Serum and plasma biomarkers assessed before and directly after treadmill running.

<p>A. CK levels assessed before and directly after treadmill running. CK levels collected before exercise were significantly (<i>P</i><0.01) lower than those collected directly after exercise in <i>mdx-Xist</i>^Δhs and <i>mdx</i> mice. This exercise induced increase was absent in wild type mice. B. CK levels determined before exercise were significantly (<i>P</i><0.01) elevated in <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice compared to wild type, but this was less pronounced for the <i>mdx-Xist</i>^Δhs mice as <i>mdx</i> mice had significantly (<i>P</i><0.05) higher CK levels. C. Plasma collected directly after exercise contained extremely high CK levels, in both <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice but not in wild type mice. D. Serum levels of MMP-9 were elevated in <i>mdx</i> mice compared to <i>Xist</i>^Δhs mice at both 8 and 14 weeks of age, while levels were normalized in <i>mdx-Xist</i>^Δhs mice. E. TIMP-1 levels were elevated in serum of both <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. In wild type mice we found an age related increase of the serum TIMP-1 level. Interestingly, at the age of 8 weeks, levels of <i>mdx-Xist</i>^Δhs mice were significantly lower than those of <i>mdx</i> mice. F. <i>Mdx</i> mice had significantly elevated OPN levels compared to both wild type and <i>mdx-Xist</i>^Δhs at 8 weeks of age. Single asterisks indicate a <i>P</i><0.05, average dystrophin level of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Dystrophin expression pattern and morphological examination of del52hDMD/<i>mdx</i> mouse lines.

<p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with either GTX (human and mouse specific) or Mandys106 (human specific). Wild type expression levels of human dystrophin were observed in hDMD/<i>mdx</i> mice. Notably, del52hDMD/<i>mdx</i>#37 mice expressed traces of human dystrophin, in both cardiac and skeletal muscle, while this was not observed in del52hDMD/<i>mdx</i>#35 and <i>mdx</i>(BL6) mice. <b>B</b>) Sections of the heart and quadriceps stained with human specific dystrophin antibodies. Expression of human dystrophin is at wild type level in hDMD/<i>mdx</i> mice as anticipated. Both C57BL/6J, <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice did not express human dystrophin. Interestingly, in most fibers of del52hDMD/<i>mdx</i>#37 mice, human dystrophin was expressed at low levels. Haematoxylin and eosin staining revealed signs of degeneration and regeneration in the quadriceps of both del52hDMD/<i>mdx</i> strains, as evident by variation in fiber size, centralized nuclei and patches of fibrosis and inflammation. Overall pathology appeared to be slightly less extensive in del52hDMD/<i>mdx</i>#37 mice compared to <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice. <b>C</b>) Almost no centralized nuclei were found in wild type mice, while half of the myofibers in <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice had centrally located nuclei. The percentage in del52hDMD/<i>mdx</i>#37 mice was with 26% significantly lower. Data were based on manual counts of 5 randomly taken pictures of 2 males and 2 females per genotype. Asterisks indicate <i>P</i><0.01.</p

Confirmational analysis of offspring from blastocyst transplanted mice.

<p>Pups were analysed for chimerism by multiplex PCR and MCA. <b>A</b>) Four of the pups derived after transplantation of blastocysts injected with ES cells of clone 9B4 showed presence of the exon 52 deleted <i>hDMD</i> gene (lines 1, 4, 8 and 11). <b>B</b>) Melting curve analysis revealed that all male pups were also chimeric for the <i>mdx</i> point mutation.</p

ViM treatment results in exon skipping and subsequent dystrophin restoration.

<p><b>A</b>) Nested PCR revealing exon skipping upon local 51ViM or 53ViM treatment in right and left gastrocnemius and triceps muscle (respectively GR, GL and TR, TL) of two del52hDMD<i>/mdx</i>#35 mice. We confirmed by Sanger sequencing that the upper band in the untreated del52hDMD<i>/mdx</i> samples involves a cryptic splicing event that is occasionally observed in untreated mice of this strain. It contains exon 51, part of intron 51, the last 101 nucleotides of exon 52, exon 53 and multiple stop codons. The arrows indicate the expected heights of fragments lacking exon 51 (700 bp) or exon 53 (721 bp). <b>B</b>) Murine specific nested PCR confirmed that the exon 51 ViM induced low levels of mouse exon 51 skipping. The exon 53 ViM only resulted in exon 53 skipping in the human transcript as no skipping band was seen in the PCR performed with mouse-specific primers (expected size 483 bp). Sanger sequence confirmed that the smaller fragment obtained in the ViM exon 51 treated muscles contained the boundary of exon 50–52. Human ctrl; healthy human control sample. The arrow indicates the expected height of fragments lacking exon 51 (463 bp). <b>C-D</b>) Exon skipping resulted in the restored dystrophin expression in gastrocnemius (C) and triceps (D) of 51ViM and 53ViM treated mice.</p

PCR analysis of targeted clones and confirmation of deletion exon 52 on RNA level.

