Targeted Exon Skipping in Transgenic hDMD Mice: A Model for Direct Preclinical Screening of Human-Specific Antisense Oligonucleotides

Abstract

The therapeutic potential of frame-restoring exon skipping by antisense oligonucleotides (AONs) has recently been demonstrated in cultured muscle cells from a series of Duchenne muscular dystrophy (DMD) patients. To facilitate clinical application, in vivo studies in animal models are required to develop safe and efficient AON-delivery methods. However, since exon skipping is a sequence-specific therapy, it is desirable to target the human DMD gene directly. We therefore set up human sequence-specific exon skipping in transgenic mice carrying the full-size human gene (hDMD). We initially compared the efficiency and toxicity of intramuscular AON injections using different delivery reagents in wild-type mice. At a dose of 3.6 nmol AON and using polyethylenimine, the skipping levels accumulated up to 3% in the second week postinjection and lasted for 4 weeks. We observed a correlation of this long-term effect with the intramuscular persistence of the AON. In regenerating myofibers higher efficiencies (up to 9%) could be obtained. Finally, using the optimized protocols in hDMD mice, we were able to induce the specific skipping of human DMD exons without affecting the endogenous mouse gene. These data highlight the high sequence specificity of this therapy and present the hDMD mouse as a unique model to optimize human-specific exon skipping in vivo.

Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles

“…This variability has been demonstrated in 2OMeAO-induced skipping of a variety of human exons in tissue culture and in a mouse carrying the human genomic dystrophin gene. But these studies have also found that a large proportion of exons are readily skipped by the use of appropriate AO sequences (21,22).…”

Comparative Analysis of Antisense Oligonucleotide Sequences for Targeted Skipping of Exon 51 During Dystrophin Pre-mRNA Splicing in Human Muscle

“…As a proof of principle, the AOs were also tested on muscle explants from DMD patients to study their effect in an ex vivo structure, as such explants have been described as a putative model in which to test gene therapy models McClorey et al, 2006a). These experiments were complemented by the intramuscular administration of PMO versions of the two most promising sequences into the gastrocnemius muscle of a mouse model transgenic for the entire human dystrophin locus (Bremmer-Bout et al, 2004). All these approaches suggested that one of the AOs (AO B30, ϩ66ϩ95) more robustly induced exon skipping, and this sequence has now been chosen for the phase I/IIa trial which is underway in the United Kingdom.…”

Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model

“…Subsequently, the same approach was successfully used in the mdx mouse model by intramuscular or i.v. delivery of AONs (11)(12)(13)(14). The antisense approach resulted in very efficient rescue of the dystrophin protein even though the effect of AONs is limited in time, thus requiring multiple administrations.…”