Enhanced in vivo delivery of antisense oligonucleotides to restore dystrophin expression in adult mdx mouse muscle

Abstract

The use of antisense oligonucleotides (AOs) to induce exon skipping leading to generation of an in-frame dystrophin protein product could be of bene¢t in around 70% of Duchenne muscular dystrophy patients. We describe the use of hyaluronidase enhanced electrotransfer to deliver uncomplexed 2P P-Omethyl modi¢ed phosphorothioate AO to adult dystrophic mouse muscle, resulting in dystrophin expression in 20^30% of ¢bres in tibialis anterior muscle after a single injection. Although expression was transient, many of the corrected ¢bres initially showed levels of dystrophin expression well above the 20% of endogenous previously shown to be necessary for phenotypic correction of the dystrophic phenotype.

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

“…The microdystrophin line shows a level of dystrophin expression at the sarcolemma similar to that seen in the minidystrophin line when using rabbit polyclonal DysC3750 over a range of dilutions. 20,28 As shown in Figure 2b, at a dilution of 1:2000, the endogenous signal in the C57/Bl10 wild-type mouse tissue and in revertant fibres in the mdx is still clearly visible, but staining of the transgene product is not detectable in the transgenic line (Figure 2b). …”

“…20 For the detection of revertant fibres or transfected dystrophin-positive fibres above the endogenous transgene expression levels, DysC3750 was titred down to a dilution of 1:2000, a level at which this antibody shows no dystrophin detection in the minidystrophin line. 28 Primary antibodies were diluted in PBST plus 1% FCS. Bound antibody was detected with an appropriate biotinylated species specific secondary antibody (Dako, Ely, UK) at 1:500 in PBST, followed by a 30 min incubation with horseradish peroxidaseconjugated streptavidin/biotin complex (ABC-HRP, Vector Laboratories, Peterborough, UK).…”

“…After a 5 min centrifugation at 13 000 rpm in a bench centrifuge to pellet myofilaments, the supernatants were solubilized in 10% SDS sample buffer and fractionated on a 6% polyacrylamide gel as described previously. 27,28 Proteins were transferred to PVDF membrane and equality of protein transfer confirmed by staining the membrane with a 0.1% solution of amido black. For the analysis of humoral immune responses, strips of membrane from blots containing paired tracks of either AD17 and mdx or C57BL10 and mdx extracts were prepared.…”

Gene Therapy Progress and Prospects: Electroporation and other physical methods

“…We have demonstrated that electroporation can also be used to deliver oligonucleotides to muscle, specifically for antisense-mediated exon skipping in dystrophic skeletal muscle. 20 Electroporation has been extensively tested for gene transfer into tumours. Even the transfer of empty plasmid vector is sufficient to cause significant tumour regression in some models.…”

Efficient Nuclear Delivery and Nuclear Body Localization of Antisense Oligo-Nucleotides using Degradable Polymersomes

“…The significant advantage of AONs is the ability to target the gene irrespective of its large size and clinical applicability as small-molecule based therapy [9]. However, efficient delivery of AONs in muscles is a great challenge and much depends on the carriers that provide protection against degradation, cell specificity and release [10][11][12].…”