RESUMEN
Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Femenino , Ratones , Animales , Distrofina/genética , Distrofina/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Músculos/metabolismo , Edición Génica , Resultado del Tratamiento , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Modelos Animales de EnfermedadRESUMEN
Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.
Asunto(s)
Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/química , Ácido Palmítico/química , Animales , Terapia Genética/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Oligonucleótidos Antisentido/efectos adversos , Oligonucleótidos Antisentido/farmacocinética , Distribución TisularRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1008690.].
RESUMEN
OBJECTIVES: Duchenne muscular dystrophy is associated with various degrees of cognitive impairment and behavioral disturbances. Emotional and memory deficits also constitute reliable outcome measures to assess efficacy of treatments in the mdx mouse lacking the muscle and neuronal full-length dystrophins. The present study aimed to evaluate whether these deficits could be alleviated by the restoration of brain dystrophin. METHODS: We performed intracerebroventricular administration of a new potent tricyclo-DNA antisense oligonucleotide (tcDNA-ASO) containing a full phosphodiester backbone conjugated to a palmitic acid moiety (tcDNA-ASO), designed to skip the mutated exon 23 of mdx mice. RESULTS: We first show that the tcDNA-ASO rescues expression of brain dystrophin to 10-30% of wild-type levels and significantly reduces the abnormal unconditioned fear responses in mdx mice in a dose-dependent manner, 5 weeks post-injection. Exon skipping efficiency, ASO biodistribution, protein restoration and effect on the fear response were optimal with a dose of 400 µg at 6-7 weeks post-injection, with synaptic-like expression in brain tissues such as the hippocampus and amygdala. Furthermore, this dose of tcDNA-ASO restored long-term memory retention of mdx mice in an object recognition task, but only had minor effects on fear conditioning. INTERPRETATION: These results suggest for the first time that postnatal re-expression of brain dystrophin could reverse or at least alleviate some cognitive deficits associated with Duchenne muscular dystrophy. ANN NEUROL 2022;92:213-229.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Exones/genética , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos , Oligonucleótidos Antisentido , Distribución TisularRESUMEN
Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.
Asunto(s)
Factor IX/genética , Hemofilia B/genética , Mutación con Pérdida de Función , ARN Mensajero/genética , Supresión Genética , Regiones no Traducidas 3' , Animales , Células CHO , Cricetinae , Cricetulus , Factor IX/metabolismo , Células HEK293 , Células HeLa , Humanos , Oligonucleótidos Antisentido/genética , Fenotipo , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a neurodevelopmental disorder primarily caused by the loss of the full-length Dp427 dystrophin in both muscle and brain. The basis of the central comorbidities in DMD is unclear. Brain dystrophin plays a role in the clustering of central gamma-aminobutyric acid A receptors (GABAARs), and its loss in the mdx mouse alters the clustering of some synaptic subunits in central inhibitory synapses. However, the diversity of GABAergic alterations in this model is still fragmentary. In this study, the analysis of in vivo PET imaging of a benzodiazepine-binding site radioligand revealed that the global density of central GABAARs is unaffected in mdx compared with WT mice. In contrast, semi-quantitative immunoblots and immunofluorescence confocal imaging in tissue sections revealed complex and differential patterns of alterations of the expression levels and/or clustered distribution of a variety of synaptic and extrasynaptic GABAAR subunits in the hippocampus, cerebellum, cortex, and spinal cord. Hence, dystrophin loss not only affects the stabilization of synaptic GABAARs but also influences the subunit composition of GABAARs subtypes at both synaptic and extrasynaptic sites. This study provides new molecular outcome measures and new routes to evaluate the impact of treatments aimed at compensating alterations of the nervous system in DMD.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Ratones , Animales , Ratones Endogámicos mdx , Distrofina/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ratones Endogámicos C57BL , Distrofia Muscular de Duchenne/metabolismo , Ácido gamma-Aminobutírico/metabolismo , BenzodiazepinasRESUMEN
The mdx52 mouse model of Duchenne muscular dystrophy (DMD) is lacking exon 52 of the DMD gene that is located in a hotspot mutation region causing cognitive deficits and retinal anomalies in DMD patients. This deletion leads to the loss of the dystrophin proteins, Dp427, Dp260 and Dp140, while Dp71 is preserved. The flash electroretinogram (ERG) in mdx52 mice was previously characterized by delayed dark-adapted b-waves. A detailed description of functional ERG changes and visual performances in mdx52 mice is, however, lacking. Here an extensive full-field ERG repertoire was applied in mdx52 mice and WT littermates to analyze retinal physiology in scotopic, mesopic and photopic conditions in response to flash, sawtooth and/or sinusoidal stimuli. Behavioral contrast sensitivity was assessed using quantitative optomotor response (OMR) to sinusoidally modulated luminance gratings at 100% or 50% contrast. The mdx52 mice exhibited reduced amplitudes and delayed implicit times in dark-adapted ERG flash responses, particularly in their b-wave and oscillatory potentials, and diminished amplitudes of light-adapted flash ERGs. ERG responses to sawtooth stimuli were also diminished and delayed for both mesopic and photopic conditions in mdx52 mice and the first harmonic amplitudes to photopic sine-wave stimuli were smaller at all temporal frequencies. OMR indices were comparable between genotypes at 100% contrast but significantly reduced in mdx52 mice at 50% contrast. The complex ERG alterations and disturbed contrast vision in mdx52 mice include features observed in DMD patients and suggest altered photoreceptor-to-bipolar cell transmission possibly affecting contrast sensitivity. The mdx52 mouse is a relevant model to appraise the roles of retinal dystrophins and for preclinical studies related to DMD.
