RESUMEN

Limb girdle muscular dystrophy type 2D (LGMD2D) is characterized by progressive weakening of muscles in the hip and shoulder girdles. It is caused by a mutation in the α-sarcoglycan gene and results in absence of α-sarcoglycan in the dystrophin-glycoprotein complex. The activin type IIB receptor is involved in the activin/myostatin pathway, with myostatin being a negative regulator of muscle growth. In this study, we investigated the effects of sequestering myostatin by a soluble activin type IIB receptor (sActRIIB) on muscle growth in Sgca-null mice, modelling LGMD2D. Treatment was initiated at 3 weeks of age, prior to the disease onset, or at 9 weeks of age when already in an advanced stage of the disease. We found that early sActRIIB treatment resulted in increased muscle size. However, this led to more rapid decline of muscle function than in saline-treated Sgca-null mice. Furthermore, no histopathological improvements were seen after sActRIIB treatment. When initiated at 9 weeks of age, sActRIIB treatment resulted in increased muscle mass too, but to a lesser extent. No effect of the treatment was observed on muscle function or histopathology. These data show that sActRIIB treatment as a stand-alone therapy does not improve muscle function or histopathology in Sgca-null mice.

Asunto(s)

Miostatina , Sarcoglicanopatías , Receptores de Activinas/metabolismo , Activinas/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Músculo Esquelético/patología , Miostatina/genética , Sarcoglicanopatías/metabolismo , Sarcoglicanos/genética , Sarcoglicanos/metabolismo

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 fármacos

RESUMEN

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca+2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.

Asunto(s)

Astrocitos/metabolismo , Distrofina/metabolismo , Ácido Glutámico/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Astrocitos/citología , Calcio/metabolismo , Citoesqueleto/metabolismo , Distrofina/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Proyección Neuronal/fisiología , Neuronas/citología , Óxido Nítrico/metabolismo

RESUMEN

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/genética

RESUMEN

Duchenne muscular dystrophy is a severe, progressive muscle-wasting disease that is caused by mutations that abolish the production of functional dystrophin protein. The exon skipping approach aims to restore the disrupted dystrophin reading frame, to allow the production of partially functional dystrophins, such as found in the less severe Becker muscular dystrophy. Exon skipping is achieved by antisense oligonucleotides (AONs). Several chemical modifications have been tested in nonclinical and clinical trials. The morpholino phosphorodiamidate oligomer eteplirsen has been approved by the Food and Drug Administration, whereas clinical development with the 2'-O-methyl phosphorothioate (2OMePS) AON drisapersen was recently stopped. In this study, we aimed to study various aspects of 2OMePS AONs in nonclinical animal studies. We show that while efficiency of exon skipping restoration is comparable in young and older C57BL/10ScSn-Dmdmdx/J (mdx/BL10) mice, functional improvement was only observed for younger treated mice. Muscle quality did not affect exon skipping efficiency as exon skip and dystrophin levels were similar between mdx/BL10 and more severely affected, age-matched D2-mdx mice. We further report that treadmill running increases AON uptake and dystrophin levels in mdx/BL10 mice. Finally, we show that even low levels of exon skipping and dystrophin restoration are sufficient to significantly increase the survival of mdx-utrn-/- mice from 70 to 97 days.

Asunto(s)

Distrofina/genética , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/farmacología , Utrofina/genética , Animales , Ensayos Clínicos como Asunto , Distrofina/antagonistas & inhibidores , Exones/efectos de los fármacos , Exones/genética , Terapia Genética , Humanos , Ratones , Ratones Endogámicos mdx , Morfolinos/genética , Morfolinos/farmacología , Atrofia Muscular Espinal , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos/farmacología , Oligonucleótidos Antisentido/genética , Compuestos Organotiofosforados/farmacología

RESUMEN

Duchenne muscular dystrophy is a severe muscle wasting disease, characterized by a severely reduced lifespan in which cardiomyopathy is one of the leading causes of death. Multiple therapies aiming at dystrophin restoration have been approved. It is anticipated that these therapies will maintain muscle function for longer and extend the ambulatory period, which in turn will increase the cardiac workload which could be detrimental for cardiac function. We investigated the effects of voluntary running exercise in combination with low dystrophin levels on function and pathology of skeletal muscle and heart. We divided 15.5-month old female mdx (no dystrophin), mdx-XistΔhs (varying low dystrophin levels) and wild type mice (BL10-WT and XistΔhs-WT) to either a sedentary or voluntary wheel running regime and assessed muscle function at 17.5 months of age. Thereafter, a cardiac MRI was obtained, and muscle and heart histopathology were assessed. We show that voluntary exercise is beneficial to skeletal muscle and heart function in dystrophic mice while not affecting muscle pathology. Low amounts of dystrophin further improve skeletal muscle and cardiac function. These findings suggest that voluntary exercise may be beneficial for skeletal muscle and heart in DMD patients, especially in conjunction with low amounts of dystrophin.

