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

Spitali, Pietro; Heemskerk, Hans; Vossen, Rolf H. A. M.; Ferlini, Alessandra; Dunnen, Johan T. den; Hoen, Peter A C ‘t; Aartsma‐Rus, Annemieke

doi:10.1038/labinvest.2010.98

Cited by 35 publications

(26 citation statements)

References 21 publications

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“…2B). This demonstrated the bioactivity of AAV-U7-high and AAVdiluted vectors; however, nested RT-PCR is an insufficient method to quantify exon-skipping efficiency (Spitali et al, 2010). Importantly, only high titer AAV-U7, but not diluted AAV-U7, restored dystrophin protein expression to detectable levels (Fig.…”

Section: Restoration Of Dystrophin Expressionmentioning

confidence: 88%

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Combined Effect of AAV-U7-Induced Dystrophin Exon Skipping and Soluble Activin Type IIB Receptor in mdx Mice

et al. 2012

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Adeno-associated virus (AAV)-U7-mediated skipping of dystrophin-exon-23 restores dystrophin expression and muscle function in the mdx mouse model of Duchenne muscular dystrophy. Soluble activin receptor IIB (sActRIIB-Fc) inhibits signaling of myostatin and homologous molecules and increases muscle mass and function of wild-type and mdx mice. We hypothesized that combined treatment with AAV-U7 and sActRIIB-Fc may synergistically improve mdx muscle function. Bioactivity of sActRIIB-Fc on skeletal muscle was first demonstrated in wild-type mice. In mdx mice we show that AAV-U7-mediated dystrophin restoration improved specific muscle force and resistance to eccentric contractions when applied alone. Treatment of mdx mice with sActRIIB-Fc increased body weight, muscle mass and myofiber size, but had little effect on muscle function. Combined treatment stimulated muscle growth comparable to the effect of sActRIIB-Fc alone and dystrophin rescue was similar to AAV-U7 alone. Moreover, combined treatment improved maximal tetanic force and the resistance to eccentric contraction to similar extent as AAV-U7 alone. In conclusion, combination of dystrophin exon skipping with sActRIIB-Fc brings together benefits of each treatment; however, we failed to evidence a clear synergistic effect on mdx muscle function.

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Section: Restoration Of Dystrophin Expressionmentioning

confidence: 88%

“…Next, cDNA was generated from 100 ng total RNA using Bioscript reverse transcriptase enzyme (Bioline) and random hexamer primers. Nested PCR was performed with primers detecting both full-length dystrophin transcript and the skipped product, as previously described (Spitali et al, 2010).…”

Section: Reverse-transcription Pcrmentioning

confidence: 99%

Combined Effect of AAV-U7-Induced Dystrophin Exon Skipping and Soluble Activin Type IIB Receptor in mdx Mice

et al. 2012

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“…Total RNA was extracted and reverse transcription-PCR analysis was performed as described with a single round of PCR. 38 PCR products were visualized on 2% agarose gels and quantified with the Agilent 2100 Bioanalyzer (Agilent, Amstelveen, the Netherlands).…”

Section: Methodsmentioning

confidence: 99%

Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

Winter

Heemskerk

Karnaoukh

et al. 2012

Molecular Therapy - Nucleic Acids

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Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD) is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs). Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype). Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK) levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

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“…The intensity of PCR bands was analyzed by using ImageJ software (http://rsbweb.nih.gov/ij/), and skipping efficiency was calculated by using the following formula [(the intensity of skipped band) / (the intensity of skipped band + the intensity of unskipped band)]. 51 After the resulting PCR bands were extracted using a gel extraction kit (Qiagen), direct sequencing of PCR products was performed by the Biomatrix Laboratory (Chiba, Japan).…”

Section: Methodsmentioning

confidence: 99%

In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

et al. 2010

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A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5' and 3' splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.

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

Cited by 35 publications

References 21 publications

Combined Effect of AAV-U7-Induced Dystrophin Exon Skipping and Soluble Activin Type IIB Receptor in mdx Mice

Combined Effect of AAV-U7-Induced Dystrophin Exon Skipping and Soluble Activin Type IIB Receptor in mdx Mice

Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

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