RESUMO
The emergence of adeno-associated virus (AAV)-based gene therapy has brought hope to patients with severe monogenic disorders. However, immune responses to AAV vectors and transgene products present challenges that require effective immunosuppressive strategies. This systematic review focuses on the immunosuppressive protocols used in 38 clinical trials and 35 real-world studies, considering a range of monogenic diseases, AAV serotypes, and administration routes. The review underscores the need for a deeper understanding of immunosuppressive regimens to enhance the safety and effectiveness of AAV-based gene therapy. Characterizing the immunological responses associated with various gene therapy treatments is crucial for optimizing treatment protocols and ensuring the safety and efficacy of forthcoming gene therapy interventions. Further research and understanding of the impact of immunosuppression on disease, therapy, and route of administration will contribute to the development of more effective and safer gene therapy approaches in the future.
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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.
Assuntos
Distrofina/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Linhagem Celular , Distrofina/genética , Camundongos , Mioblastos Esqueléticos/metabolismo , Utrofina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Distrofina/metabolismo , MicroRNAs/metabolismo , Sarcolema/metabolismo , Animais , Criança , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Sarcolema/patologiaRESUMO
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.
Assuntos
Monofosfato de Citidina/genética , Distrofina/deficiência , Morfolinos/uso terapêutico , Animais , Cardiomiopatia Dilatada/genética , Carnitina O-Palmitoiltransferase/genética , Fator de Crescimento do Tecido Conjuntivo/análise , Monofosfato de Citidina/fisiologia , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Éxons , Terapia Genética/métodos , Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Oxigenases de Função Mista/metabolismo , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , NADPH Oxidase 4/análise , Oligonucleotídeos Antissenso/genética , Peptídeos/genética , Fenótipo , Volume Sistólico , Proteína Desacopladora 3/genética , Função Ventricular DireitaRESUMO
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.
Assuntos
Peptídeos Penetradores de Células/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Distrofia Muscular de Duchenne/tratamento farmacológico , Doenças Neuromusculares/tratamento farmacológico , Peptídeos Penetradores de Células/genética , Humanos , Morfolinos/genética , Morfolinos/uso terapêutico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/uso terapêuticoRESUMO
Physiology and behaviour are critically dependent on circadian regulation via a core set of clock genes, dysregulation of which leads to metabolic and sleep disturbances. Metabolic and sleep perturbations occur in spinal muscular atrophy (SMA), a neuromuscular disorder caused by loss of the survival motor neuron (SMN) protein and characterized by motor neuron loss and muscle atrophy. We therefore investigated the expression of circadian rhythm genes in various metabolic tissues and spinal cord of the Taiwanese Smn-/-;SMN2 SMA animal model. We demonstrate a dysregulated expression of the core clock genes (clock, ARNTL/Bmal1, Cry1/2, Per1/2) and clock output genes (Nr1d1 and Dbp) in SMA tissues during disease progression. We also uncover an age- and tissue-dependent diurnal expression of the Smn gene. Importantly, we observe molecular and phenotypic corrections in SMA mice following direct light modulation. Our study identifies a key relationship between an SMA pathology and peripheral core clock gene dysregulation, highlights the influence of SMN on peripheral circadian regulation and metabolism and has significant implications for the development of peripheral therapeutic approaches and clinical care management of SMA patients.
