RESUMEN
Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast-myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2'-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.
Asunto(s)
Distrofia Miotónica/genética , Oligonucleótidos/genética , ARN/genética , Alelos , Animales , Proteínas CELF1 , Silenciador del Gen , Ratones , Modelos Genéticos , Músculo Esquelético/metabolismo , Mutación , Mioblastos/metabolismo , Distrofia Miotónica/terapia , Oligonucleótidos/química , Oligonucleótidos Antisentido/genética , Interferencia de ARN , Empalme del ARN , Proteínas de Unión al ARN/genéticaRESUMEN
BACKGROUND: Antisense-mediated exon skipping is a putative treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the disrupted DMD reading frame is restored, allowing generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. In vivo studies are mainly performed using 2'-O-methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds were never directly compared. METHODS: mdx and humanized (h)DMD mice were injected intramuscularly and intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human exons 44, 45, 46 and 51. RESULTS: Intramuscular injection showed that increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for mouse exon 23. Although PMO induced more mouse exon 23 skipping, PMO and 2OMePS were more comparable for human exons. After intravenous administration, exon skipping and novel protein was shown in the heart with both chemistries. Furthermore, PMO showed lower intramuscular concentrations with higher exon 23 skipping levels compared to 2OMePS, which may be due to sequestration in the extracellular matrix. Finally, two mismatches rendered 2OMePS but not PMO AONs nearly ineffective. CONCLUSIONS: The results obtained in the present study indicate that increasing AON length improves skipping efficiency in some but not all cases. It is feasible to induce exon skipping and dystrophin restoration in the heart after injection of 2OMePS and unconjugated PMO. Furthermore, differences in efficiency between PMO and 2OMePS appear to be sequence and not chemistry dependent. Finally, the results indicate that PMOs may be less sequence specific than 2OMePS.
Asunto(s)
Exones/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Distrofia Muscular de Duchenne , Oligonucleótidos Antisentido , Oligonucleótidos Fosforotioatos , Animales , Secuencia de Bases , Humanos , Ratones , Ratones Endogámicos mdx , Datos de Secuencia Molecular , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Miocardio/citología , Miocardio/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Fosforotioatos/administración & dosificación , Oligonucleótidos Fosforotioatos/genéticaRESUMEN
We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b. These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.