title: Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy.
abstract: The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage ('click chemistry') in the other. The most active bi-specific CPP-PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP-PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Fazel
authors.initials: F
authors.lastname: Shabanpoor
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.
authors.forename: Graham
authors.initials: G
authors.lastname: McClorey
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Amer F
authors.initials: AF
authors.lastname: Saleh
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Järver
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK mgait@mrc-lmb.cam.ac.uk.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
mesh_terms: D030301:Activin Receptors, Type II / Q000235:genetics; D000818:Animals; D002470:Cell Survival; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D016189:Dystrophin / Q000235:genetics; D005091:Exons; D051379:Mice; D018101:Mice, Inbred mdx; D060172:Morpholinos / Q000138:chemical synthesis / Q000737:chemistry*; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D012326:RNA Splicing
chemical_list: D057846:Cell-Penetrating Peptides; D016189:Dystrophin; D060172:Morpholinos; D030301:Activin Receptors, Type II; C425584:activin receptor type II-B