Comparative Analysis of Antisense Oligonucleotide Sequences for Targeted Skipping of Exon 51 During Dystrophin Pre-mRNA Splicing in Human Muscle

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin

In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy

“…The earliest demonstrations of successful AO-induced exon skipping of the dystrophin were demonstrated in vitro in human lymphoblastoid and mdx mouse muscle cells [18,21], skipping DMD exons 19 and exons 22-30, respectively. Subsequently, numerous in vitro exon skipping studies have been undertaken demonstrating precise skipping of DMD exon 23 in mdx mouse muscle cells [29] with protein correction in mdx mice in vivo [6]; successful DMD exon 46 skipping in patient-derived muscle cells [22]; further mdx exon 23 2 OMePS sequence refinement and evaluation of AO concentrations [30]; evaluation of 'leashed' PMO AOs [23]; the comparative effects of different 2 OMePS, PMO, PNA and LNA AOs for skipping DMD exon 46 in human muscle cells [2]; the influence of AO length on AO splice correcting efficacy [31]; optimization and selection of a human DMD exon 51 skipping AO for clinical trial [32]; and the exon skipping activity of PNA and PNApeptide AOs in mdx muscle cells [8,13,14]. Despite these and other in vitro studies, little agreement exists on the optimal parameters for in vitro AO screening, no studies to date have compared across different AO backbone chemistries and AO-peptide modifications, and little if any data exist on the value of in vitro screens as predictors of in vivo AO activity across a wide range of AO compounds.…”

Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino

“…Similar to that observed in the skeletal muscles, dystrophin expression improved further with the increase in doses. A measure of (1)(2)(3)(4)(5)(6), heart tissues (7-11) and cadiomyocytes (12)(13)(14). 1, sample from control TA muscle; 12, freshly isolated cardiomyocytes without treatment; 2, 3, 7, 8 and 13, 2 days after 50 mg ml -1 morpholinoE23 treatment; 4, 5, 9, 10 and 14, 5 days after the treatment; 6 and 11, 7days after the treatment.…”

“…11 Furthermore, effective restoration of reading frame and production of dystrophin have now been achieved in Phase I clinical trials targeting human dystrophin exon 51 in muscles of DMD patients by local injections with both 2 0 O methyl phosphorothioate AONs and morpholino oligomers (personal communication). 12,13 DMD affects body-wide muscles including the cardiac muscle. As DMD patients live longer because of improved multidisciplinary patient care, rescuing dystrophin expression in cardiac muscle becomes more critical for their longevity and quality of life.…”

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

“…However, different ways to quantify exon-skipping levels are used by different groups, making it difficult to compare results between labs. [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.…”

“…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.…”