<p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as template in a multiplex PCR. Here the exons 46, 51 and 52 of the <i>hDMD</i> gene were analysed where exon 46 and 51 are positive controls and exon 52 the target to be deleted. <b>A</b>) An example is shown where candidate samples 2 and 5 are of interest because they are negative for exon 52 but positive for the control exons. <b>B</b>) For a large number of clones additional fragments were found for exon 52, suggesting non-homologues end joining (NHEJ) of TALEN induced double stranded breaks <b>C</b>) Representative image of LR-PCR performed on DNA of sub-clones of four exon 52 negative clones (9B4, 10H2, 11C9 and 11E7). LR-PCR was performed with primers targeting intron 51 (outside the targeting arm) and blasticidin (only present after homologous recombination), to rule out loss of PCR primer recognition sites by NHEJ and to confirm true targeting. <b>D</b>) RT-PCR was performed for RNA isolated from embryoid bodies of selected clones. The different fragments were isolated, purified and Sanger sequence analysed. In the wild type situation exon 52 was present, whereas in the properly targeted clones exon 52 was not present. This confirmed the exon 52 deletion on RNA level.</p

Expression of several genes involved in disease pathology.

<p>A and B. For most biomarkers a clear dystrophin level dependent restoration of gene expression levels was observed in the <i>mdx-Xist</i>^Δhs mice, where intermediate dystrophin levels resulted in low expression of genes involved in disease pathology. For some genes, dystrophin levels <15% were enough to reduce gene expression while for other genes >30% was necessary. C. In the heart, even dystrophin levels of <15% decreased expression of fibrotic biomarkers like <i>Timp-1</i>. Since the mice were young, no difference in expression levels in heart function was observed, except for <i>Nppa</i>. Double asterisks indicate a <i>P</i><0.01.</p

Muscle fiber size, degree of central nucleation and percentage of fibrotic/necrotic tissue.

<p>A. Regenerating and hypertrophic fibers were mainly observed in the <i>mdx</i> mice. A dystrophin level depend trend towards wild type distribution was observed for <i>mdx-Xist</i>^Δhs mice where <15% dystrophin already resulted in improvement. B. Dystrophin levels between 15–30% and >30% resulted in a reduction of the percentage of centralized nuclei of 40% and 60% respectively. C. The quadriceps of all mice was significantly more severely affected compared to <i>Xist</i>^Δhs mice. D. The diaphragm was the most severely affected muscle with on average 20% fibrotic/necrotic tissue in <i>mdx</i> mice. All mice were significantly more severely affected compared to <i>Xist</i>^Δhs mice. <i>Mdx-Xist</i>^Δhs mice with >30% dystrophin had less fibrotic/necrotic tissue than <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice with <15% dystrophin, but this difference was only significant between both <i>mdx-Xist</i>^Δhs groups. # indicates a significant difference of that bar with all the other groups. Single asterisks indicate a <i>P</i><0.05 and double asterisks indicate a <i>P</i><0.01.</p

<i>Mdx-Xist</i>^Δhs mice.

<p>A. To breed mice with low dystrophin levels, female <i>Xist</i>^Δhs mice, carrying a mutation in the <i>Xist</i> promoter which coordinates X-inactivation, were crossed with dystrophin negative <i>mdx</i> males. During embryogenesis, the maternal X-chromosome encoding a functional dystrophin gene will be preferentially (60–90%) inactivated as a result of the mutated <i>Xist</i> promoter. The <i>Xist</i>^Δhs mice were a kind gift from N. Brockdorff (MRC Clinical Sciences Center, London, UK, current affiliation Department of Biochemistry, University of Oxford, UK). B. Picture of a representative Western blot. The percentage of dystrophin was determined for the quadriceps of all <i>mdx-Xist</i>^Δhs mice by Western blot (2–9 technical replicates per mouse). The percentage of individual mice was determined using a concentration curve made from wild type samples. Myosin was used as a loading control. C. Skewed X-inactivation resulted in dystrophin levels of 3–47% (mean 22.7, stdev 12.1, <i>n</i> = 24) (as determined by Western blot) in the female <i>mdx-Xist</i>^Δhs offspring. Each bar represents the dystrophin level of an individual mouse. The dystrophin levels of the individual mice belonging to the three dystrophin groups can be appreciated from this graph. D. Dystrophin levels determined by Western blot and manual counting of dystrophin positive fibers demonstrate a strong correlation (R = 0.97). E. Longitudinal sections of a quadriceps stained with dystrophin (green) and spectrin (red). From the pictures it can be appreciated that dystrophin expression is not homogeneously expressed across the fiber but rather confined to certain nuclear domains.</p

Exercise induced histopathology and biomarker gene expression.

<p>A. Wild type mice were less severely affected than <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Due to high variation between individual <i>mdx</i> mice, the difference between <i>mdx</i> and wild type mice was not significant. <i>Mdx-Xist</i>^Δhs mice had slightly less fibrotic tissue than <i>mdx</i> mice. B. No difference in expression of genes involved in disease pathology was found between <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Both mouse strains did differ significantly from wild type mice, of which the expression levels were low. Single and double asterisks indicate a <i>P</i><0.05 and <i>P</i><0.01, respectively. # Indicates a significant difference from all other groups, average dystrophin levels of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Functional performance measured directly after treadmill exercise.

<p>A. Longest hanging time with the two limb hanging test was achieved by wild type and <i>mdx-Xist</i>^Δhs mice which both performed significantly (<i>P</i><0.001) better than <i>mdx</i> mice. B. Fore limb grip strength of <i>mdx-Xist</i>^Δhs mice was significantly (<i>P</i><0.001) better than that of <i>mdx</i> mice. C. Wild type mice outperformed <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice on the rotarod, while no significant difference was observed between <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Average dystrophin level of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p