Asunto(s)
Distrofia Muscular de Duchenne/fisiopatología , Percepción Visual/fisiología , Animales , Electrorretinografía , Ratones , Ratones Endogámicos mdx , Transmisión Sináptica/fisiologíaRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by progressive muscle wasting that has currently no cure. Exon-skipping strategy represents one of the most promising therapeutic approaches that aim to restore expression of a shorter but functional dystrophin protein. The antisense field has remarkably progress over the last years with recent accelerated approval of the first antisense oligonucleotide-based therapy for DMD, Exondys 51, though the therapeutic benefit remains to be proved in patients. Despite clinical advances, the poor effective delivery to target all muscle remains the main hurdle for antisense drug therapy. This review describes the antisense-based exon-skipping approach for DMD, from proof-of-concept to first marketed drug. We discuss the main obstacles to achieve a successful exon-skipping therapy and the latest advances of the international community to develop more powerful chemistries and more sophisticated delivery systems in order to increase potency, bioavailability and safety. Finally, we highlight the importance of collaborative efforts and early dialogue between drug developers and regulatory agencies in order to overcome difficulties, find appropriate outcome markers and collect useful data.
Asunto(s)
Empalme Alternativo/genética , Distrofia Muscular de Duchenne/genética , ARN sin Sentido/uso terapéutico , Animales , Distrofina/genética , Exones/genética , Terapia Genética , Humanos , Morfolinos/genética , Morfolinos/farmacología , Oligonucleótidos Antisentido/genética , Empalme del ARN/genética , ARN sin Sentido/genéticaRESUMEN
Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.
Asunto(s)
Nanopartículas/química , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinética , Receptores Depuradores de Clase A/metabolismo , Animales , Secuencia de Bases , Línea Celular , Exones , Terapia Genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Micelas , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Receptores Depuradores de Clase A/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.
Asunto(s)
Dependovirus/genética , Distrofina/genética , Exones , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , ARN Nuclear Pequeño/genética , Utrofina/genética , Animales , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Distrofina/metabolismo , Terapia Genética , Ratones , Distrofia Muscular de Duchenne/patología , Fenotipo , ARN Nuclear Pequeño/uso terapéutico , Utrofina/metabolismoRESUMEN
In the context of future adeno-associated viral (AAV)-based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment.
Asunto(s)
Empalme Alternativo , Dependovirus/genética , Vectores Genéticos/genética , Genoma Viral , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , ARN Nuclear Pequeño/genética , Animales , Cardiotoxinas/farmacología , Dependovirus/metabolismo , Distrofina/genética , Distrofina/metabolismo , Exones , Expresión Génica , Terapia Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapiaRESUMEN
Antisense oligonucleotides (ASO) are very promising drugs for numerous diseases including neuromuscular disorders such as Duchenne muscular dystrophy (DMD). Several ASO drugs have already been approved by the US Food and Drug Administration for DMD and global efforts are still ongoing to improve further their potency, notably by developing new delivery systems or alternative chemistries. In this context, a recent study investigated the potential of different chemically modified ASO to induce exon-skipping in mouse models of DMD. Importantly, the authors reported a strong discrepancy between exon-skipping and protein restoration levels, which was mainly owing to the high affinity of locked nucleic acid (LNA) modifications to the target RNA, thereby interfering with the amplification of the unskipped product and resulting in artificial overamplification of the exon-skipped product. These findings urged us to verify whether a similar phenomenon could occur with tricyclo-DNA (tcDNA)-ASO that also display high-affinity properties to the target RNA. We thus ran a series of control experiments and demonstrate here that exon-skipping levels are not overestimated owing to an interference of tcDNA-ASO with the unskipped product in contrast to what was observed with LNA-containing ASO.
