Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study

Abstract

Background: Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder.

Methods: We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250.

Findings: Two patients received 0.09 mg AVI-4658 in 900 microL (0.9%) saline and five patients received 0.9 mg AVI-4658 in 900 microL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44-79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26.4%), and the mean intensity was 17% (range 11-21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0.002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot.

Interpretation: Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD.

Funding: UK Department of Health.

Figure 1

Deletions and predicted results of exon skipping in the patients who were studied (A) Pre-mRNA transcripts and dystrophin protein products from full length DMD, in patients with Duchenne muscular dystrophy, and predicted protein sequences after exon skipping. (I) The normal dystrophin gene produces the full length dystrophin product. (II) Patients 1 and 2 had a deletion in exon 50 that disrupts the open reading frame, leading to a truncated and unstable dystrophin. (III) Skipping of exon 51 restores the reading frame, producing a truncated but functional dystrophin that lacks exons 50 and 51. (IV) Patient 7 is missing exons 49 and 50. (V) Patients 3 and 4 are missing exons 48–50. (VI) Patients 5 and 6 are missing exons 45–50. All the truncated dystrophins produced after skipping of exon 51 are missing the hinge 3 region and some of the rod domain but have been associated with the milder BMD phenotype., (B) Structure of the phosphorodiamidate morpholino modification of the antisense oligomer.

Figure 2

Procedure for prescreening of patients before injection of AVI-4658. Patient 3 is shown as an example; similar results were obtained for all patients. (A) Transverse MRI of the lower leg and coronal MRI of the extensor digitorum brevis muscle (arrow) confirmed the suitability of the muscle. (B) Skin fibroblasts from all patients were forced into myogenic differentiation and treated with an AVI-4658 congener to confirm exon skipping and dystrophin production. RT-PCR analysis shows two bands: the high molecular weight band corresponds to the unskipped transcript (including exons 46, 47, 51, and 52) and the low molecular weight band corresponds to the transcript fragment with size specific skipping of exon 51. (C) Exon 51 skipping was confirmed by sequencing.

Figure 3

Dystrophin expression in patients treated with high-dose AVI-4658 Transverse sections of treated and contralateral EDB muscles that were immunostained for dystrophin with MANDYS106. (A) Low-power micrograph of a whole section taken with ×10 objective lens shows widespread expression of dystrophin in fibres from the treated muscle in patient 4. (B) Higher magnification (×20 objective lens) of dystrophin immunolabelling in treated and untreated sections in patients 3–7. Scale bars=100 μm.

Figure 4

Intensity of dystrophin expression in patients treated with high-dose AVI-4658 relative to control (A) Mean random intensity measurements. (B) Measurement of mean dystrophin intensity in positive fibres: intensity measurements exclusively targeted to 100 dystrophin-positive and 100 dystrophin-negative fibres within the same area in patient 4. Bars are SEM.

Figure 5

Exon 51 skipping in amplified RNA from treated muscles (A) RT-PCR analysis of RNA extracted from treated (X), untreated (O), and control (C) muscle sections detects shorter transcript fragments in the treated muscles, with sizes that correspond to the specific skipping of exon 51. (B) Exon 51 skipping was confirmed by sequencing. (C) Western blot analysis of homogenates of treated and untreated muscle (20×10 μm sections) and control muscle (2×10 μm sections [to avoid overexposure]) shows dystrophin expression in extracts from the control muscles (C) and treated (X) extensor digitorum brevis but not in the contralateral muscles (O). Loading was monitored with protogold. Low dose=0·09 mg AV-4658. High dose=0·9 mg AV-4658.