RESUMO
The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD.
Assuntos
Distrofina/genética , Éxons , Miostatina/genética , Oligonucleotídeos Antissenso/química , Fases de Leitura Aberta , Processamento Alternativo , Animais , Animais Recém-Nascidos , Arginina/química , Diafragma/metabolismo , Modelos Animais de Doenças , Distrofina/metabolismo , Terapia Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Morfolinos/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Miostatina/metabolismo , Necrose , Peptídeos/química , Fases de LeituraRESUMO
The emergence of variant fowlpox viruses (FWPVs) and increasing field use of recombinants against avian influenza H5N1 emphasize the need to monitor vaccines and to distinguish them from field strains. Five commercial vaccines, two laboratory viruses and two European field isolates were characterized by PCR and sequencing at 18 loci differing between attenuated FP9 and its pathogenic progenitor. PCR failed to discriminate between the viruses and sequence determination revealed no significant differences at any locus, except for a polymorphic locus encompassed by deletion 24 (9.3 kbp) in FP9. Surprisingly, 'novel' previously unreported sequence (spanning 1.2 kbp) was found in both European field isolates and three of the vaccines. It was absent from the other two vaccines, removed by a 1.2 kbp deletion identical to that surprisingly also observed in the completely sequenced genome of FPV USDA. This locus (H9) adds a potentially useful tool for discriminating between FWPV field isolates and vaccines.