RESUMEN
Duchenne muscular dystrophy is an X-linked, recessive muscular dystrophy in which the absence of the dystrophin protein leads to fibrosis, inflammation and oxidative stress, resulting in loss of muscle tissue. Drug repurposing, i.e. using drugs already approved for other disorders, is attractive as it decreases development time. Recent studies suggested that simvastatin, a cholesterol lowering drug used for cardiovascular diseases, has beneficial effects on several parameters in mdx mice. To validate properly the effectiveness of simvastatin, two independent labs tested the effects of 12-week simvastatin treatment in either young (starting at 4 weeks of age) or adult (starting at 12 weeks of age) mdx mice. In neither study were benefits of simvastatin treatment observed on muscle function, histology or expression of genes involved in fibrosis, regeneration, oxidative stress and autophagy. Unexpectedly, although the treatment protocol was similar, simvastatin plasma levels were found to be much lower than observed in a previous study. In conclusion, in two laboratories, simvastatin did not ameliorate disease pathology in mdx mice, which could either be due to the ineffectiveness of simvastatin itself or due to the low simvastatin plasma levels following oral administration via the food.
Asunto(s)
Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Simvastatina/farmacología , Animales , Modelos Animales de Enfermedad , Fibrosis/fisiopatología , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/efectos de los fármacosRESUMEN
Limb girdle muscular dystrophy (LGMD) types 2D and 2F are caused by mutations in the genes encoding for α- and δ-sarcoglycan, respectively, leading to progressive muscle weakness. Mouse models exist for LGMD2D (Sgca-/-) and 2F (Sgcd-/-). In a previous natural history study, we described the pathology in these mice at 34 weeks of age. However, the development of muscle pathology at younger ages has not been fully characterised yet. We therefore performed a study into age-related changes in muscle function and pathology by examining mice at different ages. From 4 weeks of age onwards, male mice were subjected to functional tests and sacrificed at respectively 8, 16 or 24 weeks of age. Muscle histopathology and expression of genes involved in muscle pathology were analysed for several skeletal muscles, while miRNA levels were assessed in serum. In addition, for Sgcd-/- mice heart pathology was assessed. Muscle function showed a gradual decline in both Sgca-/- and Sgcd-/- mice. Respiratory function was also impaired at all examined timepoints. Already at 8 weeks of age, muscle pathology was prominent, and fibrotic, inflammatory and regenerative markers were elevated, which remained relatively constant with age. In addition, Sgcd-/- mice showed signs of cardiomyopathy from 16 weeks of age onwards. These results indicate that Sgca-/- and Sgcd-/- are relevant disease models for LGMD2D and 2F.
Asunto(s)
Músculo Esquelético/patología , Sarcoglicanopatías/patología , Envejecimiento , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Músculo Esquelético/metabolismo , Sarcoglicanopatías/genética , Sarcoglicanos/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disorder caused by reading frame disrupting mutations in the DMD gene. Exon skipping is a therapeutic approach for DMD. It employs antisense oligonucleotides (AONs) to restore the disrupted open reading frame, allowing the production of shorter, but partly functional dystrophin protein as seen in less severely affected Becker muscular dystrophy patients. To be effective, AONs need to be delivered and effectively taken up by the target cells, which can be accomplished by the conjugation of tissue-homing peptides. We performed phage display screens using a cyclic peptide library combined with next generation sequencing analyses to identify candidate muscle-homing peptides. Conjugation of the lead peptide to 2'-O-methyl phosphorothioate AONs enabled a significant, 2-fold increase in delivery and exon skipping in all analyzed skeletal and cardiac muscle of mdx mice and appeared well tolerated. While selected as a muscle-homing peptide, uptake was increased in liver and kidney as well. The homing capacity of the peptide may have been overruled by the natural biodistribution of the AON. Nonetheless, our results suggest that the identified peptide has the potential to facilitate delivery of AONs and perhaps other compounds to skeletal and cardiac muscle.
