Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study

Abstract

Background: We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy.

Method: We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5-15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597.

Findings: 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1-10·6) to 16·4% (10·8-22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts.

Interpretation: The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy.

Funding: UK Medical Research Council; AVI BioPharma.

Figure 1

Patients recruited to the trial, their assignment to cohorts, and the dose-escalation scheme Each full red box represents a time interval of 12 weeks' dosing. Arrows show the timepoints at which the data safety monitoring board met with clinical investigators and the sponsor to review safety before subsequent dose escalations. *Patient withdrawn from study after seven doses.

Figure 2

Plasma pharmacokinetics of AVI-4658 Mean plasma concentrations of AVI-4658 versus nominal elapsed time averaged across weeks 1, 6, and 12. Area under the curve (AUC) over 24 h accounted for greater than 95% of AUC_0–∞, suggesting that most of the drug eliminated from the plasma was cleared within 24 h. AVI-4658 plasma exposure increased in a nearly proportional manner with dose for maximum concentration, AUC_0–24, and AUC_0–∞. Error bars show SDs.

Figure 3

Dystrophin protein expression in the seven patients who responded to treatment (A) Transverse sections of treated (post) and untreated (pre) muscle specimens immunolabelled with MANDYS106 antibody. (B) Post-treatment biopsy samples from participants P15 and P18; low-magnification images showing widespread and patchy dystrophin expression (arrows). (C) Western blotting of pretreatment and post-treatment muscle biopsy samples with antidystrophin Dys1 (exon 26–30) and antisarcomeric α-actinin antibodies; an average of 150 μg of total patient proteins was loaded per lane. CT=μg control muscle extract.

Figure 4

Functional analysis of restored dystrophin (A) Expression of dystrophin, α-sarcoglycan, and neuronal NOS in post-treatment muscle biopsy samples from participants P18 and P19 was quantified relative to control muscle in 40 dystrophin-positive or dystrophin-negative muscle fibres and normalised to β-spectrin expression. To overcome high background seen with the neuronal NOS antibody, the average background intensity of neuronal NOS-negative membranes was subtracted from control and patient values. For participant P19, there was no difference in α-sarcoglycan intensity between dystrophin-positive and dystrophin-negative fibres in the post-treatment muscle biopsy sample and neuronal NOS showed only a small increase in dystrophin-positive fibres (paired two-tailed t test, p=0·0007). For participant P18, neuronal NOS and α-sarcoglycan intensity was significantly increased in the dystrophin-positive fibres (neuronal NOS mean intensity as percentage of control: dystrophin-negative fibres 7% [SD 7], dystrophin-positive fibres 28% [SD 17], paired two-tailed t test, p≤0·0001; α-sarcoglycan mean intensity as percentage of control: dystrophin-negative fibres 45% [SD 16], dystrophin-positive fibres 75% [SD 31], p<0·0001). (B) Sarcolemmal restoration of the dystrophin-associated glycoprotein complex by AVI-4658. Post-treatment muscle biopsy samples from participants P19 and P18 were stained with antibodies against dystrophin (exon 43, MANDYS106), α-sarcoglycan, neuronal NOS, and β-spectrin. The arrows show the same dystrophin-positive fibre in each panel. In P19 (deletion of exons 45–50) the fibre shown by the arrow has increased α-sarcoglycan sarcolemmal expression, but not neuronal NOS because this patient is deleted for part of the dystrophin neuronal NOS binding site. (C) Inflammatory infiltrates quantification on pretreatment and post-treatment muscle samples. Muscle sections were incubated with antibodies (DAKO, UK) raised against human CD3 (pan T cell), CD4 (helper T cell) and CD8 (killer T cell). For each section, the number of CD-positive cells was represented as a percentage of the total number of muscle fibres. Patients with pretreatment and post-treatment values of zero are not represented. NOS=nitric oxide synthase.