Summary of additional descriptive parameters.

<p>Summary of additional descriptive parameters.</p

^†Predicted skip is shown for 2’O-Methyl oligos categorised as ‘good’ or not, based on the level stratifications reported for each study (Aartsma-Rus, >25%; Dwi Pramono, >27.5%; Harding, >30%).

<p>^†Predicted skip is shown for 2’O-Methyl oligos categorised as ‘good’ or not, based on the level stratifications reported for each study (Aartsma-Rus, >25%; Dwi Pramono, >27.5%; Harding, >30%).</p

Predictive modelling for PMO oligonucleotides.

<p>(A) Descriptive parameters are added consecutively to the model based on their predictive power (except parameter 1, which is the intercept). K-fold R² (green triangles), an indicator of the likelihood of the model to be predictive of new datasets, increases markedly with the addition of dG50 (binding energy of the oligo to a region encompassing the target site and 50 base flanks), DfA, the distance of the target from the upstream splice acceptor site, and Malueka type, the category of the exon based on splice-related descriptors defined by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120058#pone.0120058.ref038" target="_blank">38</a>]. R² (blue squares) is improved by the addition of further parameters but K-fold R² is only slightly increased. (B) Percentage skipping as reported (Observed skip) against the percentage skipping, as predicted by a standard least squares model for the skipping efficacy of PMO oligos, based on the parameters: dG50, DfA, Malueka type, and the factorial of dG50 x DfA. A line of best fit is marked (red line) with 0.05 significance curves (dashed red lines), and average of observed (dashed blue line). (C) Confusion matrix showing the numbers of correct and incorrect predictions of skipping efficacy greater or less than 30% by an ordinal logistic model constructed using the same parameters (green background = correctly predicted; red = incorrectly predicted).</p

Prospective testing of new PMO oligo sequences targeting exons 44 and 53 of the human dystrophin transcript.

<p>(A) Representative gels showing RT-PCR of the native and exon-44-skipped (252 bp) or exon-53-skipped (190 bp) transcripts following exon skipping treatments in cell lines derived from a DMD patient harbouring targetable mutations in exons 44 or 53. M: 100 bp ladder, NT: non-treated, Mock: random 31-mer; test oligos are numbered according to their distance from the acceptor site. As a loading control, GAPDH is shown. (B) Western blots using an anti-dystrophin C-terminal antibody, showing rescued truncated dystrophin protein. A calibration curve of full-length dystrophin from normal control cells was loaded for comparison; Mock: random 31-mer PMO; test oligos are numbered according to their distance from the acceptor site; MyHC: Myosin Heavy Chain loading control. (C-E, F-H) Quantification of RNA and protein levels for exons 44 and 53, respectively. (C, F) Observed skipping efficacy (skipped transcript as a percentage of total non-skipped plus skipped transcript) is plotted against predicted values. R² values are shown for each repeat. (D-E, G-H) Plots of predicted skipping efficacy against distance from exon acceptor site, showing observed skipped transcript levels (D, G) or observed protein levels (E, H). Values for predicted skip are normalized to the average value of the observed skips or of the observed protein levels, to allow for inter-study variation in general skipping efficacy. Distance from acceptor is given for the first (5’-most) base of the target site. A moving average is shown over 15 bases (in this way, the moving average indicates the value for the mid-point of each target site).</p

Binding energetics and RNA folding accessibility.

<p>(A) The energy (dG) of binding was calculated for the oligo to the target region and flanking regions of various extents: flanks of 50 (dG50), 100 (dG100), or 200 (dG200) bases around the target site, or flanks extending from the 5’ end of the exon to 10 bases downstream of the target 3’ end (dG_et+10). (B) Accessibility (the likelihood of bases being unpaired) of the target site within the predicted structure of the folded RNA: (1) target site; (2) target site normalized to oligo length; (3) the 15 bases of the target’s 3’ end; (4) the 8 bases of the target’s 3’ end; (5) the most accessible 8 bases of the target site.</p

Predictive formulas derived for PMO (Popplewell) data applied across 2’O Methyl data (Aartsma-Rus, Dwi Pramono, Harding, and Wilton).

<p>Predicted skip is shown for oligos categorised as ‘good’ or not, based on the level stratifications reported for each study (Aartsma-Rus, >25%; Dwi Pramono, >27.5%; Harding, >30%; Popplewell, >30%; Wilton, >30%).</p

Screening across exons for predicted exon skipping and comparison with published data (from [29]).

<p>Distance from acceptor is given for the first (5’-most) base of the target site. Observed and predicted percentage skipping are shown for 30-mer and 25-mer oligonucleotides, with moving averages shown over 15 and 13 bases, respectively (in this way, the moving average indicates the value for the mid-point of each target site).</p

Mutation analysis and exon skipping approach.

<p>(A) Cell line GM05162 harbors an out-of-frame deletion mutation of <i>dystrophin</i> exons 46–50, which requires skipping 6 exons via PMOs to correct the reading frame. Sides of schematic boxes represent the codon phase. (B) Cell line GM05017 harbors an out-of-frame deletion mutation of <i>dystrophin</i> exons 45–50, which requires skipping 5 exons via PMOs to correct the reading frame. (C) Structure of full-length dystrophin and exons 45-55-deleted dystrophin. The truncated dystrophin generated by exons 45–55 skipping contains a hybrid rod repeat (yellow bars) of rods 17 and 22. Actin bind, actin-binding domain; H1-4, hinge domain 1–4; CRD, cysteine-rich domain; CTD, C-terminal domain.</p

PMO sequences used for exons 45–55 skipping.

<p>PMO sequences used for exons 45–55 skipping.</p

Multi-exon skipping of <i>dystrophin</i> exons 45–55 in transdifferentiated DMD patient cells.

<p>(A) RT-PCR for <i>dystrophin</i> following cocktail PMO transfection in transdifferentiated DMD patient cells. Cells were treated with 1, 3, or 10 μM each PMO. Expected molecular weight of <i>dystrophin</i> exons 45–55 skipped mRNA is 308 bp. (B) Representative immunocytochemistry of transduced DMD fibroblasts following PMO cocktail transfection. Nuclei counterstained with DAPI. Scale bars: 100 μm.</p