RESUMEN
Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.
Asunto(s)
Distrofia Muscular de Duchenne , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/química , Animales , Exones , Ratones , Músculo Esquelético , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Fosforotioatos/farmacología , Empalme del ARN/efectos de los fármacosRESUMEN
Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah-/-;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah-/-;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah-/-;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah-/-;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah-/-;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics.
Asunto(s)
Citidina Monofosfato/genética , Distrofina/deficiencia , Morfolinos/uso terapéutico , Animales , Cardiomiopatía Dilatada/genética , Carnitina O-Palmitoiltransferasa/genética , Factor de Crecimiento del Tejido Conjuntivo/análisis , Citidina Monofosfato/fisiología , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Exones , Terapia Genética/métodos , Corazón/fisiopatología , Masculino , Ratones , Ratones Endogámicos mdx , Oxigenasas de Función Mixta/metabolismo , Distrofia Muscular de Duchenne/genética , Miocardio/metabolismo , NADPH Oxidasa 4/análisis , Oligonucleótidos Antisentido/genética , Péptidos/genética , Fenotipo , Volumen Sistólico , Proteína Desacopladora 3/genética , Función Ventricular DerechaRESUMEN
In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients.
Asunto(s)
Distrofina/genética , Terapia Genética , Morfolinos/administración & dosificación , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/administración & dosificación , Animales , Dependovirus/genética , Exones/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Humanos , Ratones Endogámicos mdx , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Sarcolema/efectos de los fármacos , Sarcolema/patologíaRESUMEN
Therapy-responsive biomarkers are an important and unmet need in the muscular dystrophy field where new treatments are currently in clinical trials. By using a comprehensive high-resolution mass spectrometry approach and western blot validation, we found that two fragments of the myofibrillar structural protein myomesin-3 (MYOM3) are abnormally present in sera of Duchenne muscular dystrophy (DMD) patients, limb-girdle muscular dystrophy type 2D (LGMD2D) and their respective animal models. Levels of MYOM3 fragments were assayed in therapeutic model systems: (1) restoration of dystrophin expression by antisense oligonucleotide-mediated exon-skipping in mdx mice and (2) stable restoration of α-sarcoglycan expression in KO-SGCA mice by systemic injection of a viral vector. Following administration of the therapeutic agents MYOM3 was restored toward wild-type levels. In the LGMD model, where different doses of vector were used, MYOM3 restoration was dose-dependent. MYOM3 fragments showed lower inter-individual variability compared with the commonly used creatine kinase assay, and correlated better with the restoration of the dystrophin-associated protein complex and muscle force. These data suggest that the MYOM3 fragments hold promise for minimally invasive assessment of experimental therapies for DMD and other neuromuscular disorders.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Conectina/metabolismo , Distrofias Musculares/metabolismo , Proteómica , Adolescente , Adulto , Animales , Biomarcadores , Estudios de Casos y Controles , Niño , Preescolar , Conectina/sangre , Creatina Quinasa , Modelos Animales de Enfermedad , Humanos , Espectrometría de Masas , Ratones , Ratones Endogámicos mdx , Distrofias Musculares/sangre , Distrofias Musculares/terapia , Distrofia Muscular de Duchenne/sangre , Distrofia Muscular de Duchenne/metabolismo , Proteómica/métodos , Resultado del Tratamiento , Adulto JovenRESUMEN
Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.
Asunto(s)
Péptidos de Penetración Celular/genética , Distrofina/biosíntesis , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/genética , Animales , Péptidos de Penetración Celular/administración & dosificación , Modelos Animales de Enfermedad , Distrofina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Terapia Genética , Humanos , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/administración & dosificaciónRESUMEN
Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle.
Asunto(s)
Distrofina/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Terapia Genética , Masculino , Ratones , Ratones Endogámicos mdx , MicroARNs/metabolismo , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatología , Distrofia Muscular de Duchenne/terapia , Mutación , Proteómica , ARN Mensajero/metabolismoRESUMEN
The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage ('click chemistry') in the other. The most active bi-specific CPP-PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP-PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.
