Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

<p>Abstract</p> <p>Background</p> <p>Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs) targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts.</p> <p>Methods</p> <p>Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed.</p> <p>Results</p> <p>For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62), by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons.</p> <p>Conclusion</p> <p>The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.</p

Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy

<div><p>Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.</p></div

Dystrophin levels in skeletal muscle samples derived from DMD patients.

<p>Panel of 17 gastrocnemius, biceps and tibialis muscle samples from DMD patients, analysed for dystrophin using ab154168 and Mandys106 and normalized to α-actinin (n = 2). Protein loading was 1.25 μg. Muscle types are indicated between brackets: G = gastrocnemius; B = biceps; T = tibialis and M = miscellaneous.</p

Workflow of Wes procedure.

<p>The required steps and time to complete a Wes run and analysis are indicated.</p

Assay variation using ab154168 and Mandys106.

<p>Assay variation using ab154168 and Mandys106.</p

Comparison of dystrophin detection by Western blot and Wes in skeletal muscle samples from a healthy control, a BMD patient and 3 DMD patients.

<p>Western blot analysis of a healthy control, a BMD and 3 DMD samples. Expected molecular weights: BMD del45-47: 410 kDa, DMD del45-54: 367 kDa, DMD del43: 422 kDa, DMD del45-52: 381 kDa. A) A dystrophin signal is visible of the expected size (427 kDa) in the healthy control and BMD samples when loading 20 μg per lane, but not for the DMD samples. B) Loading of 10x more material (200 μg per lane) results in a weak dystrophin signal for 2 out of the 3 DMD samples using ab154168 and no signal using Mandys106. The lower panel shows the α-actinin signal. Note that the middle DMD sample has a deletion of exon 43 (epitope for Mandys106), so it is not expected to produce a signal. The additional bands of lower molecular weight are either degradation products and/or dystrophin isoforms. C) Wes analysis of the same samples: At 1 μg total protein (5 μl of a 200 μg/ml dilution) clear signals are detected at 300 kDa for all but the lowest DMD sample using ab154168. The bottom graphs show the DMD data again, only with adjusted y-axes, to zoom in on the grey shaded areas of the upper graphs. D) Virtual blot and electropherogram representation of a typical full length dystrophin signal in a healthy control sample detected by Wes. The dystrophin signal is detected using 4 different antibodies: Dys-1 (1/25 antibody, loaded 0.04 μg protein), Manex59B (1/100 antibody, loaded 0.15 μg protein), ab154168 (1/1000 antibody, loaded 0.13 μg protein) and Mandys106 (1/50 antibody, loaded 0.13 μg protein). E) An overview of the dystrophin antibody epitopes recognised by the different antibodies and the exons coding for the epitopes. The arrows indicate the promotors of the different dystrophin isoforms (Dp427: brain (B), muscle (M) and Purkinje (P); Dp260, Dp140, Dp116 and Dp71).</p

Dystrophin levels in skeletal muscle samples derived from BMD patients.

<p>Panel of 25 tibialis samples from BMD patients, analysed for dystrophin using ab154168 and Mandys106 and normalized to α-actinin (n = 2). Protein loading was 0.125 μg. A) Dystrophin levels (divided by α-actinin) expressed as a percentage of healthy control (% CTRL). The 2 BMD samples indicated by an arrow are depicted in B. B) Electropherogram showing the molecular weight shift of the peaks for mutant dystrophin with different sizes derived from the 2 BMD samples (A and B) indicated by an arrow in A, compared to a control sample with full-length dystrophin. BMD A (del 45–55: calculated MW = 358 kDa, expected apparent MW on Wes is 279 kDa; BMD B (del 45–48): calculated MW = 401 kDa, expected apparent MW on Wes is 294 kDa.</p

Overview of dystrophin antibodies.

<p>Overview of dystrophin antibodies.</p

Optimization of antibody dilution for Mandys106 and ab154168 and determining linearity of quantification.

<p>Skeletal muscle samples from a healthy control and a DMD patient with high trace dystrophin and a DMD patient with low or complete absence of dystrophin were used for optimisation and to determine the linear range of quantification. A and B) Dystrophin detected in healthy control (25 μg/ml) and DMD (250 μg/ml) samples using different antibody dilutions. C and D) Dystrophin detected in different amounts of healthy muscle lysate, using the antibody concentrations established in -Aand B (Mandys106: 1/50; ab154168: 1/1000). The small squares at the bottom right of graphs C and D represent a zoomed-in section of the low protein concentration range, to show signal linearity between 0 and 0.2 μg total protein. The arrows indicate the dilutions/concentrations selected for further experiments.</p