%

“…Relative quantification of RT-PCR products can be performed using bioanalyzer technology to assess exon-skipping percentages. 5,6 Alternatively, densitometric analysis to assess the intensity of fragments in the agarose gel has been used. 10 This could, however, lead to a bias since the longer unskipped fragments bind more intercalating ethidium bromide than the shorter skipped fragments, leading to a brighter signal and underestimation of exon-skipping percentages.…”

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Accurate quantification of dystrophin mRNA and exon skipping levels in Duchenne Muscular Dystrophy

Spitali

Heemskerk

Vossen

et al. 2010

Laboratory Investigation

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Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains B1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model.

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Splicing Modulation as Therapy for Human Genetic Disease

Srirangalingam

Khoo

2015

Encyclopedia of Life Sciences

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The sequencing of the human genome has led to a greater appreciation of the contribution that genetic abnormalities and splicing errors make to the burden of human disease. The modulation of splicing using antisense oligonucleotides provides an attractive therapeutic option for tailored interventions in rare genetic disease. In this review, we outline the various approaches to splicing modulation and provide examples of each strategy. Finally, we address some of the practical issues facing the field in trying to bring this technology towards clinical application using the treatment of Duchenne muscular dystrophy and spinal muscular atrophy as examples. Key Concepts Ninety‐five percent of human genes undergo splicing. An estimated 50% of all human genetic diseases result from splicing abnormalities. Splicing modulation using antisense oligonucleotide (ASO) technology offers the future prospect of personalised medicine tailored to specific mutations. Strategies to modulate splicing include suppression of an aberrant splice isoform, switching between known splicing variants or generating a novel variant with a potential therapeutic attribute. ASO chemistries are varied and have been optimised to improve target binding and resist nuclease degradation. ASO targets include all pre‐mRNA sites which influence the splicing process. Splicing events can be complex and unpredictable. Therefore an iterative method of design and test is required to optimise ASO efficacy. ASO side‐effects are likely to stem from the ASO chemistry, off‐target effects as the result of nonspecific binding, and ASO distribution to organs when given in vivo. Optimising tissue delivery of ASO remains a significant challenge.

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In vivo comparison of 2′‐O‐methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping

Cited by 162 publications

References 33 publications

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

Accurate quantification of dystrophin mRNA and exon skipping levels in Duchenne Muscular Dystrophy

Splicing Modulation as Therapy for Human Genetic Disease

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