Asunto(s)
Péptidos de Penetración Celular/química , Morfolinos/química , Distrofia Muscular de Duchenne/genética , Empalme del ARN , Receptores de Activinas Tipo II/genética , Animales , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Distrofina/genética , Exones , Ratones , Ratones Endogámicos mdx , Morfolinos/síntesis químicaRESUMEN
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
Extracellular microRNAs (miRNAs) are promising biomarkers of the inherited muscle wasting condition Duchenne muscular dystrophy, as they allow non-invasive monitoring of either disease progression or response to therapy. In this study, serum miRNA profiling reveals a distinct extracellular miRNA signature in dystrophin-deficient mdx mice, which shows profound dose-responsive restoration following dystrophin rescue. Extracellular dystrophy-associated miRNAs (dystromiRs) show dynamic patterns of expression that mirror the progression of muscle pathology in mdx mice. Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature. Similarly, extracellular dystromiRs were elevated following experimentally-induced skeletal muscle injury and regeneration in non-dystrophic mice. Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes. In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.
Asunto(s)
MicroARNs/sangre , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/sangre , Animales , Biomarcadores/sangre , Proteínas Sanguíneas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/lesiones , Músculo Esquelético/fisiología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/terapia , RegeneraciónRESUMEN
Oligonucleotides are rapidly emerging as powerful therapeutics for hard to treat diseases. Short single-stranded oligonucleotides can base pair with target RNA and alter gene expression, providing an attractive therapeutic approach at the genetic level. Whilst conceptually appealing, oligonucleotides require chemical modification for clinical use. One emerging approach is to substitute the phosphodiester backbone with other chemical linkages such as triazole. The triazole linkage is inherently resistant to enzymatic degradation, providing stability in vivo, and is uncharged, potentially improving cell-penetration and in vivo distribution. Triazole linkages, however, are known to reduce RNA target binding affinity. Here we show that by attaching pyrene or anthraquinone to the ribose sugar on the 5'-side of the triazole, it is possible to recover duplex stability and restore the splice switching ability of triazole-containing oligonucleotides.
RESUMEN
Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young's modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD.
RESUMEN
Oligonucleotides that target mRNA have great promise as therapeutic agents for life-threatening conditions but suffer from poor bioavailability, hence high cost. As currently untreatable diseases come within the reach of oligonucleotide therapies, new analogues are urgently needed to address this. With this in mind we describe reduced-charge oligonucleotides containing artificial LNA-amide linkages with improved gymnotic cell uptake, RNA affinity, stability and potency. To construct such oligonucleotides, five LNA-amide monomers (A, T, C, 5mC and G), where the 3'-OH is replaced by an ethanoic acid group, are synthesised in good yield and used in solid-phase oligonucleotide synthesis to form amide linkages with high efficiency. The artificial backbone causes minimal structural deviation to the DNA:RNA duplex. These studies indicate that splice-switching oligonucleotides containing LNA-amide linkages and phosphorothioates display improved activity relative to oligonucleotides lacking amides, highlighting the therapeutic potential of this technology.
Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos Fosforotioatos , Amidas , Exones , Oligonucleótidos Antisentido/genética , ARN/químicaRESUMEN
The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 trans-signalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs.
Asunto(s)
Vesículas Extracelulares , Distrofia Muscular de Duchenne , Animales , Interleucina-6 , Ratones , Fibras Musculares Esqueléticas , Transducción de SeñalRESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/genética , Distrofina/genética , Morfolinos/farmacología , Distrofia Muscular de Duchenne/terapia , Telomerasa/genética , Animales , Línea Celular , Perros , Exones/genética , Terapia Genética , Humanos , Ratones , Morfolinos/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Mioblastos/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Péptidos/genética , Péptidos/farmacología , Sitios de Empalme de ARN/genéticaRESUMEN
Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.
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Distrofina/metabolismo , Factor de Respuesta Sérica/metabolismo , Transducción de Señal , Actinas/metabolismo , Animales , Línea Celular , Distrofina/genética , Ratones , Mioblastos Esqueléticos/metabolismo , Utrofina/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Conventional gene therapy has focused largely on gene replacement in target cells. However, progress from basic research to the clinic has been slow for reasons relating principally to the challenges of heterologous DNA delivery and regulation in vivo. Alternative approaches targeting RNA have the potential to circumvent some of these difficulties, particularly as the active therapeutic molecules are usually short oligonucleotides and the target gene transcript is under endogenous regulation. RNA-based strategies offer a series of novel therapeutic applications, including altered processing of the target pre-mRNA transcript, reprogramming of genetic defects through mRNA repair, and the targeted silencing of allele- or isoform-specific gene transcripts. This review examines the potential of RNA therapeutics, focusing on antisense oligonucleotide modification of pre-mRNA splicing, methods for pre-mRNA trans-splicing, and the isoform- and allele-specific applications of RNA interference.
