The status of exon skipping as a therapeutic approach to duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level.

Figure 1

Chemistries of antisense oligomers. (a) 2′-O-Methylphosphorothioate (2′OMePS AON); (b) 2′-O-methoxyethyl phosphorothioate; (c) locked nucleic acid (LNA); (d) peptide nucleic acid (PNA); (e) phosphorodiamidate morpholino oligomers (PMO); (f) AcHN-(RXRRBR)2XB peptide-tagged PMO (R, arginine, X, 6-aminohexanoic acid and B, ®- alanine) (PPMO); G, octa-guanidine PMO.

Figure 2

Restoration of dystrophin in cardiac muscles of mdx mice after six intravenous injections (at biweekly intervals) of 30 mg/kg of the PPMOE23 targeting mouse dystrophin exon 23. Muscles were examined 2 weeks after the last injection. Left panel, muscles from heart of normal C57BL/6. Middle panel, muscle from heart of control mdx mouse. Right panel, PPMO-treated mdx. Dystrophin was detected by immunochemistry with the polyclonal rabbit antidystrophin antibody, P7, and visualized with Alexa 594 tagged goat-anti-rabbit Igs. Blue nuclear staining with DAPI.

Figure 3

Diagram to illustrate the exon-skipping strategy to restore open reading frame at the mutation site in the CXMD dystrophic dog. A point mutation in the acceptor splice site in intron 6 preceeding exon 7 (X) leads to exclusion of exon 7 from the transcript and loss of open reading frame when exon 6 is spliced to exon 8. To restore open reading frame, requires the loss of at least two further exons: 6 and 8. In the event, exon 9 is also excluded from the transcript but, because it contains a whole number of codon triplets, this does not disrupt the translation of the resultant mRNA. CXMD, canine X-linked muscular dystrophy.