Asunto(s)

Corazón/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Miocardio/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Western Blotting , Femenino , Masculino , Actividad Motora/fisiología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo

RESUMEN

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/química

RESUMEN

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/métodos

RESUMEN

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/farmacocinética

RESUMEN

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

Antisense oligonucleotides (AONs) are being developed as RNA therapeutic molecules for Duchenne muscular dystrophy. For oligonucleotides with the 2'-O-methyl-phosphorothioate (2OMePS) RNA chemistry, proof of concept has been obtained in patient-specific muscle cell cultures, the mouse and dog disease models, and recently by local administration in Duchenne patients. To further explore the pharmacokinetic (PK)/pharmacodynamic (PD) properties of this chemical class of oligonucleotides, we performed a series of preclinical studies in mice. The results demonstrate that the levels of oligonucleotides in dystrophin-deficient muscle fibers are much higher than in healthy fibers, leading to higher exon-skipping levels. Oligonucleotide levels and half-life differed for specific muscle groups, with heart muscle showing the lowest levels but longest half-life (approximately 46 days). Intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) delivery methods were directly compared. For each method, exon-skipping and novel dystrophin expression were observed in all muscles, including arrector pili smooth muscle in skin biopsies. After i.v. administration, the oligonucleotide peak levels in plasma, liver, and kidney were higher than after s.c. or i.p. injections. However, as the bioavailability was similar, and the levels of oligonucleotide, exon-skipping, and dystrophin steadily accumulated overtime after s.c. administration, we selected this patient-convenient delivery method for future clinical study protocols.

Asunto(s)

Oligonucleótidos Fosforotioatos/farmacología , ARN sin Sentido/farmacología , Animales , Western Blotting , Modelos Animales de Enfermedad , Distrofina/administración & dosificación , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos mdx , Oligonucleótidos Fosforotioatos/farmacocinética , ARN sin Sentido/farmacocinética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

To assess the effect of potential therapeutic agents in dystrophic mice it is useful to have a functional test regime that does not affect the natural disease progression of mdx mice with dystrophinopathy. We determined the effect of a 12 week test regime consisting of fore limb grip strength, rotarod analysis and two and four limb hanging wire tests on the disease progression of 4-week-old mdx mice. Mice performed the different functional tests on consecutive days on a weekly basis. No difference was found in serum creatine kinase levels between functionally active and sedentary mice. The percentage of fibrotic/necrotic areas assessed in a semi-automated way with colour deconvolution of skeletal muscles, heart and diaphragm did not vary within muscles or between groups, nor did the gene expression levels of disease-related genes. We conclude that this test regime may be suitable for short-term functional evaluation of therapeutic approaches in the mdx mouse.

Asunto(s)

Evaluación de la Discapacidad , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/fisiopatología , Evaluación de Resultado en la Atención de Salud/métodos , Índice de Severidad de la Enfermedad , Factores de Edad , Animales , Creatina Quinasa/análisis , Creatina Quinasa/sangre , Modelos Animales de Enfermedad , Fibrosis/tratamiento farmacológico , Fibrosis/metabolismo , Fibrosis/fisiopatología , Regulación de la Expresión Génica/genética , Corazón/efectos de los fármacos , Corazón/fisiopatología , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Ratones Endogámicos mdx , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Proteínas Musculares/genética , Fuerza Muscular/efectos de los fármacos , Fuerza Muscular/genética , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/tratamiento farmacológico , Miocardio/metabolismo , Examen Neurológico/métodos , Valor Predictivo de las Pruebas , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/genética , Factores de Tiempo

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/genética

RESUMEN

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/genética

RESUMEN

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/genética

RESUMEN

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 , Termodinámica

RESUMEN

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 Secuencia

RESUMEN

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.

Asunto(s)

Exones , Oligonucleótidos Antisentido , Secuencia de Bases , Sitios de Unión , Conformación de Ácido Nucleico , Unión Proteica , Precursores del ARN/química , ARN Mensajero/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called tuberin. Although it has been shown that hamartin and tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with tuberin. One of the proteins identified was 14-3-3zeta, a member of the 14-3-3 protein family. The interaction between tuberin and 14-3-3zeta was confirmed in vitro and by co-immunoprecipitation; multiple sites within tuberin for 14-3-3zeta binding were identified; and it was determined that 14-3-3zeta associated with the tuberin-hamartin complex. Finally, it was shown that the tuberin/14-3-3zeta interaction is regulated by Akt-mediated phosphorylation of tuberin, providing insight into how tuberin may regulate phosphorylation of S6.

Asunto(s)

Proteínas Represoras/química , Tirosina 3-Monooxigenasa/química , Proteínas 14-3-3 , Animales , Sitios de Unión , Células COS , Células HeLa , Humanos , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismo , Proteína S6 Ribosómica/metabolismo , Transfección , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos Híbridos , Tirosina 3-Monooxigenasa/metabolismo