Résumé :
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For a subset of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has been reported to be efficacious to restore protein expression. The primary goal of our study was to evaluate the usage of novel biocompatible polymethyl methacrylate (PMMA) nanospheres (marked as T1) as alternative non-viral vehicle to in vivo deliver charged AONs, by testing in vivo nanoparticles capacity of both transporting AONs and inducing dystrophin rescue with improved efficiency and/or with more durable effect. Our results show that weekly intraperitoneal injections in mdx mice of 2'-O-methyl-modified full-length phosphorothioate (2OMePS) AON, recognising the splice donor of exon 23, combined with T1 nanoparticles induced dystrophin expression in both heart and skeletal muscle. Furthermore, the combination with this novel non-viral vehicle allowed the administration of a very low dose of AON, represented by 2.7 mg/kg, at least 1/50th if compared with the dosage currently described for naked AONs systemic delivery, however still resulting in significant levels of dystrophin rescue, both by immuno- as western- blotting (15-18% of dystrophin positive fibers). We also demonstrated that the protein rescue persists, at the same level, up to 60 days from the first injection. An extensive Real-Time quantitative PCR transcription analysis showed increased levels of dystrophin messenger after T1-AON complexes treatment in all the muscles analysed, including heart, varying from 2 to 3 folds if compared to mdx untreated muscles. We concluded that the T1 nanoparticles have the capacity to bind and convey AONs in almost all muscles as well as to reduce the dose of AON required to obtain dystrophin rescue, therefore representing a promising therapeutic AONs delivery system for future studies in DMD. (1) Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, ITALY. (2) Department of Histology, Microbiology, and Medical Biotechnologies; University of Padova, Padova, ITALY. (3) Department of Neurological Sciences and Vision, Section of Clinical Neurology, University of Verona, Verona, ITALY. (4) Department of Chemistry, University of Ferrara, Ferrara, ITALY. (5) Laboratory of Cell Biology, IOR, Bologna, Italy; IGM-C.N.R., Unit of Bologna, c/o IOR, Bologna, ITALY. (6) ISOF, CNR, Bologna, ITALY. (7) Department of Environmental and Life Sciences INSTM, University of Piemonte Orientale, Alessandria, ITALY.
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