Increase in Full-Length Dystrophin by Exon Skipping in Duchenne Muscular Dystrophy Patients with Single Exon Duplications: An Open-label Study

Subjects

Male subjects, aged 6 months or older, with DMD caused by duplication of exon 45, 51, or 53 were eligible for this study. Enrollment of up to 6 subjects was originally planned. Subjects aged ≥5 were required to receive corticosteroid treatment at a stable dose for the duration of the study. Key exclusion criteria included any current or prior treatment with ASOs, symptomatic cardiomyopathy, and anticipated pulmonary decompensation over the duration of the study.

Study procedures

Each subject was treated with casimersen or golodirsen (according to genotype) for a duration of 48 weeks. The study drug was administered weekly via intravenous infusion at a dose of 30 mg/kg, in keeping with FDA-approved dosing. A biopsy of the gastrocnemius was obtained at baseline and, on the contralateral side, at 48 weeks. Functional assessments and patient-reported outcome scales selected according to age and ambulatory status were administered at baseline and at 12, 24, 36, and 48 weeks. In non-ambulatory patients, these were defined in the protocol as the ACTIVE-seated workspace volume measurement and the PROMIS Upper Extremity Report [9, 10]. However, no ACTIVE-seated data was ultimately included in the analysis, as a review of raw data identified errors in acquisition, potentially attributable to equipment malfunction, at several examination time points. Pulmonary function tests were performed at the same time points as functional assessments. Echocardiograms were obtained at baseline, 12, and 24 weeks. Laboratory studies for safety monitoring were obtained regularly. This study was approved by the Nationwide Children’s Hospital Institutional Review Board. All subjects or their legal guardians provided informed consent for participation. This study was registered on ClinicalTrials.gov [NCT04179409].

DNA and RNA sequencing

DNA was extracted from muscle samples using the DNeasy Kit (Qiagen). In each subject, the target exon and flanking 300 bp intronic regions underwent amplification and Sanger sequencing to identify variants that may modify endogenous splicing or PMO binding (primer sequences provided in Supplementary Table 1).

RNA was extracted from homogenized muscle samples with the Trizol reagent (Invitrogen) and the RNA Clean and Concentrator-25 kit (Zymo). Exon skipping was quantified by RT-PCR using an adapted version of a previously published method [11]. For this analysis, amplicons were generated spanning the duplicated exon (Supplementary Table 1) and quantified by agarose gel electrophoresis.

Dystrophin quantification

Western blot was performed using a modified version of a previously published method [4]. Two tissue blocks were analyzed for each time point. Protein was extracted using 4 M urea buffer. 20μg of protein was loaded on a 3–8% polyacrylamide tris-acetate gel (Life Technologies), run at 150 V for 75 minutes, and transferred onto a polyvinylidene difluoride membrane at 30 V for 75 minutes. After blocking, membranes were incubated overnight at 4°C with a dystrophin rod domain antibody (DYS1, Leica Biosciences, 1:20 dilution), washed, then incubated with mouse IgG horseradish peroxidase-conjugated secondary antibody (GE Healthcare). A standard curve prepared from healthy control tissue was included on each blot.

Quantitative immunofluorescence analysis was performed at Flagship Biosciences, using two tissue blocks for each time point [12]. Briefly, 10μm frozen sections were co-stained for dystrophin (Mandys106 antibody, Milipore Sigma, 1:50 dilution) and laminin (ab11576 antibody, Abcam, 5.5μg/mL). Whole slide images were acquired at 20X magnification on a Pannoramic Scan II fluorescent scanner (3DHISTECH). The images were then analyzed using Flagship’s proprietary algorithm to quantify percent dystrophin positive fibers (PDPF). After exclusion of artefacts and background correction, a threshold was calculated for each subject based on the sarcolemmal membrane mean dystrophin staining intensity of the baseline biopsy. Fibers with ≥30% pixels above this threshold along their perimeter were considered positive.

Statistics

Descriptive statistics were used to analyze the data. Hypothesis testing was not performed given the small sample size.

Subjects

Three subjects were screened for inclusion and all were enrolled in the study. Enrollment was halted when no further subjects with eligible mutations, and for whom participation was feasible, could be identified. Baseline characteristics are summarized in Table 1. Subject 3 missed one casimersen infusion at Week 34. All other infusions were completed as scheduled and all subjects completed the study. Subject 2 could not complete any functional assessments due to autism spectrum disorder.

Clinical outcomes

In subjects 1 and 3, the PROMIS Upper Extremity Report score and forced vital capacity remained stable throughout the duration of the study (Fig. 1 A-B). Subject 3 reported a subjective improvement in trunk control, as demonstrated by regaining the ability to remove his T-shirt, to bend down in his wheelchair to pick a water bottle from the floor, and to sit up from a semi-recumbent position (Supplementary Video). Left ventricular ejection fraction remained stable throughout the study in all subjects (Fig. 1C).

Fig.1

Exon skipping and dystrophin expression. A–C. The PROMIS score, FVC, and ejection fraction remained stable over the duration of the study. D. There was an increase in full-length DMD transcripts (relative to total DMD transcripts) in subjects 1 and 3. Deletion transcripts (i.e. skipping both copies of the target exon) were only observed at very low frequencies. E-F. Dystrophin expression increased in all subjects, as did PDPF. G. Representative western blots from each sample are shown as a composite. A 0.25% –8% standard curve prepared from healthy control tissue was included on each blot. H. Representative regions of interest stained for dystrophin are shown, along with dystrophin positivity analysis. Scale bar = 100μm. 48w, 48 weeks; BL, baseline; FVC, forced vital capacity; PDPF, percent dystrophin positive fibers.

Dystrophin expression

RT-PCR identified up to 3 transcript isoforms in each sample, and Sanger sequencing confirmed that these corresponded to skipping of none, one, or both copies of the duplicated exon (Supplementary Figure 1). There was an increase in full-length DMD transcripts in subjects 1 and 3, rising from a mean of 7.1% of all DMD transcripts at baseline, to 15% at 48 weeks (Fig. 1 D and Table 2). Transcripts skipping both copies of the target exon rose from a mean of 0.3% at baseline (likely reflecting the noise floor of our quantification) to 0.5% of all DMD transcripts at 48 weeks. Sanger sequencing did not identify any rare variants (frequency <1% in the GNOMAD v3.1.2 database) in the vicinity of the target exons that could affect skipping efficiency [13]. Dystrophin expression measured by western blot increased in all subjects over the duration of the study, from a mean of 0.94% of normal at baseline to 5.1% at 48 weeks (Supplementary Fig. 1E and G Supplementary Figure 2, and Table 2). The increase in dystrophin expression was confirmed by a second independent analysis (SupplementaryFigure 3). PDPF also increased in all subjects, from a mean of 14% at baseline to 50% at 48 weeks ( Fig. 1F and H, Supplementary Figure 4).

Safety

Each subject experienced between 8 and 11 adverse events (Supplementary Table 2). No serious adverse events were recorded. Only 3 adverse events were deemed possibly related to the study drug: intermittent headache, renal calculi, and rash. No clinically significant laboratory abnormalities were detected.