RESUMEN
Muscular dystrophies are a heterogeneous group of genetic disorders characterized by muscle weakness and wasting. Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, and although the molecular mechanisms of the disease have been extensively investigated since the discovery of the gene in 1986, there is currently no effective treatment. However, new gene-based therapies have recently emerged with particular noted advances in using conventional gene replacement strategies, RNA-based technology and pharmacological approaches. While the proof of principle has been demonstrated in animal models, several clinical trials have recently been undertaken to investigate the feasibility of these strategies in patients. In particular, antisense-mediated exon skipping has shown encouraging results and holds promise for the treatment of dystrophic muscle. Here, we summarize the recent progress in therapeutic approaches to muscular dystrophies, with an emphasis on gene therapy and exon skipping for DMD.
Asunto(s)
Terapia Genética/métodos , Distrofias Musculares/terapia , Distrofina/genética , Exones/genética , Humanos , Distrofias Musculares/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function. In a transgenic mdx mouse, where utrophin was over expressed in the skeletal muscle and the diaphragm, but not in the heart, we found cardiac function, specifically right and left ventricular ejection fraction as measured using in vivo magnetic resonance imaging, was restored to wild-type levels. In mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that resulted in high levels of dystrophin restoration in the skeletal muscle and the diaphragm only, cardiac function was also restored to wild-type levels. In dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in the skeletal muscle and the diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. This novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice warrants further investigation for its implications on the need to directly treat the heart in DMD.
Asunto(s)
Cardiomiopatías/prevención & control , Diafragma/fisiopatología , Distrofina/metabolismo , Morfolinas/farmacología , Distrofia Muscular Animal/tratamiento farmacológico , Utrofina/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismo , Diafragma/efectos de los fármacos , Diafragma/metabolismo , Distrofina/genética , Corazón/fisiopatología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Ratones Transgénicos , Morfolinos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/fisiopatología , Volumen Sistólico , Utrofina/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD but still faces personalized medicine challenges as different mutations found in DMD patients require skipping of different exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or "hot-spot" in the central genomic region. In this study, we have developed 11 different U7 small-nuclear RNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of exons 45 to 55. We demonstrate that these constructs induce efficient exon skipping both in vitro in DMD patients' myoblasts and in vivo in human DMD (hDMD) mice and that they can be combined into a single vector to achieve a multi skipping of at least 3 exons. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using adeno-associated viral (AAV) vectors encoding multiple U7 small-nuclear RNAs (U7snRNAs), offering therefore very promising tools for clinical treatment of DMD.
Asunto(s)
Empalme Alternativo , Distrofina/genética , Exones , Vectores Genéticos , Distrofia Muscular de Duchenne/genética , ARN Nuclear Pequeño , Animales , Línea Celular , ADN sin Sentido , Dependovirus/genética , Modelos Animales de Enfermedad , Orden Génico , Humanos , Lentivirus/genética , Ratones , Ratones Transgénicos , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo , Transducción GenéticaRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.
Asunto(s)
Empalme Alternativo , Dependovirus/genética , Distrofina/genética , Distrofia Muscular Animal/terapia , Oligorribonucleótidos Antisentido/genética , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Calcio/metabolismo , Perros , Exones , Miembro Anterior/fisiopatología , Terapia Genética , Vectores Genéticos/administración & dosificación , Inyecciones Intramusculares , Datos de Secuencia Molecular , Contracción Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Fuerza Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/fisiopatología , Transcripción Genética , Utrofina/genética , Utrofina/metabolismoRESUMEN
Antisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD) and some antisense oligonucleotide (ASO) drugs have already been approved by the U.S. FDA for DMD. The potential of this therapy is still limited by several challenges including the poor distribution of ASOs to target tissues. Indeed, most of them accumulate in the kidney and tend to be rapidly eliminated after systemic delivery. We hypothesized here that preventing renal clearance of ASO using organic anion transporter (OAT) inhibitor could increase the bioavailability of ASOs and thus their distribution to target tissues and ultimately their efficacy in muscles. Mdx mice were, therefore, treated with ASO with or without the OAT inhibitor named probenecid. Our findings indicate that OAT inhibition, or at least using probenecid, does not improve the therapeutic potential of ASO-mediated exon-skipping approaches for the treatment of DMD.