Assuntos
Ritmo Circadiano/efeitos da radiação , Regulação da Expressão Gênica , Luz , Atrofia Muscular Espinal/metabolismo , Animais , Ritmo Circadiano/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Cardiac failure is a major cause of mortality in patients with Duchenne muscular dystrophy (DMD). Antisense-mediated exon skipping has the ability to correct out-of-frame mutations in DMD to produce truncated but functional dystrophin. Traditional antisense approaches have however been limited by their poor uptake into cardiac muscle. The addition of cell-penetrating peptides to antisense molecules has increased their potency and improved their uptake into all muscles, including the heart. We have investigated the efficacy of the Peptide-conjugated phosphodiamidate morpholino oligomer (P-PMO) Pip6a-PMO, for restoration of cardiac dystrophin and functional rescue in DMD mice- the mdx mouse and the less well characterised Cmah-/-mdx mouse (which carry a human-like mutation in the mouse Cmah gene as well as a mutation in DMD). In our first study male mdx mice were administered Pip6a-PMO, i.v, fortnightly from 12 to 30 weeks of age alongside mock-injected age-matched mdx and C57BL10 controls. Mice received 4 doses of 18 mg/kg followed by 8 doses of 12.5 mg/kg. The cardiac function of the mice was analysed 2 weeks after their final injection by MRI followed by conductance catheter and their muscles were harvested for dystrophin quantification. In the second study, male Cmah-/-mdx mice, received 12.5 mg/kg Pip6a-PMO, i.v fortnightly from 8 to 26 weeks and assessed by MRI at 3 time points (12, 18 and 28 weeks) alongside mock-injected age-matched mdx, C57BL10 and Cmah-/-mdx controls. The mice also underwent MEMRI and conductance catheter at 28 weeks. This allowed us to characterise the cardiac phenotype of Cmah-/-mdx mice as well as assess the effects of P-PMO on cardiac function. Pip6a-PMO treatment resulted in significant restoration of dystrophin in mdx and Cmah-/-mdx mice (37.5% and 51.6%, respectively), which was sufficient to significantly improve cardiac function, ameliorating both right and left ventricular dysfunction. Cmah-/-mdx mice showed an abnormal response to dobutamine stress test and this was completely ameliorated by PIP6a-PMO treatment. These encouraging data suggest that total restoration of dystrophin may not be required to significantly improve cardiac outcome in DMD patients and that it may be realistic to expect functional improvements with modest levels of dystrophin restoration which may be very achievable in future clinical trials.
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Peptídeos Penetradores de Células/uso terapêutico , Insuficiência Cardíaca/etiologia , Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/complicações , Animais , Modelos Animais de Doenças , Distrofina/metabolismo , Éxons/genética , Mutação da Fase de Leitura/genética , Coração/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/prevenção & controle , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Miocárdio/metabolismoRESUMO
The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn-/-;SMN2 and Smn2B/- mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling.
Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Proteínas de Ligação a DNA , Suplementos Nutricionais , Atrofia Muscular Espinal , Prednisolona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Knockout , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MyomiRs are muscle-specific microRNAs (miRNAs) that regulate myoblast proliferation and differentiation. Extracellular myomiRs (ex-myomiRs) are highly enriched in the serum of Duchenne Muscular Dystrophy (DMD) patients and dystrophic mouse models and consequently have potential as disease biomarkers. The biological significance of miRNAs present in the extracellular space is not currently well understood. Here we demonstrate that ex-myomiR levels are elevated in perinatal muscle development, during the regenerative phase that follows exercise-induced myoinjury, and concomitant with myoblast differentiation in culture. Whereas ex-myomiRs are progressively and specifically released by differentiating human primary myoblasts and C2C12 cultures, chemical induction of apoptosis in C2C12 cells results in indiscriminate miRNA release. The selective release of myomiRs as a consequence of cellular differentiation argues against the idea that they are solely waste products of muscle breakdown, and suggests they may serve a biological function in specific physiological contexts. Ex-myomiRs in culture supernatant and serum are predominantly non-vesicular, and their release is independent of ceramide-mediated vesicle secretion. Furthermore, ex-myomiRs levels are reduced in aged dystrophic mice, likely as a consequence of chronic muscle wasting. In conclusion, we show that myomiR release accompanies periods of myogenic differentiation in cell culture and in vivo. Serum myomiR abundance is therefore a function of the regenerative/degenerative status of the muscle, overall muscle mass, and tissue expression levels. These findings have implications for the use of ex-myomiRs as biomarkers for DMD disease progression and monitoring response to therapy.