Asunto(s)
Empalme Alternativo , Técnicas de Transferencia de Gen , Terapia Genética , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/genética , Péptidos Cíclicos , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Distrofina/genética , Exones , Humanos , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Biblioteca de Péptidos , Péptidos Cíclicos/químicaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.
RESUMEN
Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.
Asunto(s)
Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Fosforotioatos/química , Oligonucleótidos Fosforotioatos/uso terapéutico , Animales , Distrofina/genética , Exones , Humanos , Masculino , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mutación , Miocardio/metabolismo , Oligonucleótidos Antisentido/genética , Biblioteca de Péptidos , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/genética , Ácidos Nucleicos de Péptidos/uso terapéutico , Oligonucleótidos Fosforotioatos/genética , Reparación del Gen Blanco/métodosRESUMEN
Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy. It aims to restore the dystrophin open reading frame by skipping exons with antisense oligonucleotides (AONs) to allow production of partly functional proteins. The approach is currently tested in phase 3 clinical trials, but dosing and maintenance regimens have not yet been well studied. This study compared pharmacokinetic and pharmacodynamic effects of different 2'-O-methyl phosphorothioate RNA AON dosing and maintenance regimens in the preclinical mdx mouse model. When comparing different dosing regimens over a period of 8 weeks, higher levels of AON, exon skipping, and protein were observed in muscle after low daily doses compared with large weekly doses. Secondly, after receiving a high loading dose (1,250 mg/kg) in the first week, mice treated with maintenance injections twice weekly for 8 weeks showed higher preservation of therapeutic effects than mice receiving less or no maintenance injections. In both cases, the regimen resulting in the highest AON and exon skipping levels in muscle also resulted in high AON levels in liver and kidneys. These studies underline the importance of balancing optimal AON efficacy and tolerable levels in non-target organs, which may be fine-tuned by further optimization of AON treatment regimens.
Asunto(s)
Distrofina/genética , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Fosforotioatos/farmacología , Animales , Creatina Quinasa/sangre , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Esquema de Medicación , Cálculo de Dosificación de Drogas , Distrofina/agonistas , Distrofina/metabolismo , Exones , Terapia Genética , Humanos , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/síntesis química , Oligonucleótidos Fosforotioatos/farmacocinéticaRESUMEN
Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD) is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs). Here, we tested the safety and efficacy of subcutaneously administered 2'-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx/utrn(+/-); more severe phenotype). Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn(+/-) mice the therapeutic effect was larger: creatine kinase (CK) levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn(+/-) model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.Molecular Therapy-Nucleic Acids (2012) 1, e44; doi:10.1038/mtna.2012.38; published online 4 September 2012.
RESUMEN
Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.
Asunto(s)
Distrofina/efectos de los fármacos , Exones/efectos de los fármacos , Marcación de Gen/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Oligonucleótidos Antisentido/farmacología , Animales , Secuencia de Bases/efectos de los fármacos , Secuencia de Bases/genética , Células Cultivadas , Modelos Animales de Enfermedad , Distrofina/química , Distrofina/genética , Exones/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Morfolinas/química , Morfolinas/farmacología , Morfolinas/uso terapéutico , Morfolinos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Mutación/genética , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/uso terapéutico , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frame shifting and nonsense mutations in the dystrophin gene. Through skipping of an (additional) exon from the pre-mRNA, the reading frame can be restored. This can be achieved with antisense oligonucleotides (AONs), which induce exon skipping by binding to splice sites or splice enhancer sites. The resulting protein will be shorter but at least partially functional. So far, exon skipping has been very successful in cell cultures, in mouse and dog models, and even in a first exploratory study in patients. Current research mainly focuses on optimization of systemic AON delivery. Here we give an overview of the available mouse models. To obtain the most informative results for future clinical application, research may have to move from the currently preferred mdx mouse to mouse models more comparable to patients, such as the utrophin/dystrophin-negative mouse and the hDMD mouse models. Further, we briefly discuss two AON backbone chemistries that are currently in clinical trials for DMD exon skipping. We propose that different chemistries should be further developed in parallel in order to hasten the transfer of the exon skipping therapy to the clinic.