Asunto(s)
Distrofia Muscular de Duchenne , Transportadores de Anión Orgánico , Animales , Ratones , Ratones Endogámicos mdx , Distrofina/genética , Probenecid , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/tratamiento farmacológico , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos , ADN , Exones/genéticaRESUMEN
Nucleic acid-based therapeutics hold great promise for the treatment of numerous diseases, including neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA for DMD, but the potential of this therapy is still limited by several challenges, including the poor distribution of ASOs to target tissues, but also the entrapment of ASO in the endosomal compartment. Endosomal escape is a well recognized limitation that prevents ASO from reaching their target pre-mRNA in the nucleus. Small molecules named oligonucleotide-enhancing compounds (OEC) have been shown to release ASO from endosomal entrapment, thus increasing ASO nuclear concentration and ultimately correcting more pre-mRNA targets. In this study, we evaluated the impact of a therapy combining ASO and OEC on dystrophin restoration in mdx mice. Analysis of exon-skipping levels at different time points after the co-treatment revealed improved efficacy, particularly at early time points, reaching up to 4.4-fold increase at 72 h post treatment in the heart compared to treatment with ASO alone. Significantly higher levels of dystrophin restoration were detected two weeks after the end of the combined therapy, reaching up to 2.7-fold increase in the heart compared to mice treated with ASO alone. Moreover, we demonstrated a normalization of cardiac function in mdx mice after a 12-week-long treatment with the combined ASO + OEC therapy. Altogether, these findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping approaches offering promising perspectives for the treatment of DMD.
Asunto(s)
Distrofina , Oligonucleótidos , Animales , Ratones , Distrofina/genética , Ratones Endogámicos mdx , Precursores del ARN , Oligonucleótidos Antisentido/uso terapéutico , ADN , ExonesRESUMEN
The nucleic acid therapeutics field has made tremendous progress in the past decades. Continuous advances in chemistry and design have led to many successful clinical applications, eliciting even more interest from researchers including both academic groups and drug development companies. Many preclinical studies in the field focus on improving the delivery of antisense oligonucleotide drugs (ONDs) and/or assessing their efficacy in target tissues, often neglecting the evaluation of toxicity, at least in early phases of development. A series of consensus recommendations regarding regulatory considerations and expectations have been generated by the Oligonucleotide Safety Working Group and the Japanese Research Working Group for the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S6 and Related Issues (WGS6) in several white papers. However, safety aspects should also be kept in sight in earlier phases while screening and designing OND to avoid subsequent failure in the development phase. Experts and members of the network "DARTER," a COST Action funded by the Cooperation in Science and Technology of the EU, have utilized their collective experience working with OND, as well as their insights into OND-mediated toxicities, to generate a series of consensus recommendations to assess OND toxicity in early stages of preclinical research. In the past few years, several publications have described predictive assays, which can be used to assess OND-mediated toxicity in vitro or ex vivo to filter out potential toxic candidates before moving to in vivo phases of preclinical development, that is, animal toxicity studies. These assays also have the potential to provide translational insight since they allow a safety evaluation in human in vitro systems. Yet, small preliminary in vivo studies should also be considered to complement this early assessment. In this study, we summarize the state of the art and provide guidelines and recommendations on the different tests available for these early stage preclinical assessments.
Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos , Animales , Humanos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Preparaciones Farmacéuticas , Evaluación Preclínica de MedicamentosRESUMEN
The mdx52 mouse model recapitulates a frequent mutation profile associated with brain involvement in Duchenne muscular dystrophy. Deletion of exon 52 impedes expression of two dystrophins (Dp427, Dp140) expressed in brain, and is eligible for therapeutic exon-skipping strategies. We previously showed that mdx52 mice display enhanced anxiety and fearfulness, and impaired associative fear learning. In this study, we examined the reversibility of these phenotypes using exon 51 skipping to restore exclusively Dp427 expression in the brain of mdx52 mice. We first show that a single intracerebroventricular administration of tricyclo-DNA antisense oligonucleotides targeting exon 51 restores 5%-15% of dystrophin protein expression in the hippocampus, cerebellum, and cortex, at stable levels between 7 and 11 week after injection. Anxiety and unconditioned fear were significantly reduced in treated mdx52 mice and acquisition of fear conditioning appeared fully rescued, while fear memory tested 24 h later was only partially improved. Additional restoration of Dp427 in skeletal and cardiac muscles by systemic treatment did not further improve the unconditioned fear response, confirming the central origin of this phenotype. These findings indicate that some emotional and cognitive deficits associated with dystrophin deficiency may be reversible or at least improved by partial postnatal dystrophin rescue.