Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy

Abstract

Objective: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping.

Methods: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry.

Results: Twelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%-4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry.

Conclusion: Golodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization.

Clinicaltrialsgov identifier: NCT02310906.

Classification of evidence: This study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

Figures

Figure 1. Golodirsen study details

(A) Study design. (B) Study flow diagram (part 1). 6MWT = 6-minute walk test; DSMB = data safety monitoring board; PDPF = percentage dystrophin-positive fibers.

Figure 2. Treatment with golodirsen demonstrated an increase in de novo dystrophin protein

(A) Western blot examples performed with baseline (labeled as BL) and on treatment (Tx) paired samples from 4 patients. Numbers above Tx gel lanes indicate the percent normal control dystrophin, calculated from the standard curve on each blot (lanes denoted with 4%, 2%, 1%, 0.5%, 0.25%). In both panels, lane 0 represents baseline untreated DMD control tissue with no normal control lysate. Arrows and values above gel indicate the percent normal dystrophin measured for the indicated lane. (B) Western blot data shown as averaged dystrophin percent of normal for each individual patient at baseline (open circles) and at part 2, week 48, (solid squares), with dystrophin levels on y-axis presented as linear values. *Sample reading was above the upper limit of quantification.

Figure 3. Exon 53 skipping and dystrophin

(A) Treatment with golodirsen demonstrates an increase in skipping of exon 53 in dystrophin mRNA. Percent exon 53 skipping for individual patients at baseline (open circles) and on treatment (filled squares). Data represent mean ± SD values for 4–8 replicates. (B) Positive correlation between percent exon 53 skipping and production of dystrophin protein. Percent exon 53 skipping change from baseline was plotted against the change in dystrophin protein from baseline, as measured by Western blot (Spearman r correlation coefficient: 0.500; p = 0.011).

Figure 4. Dystrophin expression, baseline and treated part 2, week 48, biopsy samples from the same patient

(A) Example 1: Low magnification image of whole tissue section stained for laminin α2 (green, all fibers stained) and dystrophin (red). A total of 2,843 muscle fibers were present in the baseline whole-slide image (A.a) and 1,077 fibers in the part 2, week 48, image (A.b). Three highlighted regions from the whole tissue sections (green) are magnified below each image to illustrate the corresponding dystrophin staining in these regions (red). Image intensity was not enhanced and represents original captured images. Dystrophin protein at part 2, week 48, was 4.3% of normal, as measured by Western blot for this patient. (B) Example 2: Low magnification image of whole tissue section stained for laminin α2 (green, all fibers stained) and dystrophin (red). A total of 4,945 muscle fibers were present in the baseline whole-slide image (B.a) and 2,167 fibers in the part 2, week 48, image (B.b). Three highlighted regions from the whole tissue sections (green) are magnified below each image to highlight the corresponding dystrophin staining in these regions (red). Image intensity was not enhanced and represents original captured images. Dystrophin protein at part 2, week 48, was 1.91% of normal, as measured by Western blot for this patient.

Figure 5. Manual percentage dystrophin-positive fiber (PDPF) analysis

(A) Baseline values of PDPF for each patient are shown as open circles and part 2, week 48, intensity is shown as solid squares. This graphical representation provides a ready way to visualize relative changes in each patient. Data represent the mean of 3–4 replicates with SD bars. (B) Western blot protein change from baseline is plotted on the x-axis and percent dystrophin-positive fiber change from baseline as assessed by manual scoring is plotted on the y-axis. Spearman analysis shows a significant correlation (p < 0.001; r = 0.663). Each point represents a single patient.