Asunto(s)
Regulación de la Expresión Génica/genética , Enfermedades Genéticas Congénitas/terapia , Oligonucleótidos Antisentido/uso terapéutico , ARN/metabolismo , Animales , Enfermedades Genéticas Congénitas/genética , Humanos , Interferencia de ARN/fisiología , Precursores del ARN/metabolismo , Empalme del ARN/fisiologíaRESUMEN
BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. METHODS: Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-XistΔhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining. RESULTS: The restoration of ex-myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-XistΔhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-XistΔhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-XistΔhs muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci. CONCLUSIONS: Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.
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Distrofina/metabolismo , MicroARNs/metabolismo , Sarcolema/metabolismo , Animales , Niño , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Sarcolema/patologíaRESUMEN
The review starts with a historical perspective of the achievements of the Gait group in synthesis of oligonucleotides (ONs) and their peptide conjugates toward the award of the 2017 Oligonucleotide Therapeutic Society Lifetime Achievement Award. This acts as a prelude to the rewarding collaborative studies in the Gait and Wood research groups aimed toward the enhanced delivery of charge neutral ON drugs and the development of a series of Arg-rich cell-penetrating peptides called Pip (peptide nucleic acid/phosphorodiamidate morpholino oligonucleotide [PNA/PMO] internalization peptides) as conjugates of such ONs. In this review we concentrate on these developments toward the treatment of the neuromuscular diseases Duchenne muscular dystrophy and spinal muscular atrophy toward a platform technology for the enhancement of cellular and in vivo delivery suitable for widespread use as neuromuscular and neurodegenerative ON drugs.
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Péptidos de Penetración Celular/uso terapéutico , Atrofia Muscular Espinal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Enfermedades Neuromusculares/tratamiento farmacológico , Péptidos de Penetración Celular/genética , Humanos , Morfolinos/genética , Morfolinos/uso terapéutico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/patología , Ácidos Nucleicos de Péptidos/genética , Ácidos Nucleicos de Péptidos/uso terapéuticoRESUMEN
Protein-truncating mutations in the dystrophin gene lead to the most common childhood form of muscle wasting, Duchenne muscular dystrophy. Becker muscular dystrophy, a condition that typically arises from dystrophin gene lesions that do not disrupt the reading frame, clearly indicates that substantial domains of the dystrophin protein are not essential. Potential therapeutic intervention exists during pre-mRNA splicing, whereby selected exons are excised to either remove nonsense mutations or restore the reading frame around frame-shifting mutations from the mature mRNA. Appropriately designed antisense oligonucleotides (AOs), directed at amenable splicing motifs across the dystrophin gene transcript, block exon recognition and/or spliceosome assembly so that targeted exons are removed from the mature mRNA. We describe a panel of AOs designed to induce skipping of every exon within the human dystrophin gene transcript, with the exception of the first and last exons. Every exon targeted in vitro could be removed from the dystrophin mRNA, although some exons are more efficiently excluded than others. No single motif has emerged as a universal AO annealing site for redirection of dystrophin pre-mRNA processing, although the general trend is that the most efficient compounds are directed at motifs in the first half of the target exon.
Asunto(s)
Distrofina/genética , Exones/genética , Oligonucleótidos Antisentido/genética , Transcripción Genética/genética , Secuencia de Bases , Células Cultivadas , Humanos , Datos de Secuencia MolecularRESUMEN
The promise of nucleic acid based oligonucleotides as effective genetic therapies has been held back by their low bioavailability and poor cellular uptake to target tissues upon systemic administration. One such strategy to improve upon delivery is the use of short cell-penetrating peptides (CPPs) that can be either directly attached to their cargo through covalent linkages or through the formation of noncovalent nanoparticle complexes that can facilitate cellular uptake. In this review, we will highlight recent proof-of-principle studies that have utilized both of these strategies to improve nucleic acid delivery and discuss the prospects for translation of this approach for clinical application.