Assuntos
MicroRNAs/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Espaço Extracelular/genética , Humanos , Camundongos , MicroRNAs/sangue , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Especificidade de Órgãos , Cultura Primária de CélulasRESUMO
There is currently an urgent need for biomarkers that can be used to monitor the efficacy of experimental therapies for Duchenne Muscular Dystrophy (DMD) in clinical trials. Identification of novel protein biomarkers has been limited due to the massive complexity of the serum proteome and the presence of a small number of very highly abundant proteins. Here we have utilised an aptamer-based proteomics approach to profile 1,129 proteins in the serum of wild-type and mdx (dystrophin deficient) mice. The serum levels of 96 proteins were found to be significantly altered (P < 0.001, q < 0.01) in mdx mice. Additionally, systemic treatment with a peptide-antisense oligonucleotide conjugate designed to induce Dmd exon skipping and recover dystrophin protein expression caused many of the differentially abundant serum proteins to be restored towards wild-type levels. Results for five leading candidate protein biomarkers (Pgam1, Tnni3, Camk2b, Cycs and Adamts5) were validated by ELISA in the mouse samples. Furthermore, ADAMTS5 was found to be significantly elevated in human DMD patient serum. This study has identified multiple novel, therapy-responsive protein biomarkers in the serum of the mdx mouse with potential utility in DMD patients.
Assuntos
Proteínas ADAM/genética , Aptâmeros de Nucleotídeos/farmacologia , Biomarcadores Farmacológicos/sangue , Distrofina/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/farmacologia , Proteínas ADAM/sangue , Proteína ADAMTS5 , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/sangue , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Modelos Animais de Doenças , Distrofina/agonistas , Distrofina/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Monoéster Fosfórico Hidrolases/sangue , Monoéster Fosfórico Hidrolases/genética , Proteínas Quinases/sangue , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteômica/métodosRESUMO
Duchenne muscular dystrophy (DMD) is caused by absence of the integral structural protein, dystrophin, which renders muscle fibres susceptible to injury and degeneration. This ultimately results in cardiorespiratory dysfunction, which is the predominant cause of death in DMD patients, and highlights the importance of therapeutic targeting of the cardiorespiratory system. While there is some evidence to suggest that restoring dystrophin in the diaphragm improves both respiratory and cardiac function, the role of the diaphragm is not well understood. Here using exon skipping oligonucleotides we predominantly restored dystrophin in the diaphragm and assessed cardiac function by MRI. This approach reduced diaphragmatic pathophysiology and markedly improved diaphragm function but did not improve cardiac function or pathophysiology, with or without exercise. Interestingly, exercise resulted in a reduction of dystrophin protein and exon skipping in the diaphragm. This suggests that treatment regimens may require modification in more active patients. In conclusion, whilst the diaphragm is an important respiratory muscle, it is likely that dystrophin needs to be restored in other tissues, including multiple accessory respiratory muscles, and of course the heart itself for appropriate therapeutic outcomes. This supports the requirement of a body-wide therapy to treat DMD.
Assuntos
Diafragma/fisiopatologia , Modelos Animais de Doenças , Coração/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial , Western Blotting , Diafragma/diagnóstico por imagem , Diafragma/metabolismo , Distrofina/genética , Distrofina/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Morfolinos/química , Morfolinos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/genética , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Condicionamento Físico Animal/fisiologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Radiografia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Duchenne muscular dystrophy (DMD) is a genetic muscle disorder caused by mutations in the Dmd gene resulting in the loss of the protein dystrophin. Patients do not only experience skeletal muscle degeneration, but also develop severe cardiomyopathy by their second decade, one of the main causes of death. The absence of dystrophin in the heart renders cardiomyocytes more sensitive to stretch-induced damage. Moreover, it pathologically alters intracellular calcium (Ca2+) concentration, neuronal nitric oxide synthase (nNOS) localization and mitochondrial function and leads to inflammation and necrosis, all contributing to the development of cardiomyopathy. Current therapies only treat symptoms and therefore the need for targeting the genetic defect is immense. Several preclinical therapies are undergoing development, including utrophin up-regulation, stop codon read-through therapy, viral gene therapy, cell-based therapy and exon skipping. Some of these therapies are undergoing clinical trials, but these have predominantly focused on skeletal muscle correction. However, improving skeletal muscle function without addressing cardiac aspects of the disease may aggravate cardiomyopathy and therefore it is essential that preclinical and clinical focus include improving heart function. This review consolidates what is known regarding molecular pathology of the DMD heart, specifically focusing on intracellular Ca2+, nNOS and mitochondrial dysregulation. It briefly discusses the current treatment options and then elaborates on the preclinical therapeutic approaches currently under development to restore dystrophin thereby improving pathology, with a focus on the heart.