Asunto(s)
Exones/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/administración & dosificación , Oligonucleótidos Fosforotioatos/química , Animales , Modelos Animales de Enfermedad , Perros , Humanos , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Fosforotioatos/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
Antisense oligonucleotides (AONs) can interfere with mRNA processing through RNase H-mediated degradation, translational arrest, or modulation of splicing. The antisense approach relies on AONs to efficiently bind to target sequences and depends on AON length, sequence content, secondary structure, thermodynamic properties, and target accessibility. We here performed a retrospective analysis of a series of 156 AONs (104 effective, 52 ineffective) previously designed and evaluated for splice modulation of the dystrophin transcript. This showed that the guanine-cytosine content and the binding energies of AON-target and AON-AON complexes were significantly higher for effective AONs. Effective AONs were also located significantly closer to the acceptor splice site (SS). All analyzed AONs are exon-internal and may act through steric hindrance of Ser-Arg-rich (SR) proteins to exonic splicing enhancer (ESE) sites. Indeed, effective AONs were significantly enriched for ESEs predicted by ESE software programs, except for predicted binding sites of SR protein Tra2beta, which were significantly enriched in ineffective AONs. These findings compile guidelines for development of AONs and provide more insight into the mechanism of antisense-mediated exon skipping. On the basis of only four parameters, we could correctly classify 79% of all AONs as effective or ineffective, suggesting these parameters can be used to more optimally design splice-modulating AONs.
Asunto(s)
Oligonucleótidos Antisentido/genética , Empalme del ARN/genética , Secuencia de Bases , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/clasificación , TermodinámicaRESUMEN
BACKGROUND: The specific skipping of an exon, induced by antisense oligonucleotides (AON) during splicing, has shown to be a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients. As different mutations require skipping of different exons, this approach is mutation dependent. The skipping of an entire stretch of exons (e.g. exons 45 to 55) has recently been suggested as an approach applicable to larger groups of patients. However, this multiexon skipping approach is technically challenging. The levels of intended multiexon skips are typically low and highly variable, and may be dependent on the order of intron removal. We hypothesized that the splicing order might favor the induction of multiexon 45-55 skipping. METHODS: We here tested the feasibility of inducing multiexon 45-55 in control and patient muscle cell cultures using various AON cocktails. RESULTS: In all experiments, the exon 45-55 skip frequencies were minimal and comparable to those observed in untreated cells. CONCLUSION: We conclude that current state of the art does not sufficiently support clinical development of multiexon skipping for DMD.
Asunto(s)
Exones , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/genética , Western Blotting , Diferenciación Celular , Células Cultivadas , Terapia Genética , Humanos , Fibras Musculares Esqueléticas/patología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/terapia , Mioblastos Esqueléticos/patología , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de SecuenciaRESUMEN
As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional dystrophin proteins and potential conversion of severe DMD into milder Becker muscular dystrophy (BMD) phenotypes. We have previously described 37 exon-internal AONs that induce skipping of 14 DMD exons in human control myotube cultures. Here, we report 77 new AONs, effectively targeting an additional 21 exons. Of the 114 AONs thus far tested, 72 (67%) were effective. AON design initially was based on a partial overlap with predicted open secondary structures in the target RNA. We have analyzed various AON and target exon parameters in retrospect. Interestingly, we observed significantly higher SF2/ASF, SC35, and SRp40 values (as predicted by ESEfinder) for effective AONs when compared with ineffective AONs. In addition, the distance to the 3' splice site was significantly smaller for effective AONs. No other significant correlations were observed. Our results suggest that effective exon-internal AONs primarily act by blocking SR binding sites (which often correspond to open structures) and that ESEfinder may be used to refine AON design for DMD and other genes.