| This project aims to develop a gene therapy for Duchenne Muscular Dystrophy (DMD) based on 'antisense' oligonucleotides (AONs). DMD is a frequent lethal hereditary disease causing severe, progressive muscle weakness and premature death, mostly before the age of 30. No effective pharmacological or genetic therapy is available. In DMD, deletions (60%), duplications (10%) and point mutations disrupt the dystrophin reading frame, abolishing dystrophin synthesis. In-frame deletions and duplications cause the milder Becker Muscular Dystrophy (BMD). We are developing antisense-based 'exon-skipping' to restore the reading frame of DMD mutations by turning DMD deletions into the next-larger BMD form. From patients with revertant fibers it is suggested that even 3% remaining dystrophin may already improve their condition, while a restoration of dystrophin levels to 15-20% is generally thought to yield milder disease. This is a major advantage of AON-based therapy for DMD, compared with knock-down antisense applications, where efficiencies in excess of 80-95% are typically required for significant therapeutic effects. In cultured patient cells in vitro, we have identified a set of (2'O-Methyl-Phosphorothioate-RNA)AONs capable of efficiently skipping over 35 different exons, to a level of 70-90% of transcripts. Already eight of these AONs would jointly be beneficial for 80% of patients. The exon 51 AON in particular would even be therapeutic for 17% of patients. Our subsequent mouse in vivo studies, to assess skipping levels and biological persistence, show that two intramuscular injections of the AON associated with the carrier molecule polyethylenimine (PEI), spaced by 24h, causes skipping to last for more than a month, with a broad peak from 6-15 days. Mass spectrometry shows the AONs to persist for 30 days in the mouse muscle. This suggests that in future administration regimens, a bi-weekly dosage may realistically be considered. In mdx mice, the skipping obtained is threefold higher than that in control mice with intact muscle membrane, indicating that the defective membrane of dystrophic muscle may actively facilitate local availability. Specific targeting of the 2,5 Mb human DMD transgene in hDMD mice shows exclusive human-specific skipping, leaving the mouse transcript unaffected. This further underpins the high specificity of the antisense exon-skipping process. Finally, recent pilot systemic delivery in mdx has been reported by our colleagues in the UK to cause skipping of mouse exon 23 and significant reprisal of dystrophin synthesis. Together with the moderate requirement for dystrophin restoration for a therapeutic effect and the relatively small size of the therapeutic agent, this raises the prospect of sufficiently effective, non-viral systemic delivery. As in mouse no deletions/duplications exist like in DMD (the mdx mutation is a stop codon in exon 23) further in vivo development requires pilot clinical trials in human patients. The obvious first aim is in vivo proof of concept of exon skipping and restoration of dystrophin synthesis and safety of the AON administration following local delivery in patient muscle, then followed by the development of systemic administration to achieve clinical effect. In a collaboration between LUMC, Prosensa (a spin-off from the Leiden University W&N faculty), and academic neurologists from the LUMC, AMC Amsterdam, and AZ Leuven, and in a later phase the other neuromyological centers in the NL, we aim to set up a preclinical, pharmacological and clinical program toward a safe, tolerable and efficient clinical therapy. Preclinically, we will validate clinical-grade AON reagents, perform molecular and histological analysis of the clinical samples of consented participants prior to and during the trial, further refine application and biopsy technology and continue our research towards further improvement of efficiency and action spectrum of AONs. Pharmacologically we will generate a GMP batch of exon-51 AON, the most effective single-target AON thus far, and perform the required toxicology programme. Two clinical studies will be performed. First we will inject the compound intramuscularly and screen for local effects: specific exon 51 skipping and dystrophin synthesis, muscle morphology, inflammation and toxic signs, and potential expression of dystrophin antibodies. After a positive outcome, a systemic delivery study will follow, utilising the same AON, or one which has been improved for efficiency and action spectrum in our ongoing preclinical research. The toxicology programme will then be more extensive, as required for systemic delivery. The test programme for therapeutic and adverse effects will basically the same, but extended to include overall muscle morphology and function. |