Assuntos
Cardiomiopatias/patologia , Distrofia Muscular de Duchenne/patologia , Animais , HumanosRESUMO
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.
Assuntos
Peptídeos Penetradores de Células/genética , Distrofina/biossíntese , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/genética , Animais , Peptídeos Penetradores de Células/administração & dosagem , Modelos Animais de Doenças , Distrofina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/administração & dosagemRESUMO
Oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. Clinical translation has been often hampered by poor biodistribution, however. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids. This review focuses on CPP-delivery of several classes of oligonucleotides (ONs), namely antisense oligonucleotides, splice switching oligonucleotides (SSOs) and siRNAs. Two main strategies have been used to transport ONs with CPPs: covalent conjugation (which is more appropriate for charge-neutral ON analogues) and non-covalent complexation (which has been used for siRNA delivery essentially). Chemical synthesis, mechanisms of cellular internalization and various applications will be reviewed. A comprehensive coverage of the enormous amount of published data was not possible. Instead, emphasis has been put on strategies that have proven to be effective in animal models of important human diseases and on examples taken from the authors' own expertise.
Assuntos
Antibacterianos/administração & dosagem , Antivirais/administração & dosagem , Peptídeos Penetradores de Células/química , Portadores de Fármacos/química , Nanopartículas/química , Oligonucleotídeos/administração & dosagem , Sequência de Aminoácidos , Animais , Antibacterianos/farmacocinética , Antibacterianos/uso terapêutico , Antivirais/farmacocinética , Antivirais/uso terapêutico , Transporte Biológico , Linhagem Celular , Endocitose , Humanos , Dados de Sequência Molecular , Distrofias Musculares/tratamento farmacológico , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Oligonucleotídeos/farmacocinética , Oligonucleotídeos/uso terapêuticoRESUMO
Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder caused by mutations in the Dmd gene. In addition to skeletal muscle wasting, DMD patients develop cardiomyopathy, which significantly contributes to mortality. Antisense oligonucleotides (AOs) are a promising DMD therapy, restoring functional dystrophin protein by exon skipping. However, a major limitation with current AOs is the absence of dystrophin correction in heart. Pip peptide-AOs demonstrate high activity in cardiac muscle. To determine their therapeutic value, dystrophic mdx mice were subject to forced exercise to model the DMD cardiac phenotype. Repeated peptide-AO treatments resulted in high levels of cardiac dystrophin protein, which prevented the exercised induced progression of cardiomyopathy, normalising heart size as well as stabilising other cardiac parameters. Treated mice also exhibited significantly reduced cardiac fibrosis and improved sarcolemmal integrity. This work demonstrates that high levels of cardiac dystrophin restored by Pip peptide-AOs prevents further deterioration of cardiomyopathy and pathology following exercise in dystrophic DMD mice.
Assuntos
Cardiomiopatias/etiologia , Distrofina/genética , Morfolinos/administração & dosagem , Distrofia Muscular de Duchenne/complicações , Condicionamento Físico Animal/efeitos adversos , Animais , Biomarcadores , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Cardiomiopatias/prevenção & controle , Cardiomiopatias/terapia , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/prevenção & controle , Cardiomiopatia Dilatada/terapia , Modelos Animais de Doenças , Distrofina/metabolismo , Fibrose , Expressão Gênica , Humanos , Imagem Cinética por Ressonância Magnética , Camundongos , Camundongos Endogâmicos mdx , Miocárdio/metabolismo , Miocárdio/patologia , FenótipoRESUMO
BACKGROUND: MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest. RESULTS: The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 µl were used. CONCLUSIONS: Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity.