RESUMEN
BACKGROUND: The treatments currently approved for Duchenne muscular dystrophy (DMD), a progressive skeletal muscle wasting disease, address the needs of only a small proportion of patients resulting in an urgent need for therapies that benefit all patients regardless of the underlying mutation. Myostatin is a member of the transforming growth factor-ß (TGF-ß) family of ligands and is a negative regulator of skeletal muscle mass. Loss of myostatin has been shown to increase muscle mass and improve muscle function in both normal and dystrophic mice. Therefore, myostatin blockade via a specific antibody could ameliorate the muscle weakness in DMD patients by increasing skeletal muscle mass and function, thereby reducing patients' functional decline. METHODS: A murine anti-myostatin antibody, mRK35, and its humanized analog, domagrozumab, were developed and their ability to inhibit several TGB-ß ligands was measured using a cell-based Smad-activity reporter system. Normal and mdx mice were treated with mRK35 to examine the antibody's effect on body weight, lean mass, muscle weights, grip strength, ex vivo force production, and fiber size. The humanized analog (domagrozumab) was tested in non-human primates (NHPs) for changes in skeletal muscle mass and volume as well as target engagement via modulation of circulating myostatin. RESULTS: Both the murine and human antibodies are specific and potent inhibitors of myostatin and GDF11. mRK35 is able to increase body weight, lean mass, and muscle weights in normal mice. In mdx mice, mRK35 significantly increased body weight, muscle weights, grip strength, and ex vivo force production in the extensor digitorum longus (EDL) muscle. Further, tibialis anterior (TA) fiber size was significantly increased. NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume and exhibited increased circulating levels of myostatin demonstrating target engagement. CONCLUSIONS: We demonstrated that the potent anti-myostatin antibody mRK35 and its clinical analog, domagrozumab, were able to induce muscle anabolic activity in both rodents, including the mdx mouse model of DMD, and non-human primates. A Phase 2, potentially registrational, clinical study with domagrozumab in DMD patients is currently underway.
Asunto(s)
Anticuerpos/administración & dosificación , Contracción Muscular , Fuerza Muscular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Distrofia Muscular de Duchenne/tratamiento farmacológico , Miostatina/inmunología , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Humanos , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/inmunología , Distrofia Muscular de Duchenne/fisiopatología , Miostatina/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.
Asunto(s)
Atrofia Muscular Espinal/terapia , Oligonucleótidos/genética , Complejo Represivo Polycomb 2/metabolismo , ARN Largo no Codificante/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Línea Celular , Modelos Animales de Enfermedad , Exones/genética , Fibroblastos , Dosificación de Gen , Terapia Genética/métodos , Humanos , Ratones , Terapia Molecular Dirigida/métodos , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Mutación Puntual , Complejo Represivo Polycomb 2/genética , ARN Largo no Codificante/genética , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Activación Transcripcional/genética , Regulación hacia ArribaRESUMEN
Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.
Asunto(s)
Anticuerpos Monoclonales/química , Regiones Determinantes de Complementariedad/química , Miostatina/inmunología , Ingeniería de Proteínas/métodos , Animales , Anticuerpos Monoclonales/inmunología , Regiones Determinantes de Complementariedad/inmunología , Humanos , Ratones , Modelos MolecularesRESUMEN
The targeting of drugs to skeletal muscle is an emerging area of research. Driven by the need for new therapies to treat a range of muscle-associated diseases, these strategies aim to provide improved drug exposure at the site of action in skeletal muscle with reduced concentration in other tissues where unwanted side effects could occur. By interacting with muscle-specific cell surface recognition elements, both tissue localization and selective uptake into skeletal muscle cells can be achieved. The design of molecules that are substrates for muscle uptake transporters can provide concentration in m uscle tissue. For example, drug conjugates with carnitine can provide improved muscle uptake via OCTN2 transport. Binding to muscle surface recognition elements followed by endocytosis can allow even large molecules such as antibodies to enter muscle cells. Monoclonal antibody 3E10 demonstrated selective uptake into skeletal muscle in vivo. Hybrid adeno-associated viral vectors have recently shown promise for high skeletal muscle selectivity in gene transfer applications. Delivery technology methods, including electroporation of DNA plasmids, have also been investigated for selective muscle uptake. This review discusses challenges and opportunities for skeletal muscle targeting, highlighting specific examples and areas in need of additional research.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Transporte Biológico/fisiología , Sistemas de Liberación de Medicamentos/tendencias , Descubrimiento de Drogas/tendencias , Electroporación/métodos , Electroporación/tendencias , HumanosRESUMEN
Loss of muscle and bone mass with age are significant contributors to falls and fractures among the elderly. Myostatin deficiency is associated with increased muscle mass in mice, dogs, cows, sheep and humans, and mice lacking myostatin have been observed to show increased bone density in the limb, spine, and jaw. Transgenic overexpression of myostatin propeptide, which binds to and inhibits the active myostatin ligand, also increases muscle mass and bone density in mice. We therefore sought to test the hypothesis that in vivo inhibition of myostatin using an injectable myostatin propeptide (GDF8 propeptide-Fc) would increase both muscle mass and bone density in aged (24 mo) mice. Male mice were injected weekly (20 mg/kg body weight) with recombinant myostatin propeptide-Fc (PRO) or vehicle (VEH; saline) for four weeks. There was no difference in body weight between the two groups at the end of the treatment period, but PRO treatment significantly increased mass of the tibialis anterior muscle (+ 7%) and increased muscle fiber diameter of the extensor digitorum longus (+ 16%) and soleus (+ 6%) muscles compared to VEH treatment. Bone volume relative to total volume (BV/TV) of the femur calculated by microCT did not differ significantly between PRO- and VEH-treated mice, and ultimate force (Fu), stiffness (S), toughness (U) measured from three-point bending tests also did not differ significantly between groups. Histomorphometric assays also revealed no differences in bone formation or resorption in response to PRO treatment. These data suggest that while developmental perturbation of myostatin signaling through either gene knockout or transgenic inhibition may alter both muscle and bone mass in mice, pharmacological inhibition of myostatin in aged mice has a more pronounced effect on skeletal muscle than on bone.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Miostatina/uso terapéutico , Osteoporosis/tratamiento farmacológico , Sarcopenia/tratamiento farmacológico , Envejecimiento/patología , Envejecimiento/fisiología , Animales , Peso Corporal/efectos de los fármacos , Densidad Ósea/fisiología , Evaluación Preclínica de Medicamentos/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Miostatina/antagonistas & inhibidores , Miostatina/deficiencia , Miostatina/farmacología , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Osteoporosis/patología , Osteoporosis/fisiopatología , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Sarcopenia/patología , Sarcopenia/fisiopatología , Estrés Mecánico , Tibia/efectos de los fármacos , Tibia/fisiopatología , Microtomografía por Rayos X/métodosRESUMEN
Skeletal muscle atrophy can be a consequence of many diseases, environmental insults, inactivity, age, and injury. Atrophy is characterized by active degradation, removal of contractile proteins, and a reduction in muscle fiber size. Animal models have been extensively used to identify pathways that lead to atrophic conditions. We used genome-wide expression profiling analyses and quantitative PCR to identify the molecular changes that occur in two clinically relevant mouse models of muscle atrophy: hindlimb casting and Achilles tendon laceration (tenotomy). Gastrocnemius muscle samples were collected 2, 7, and 14 days after casting or injury. The total amount of muscle loss, as measured by wet weight and muscle fiber size, was equivalent between models on day 14, although tenotomy resulted in a more rapid induction of muscle atrophy. Furthermore, tenotomy resulted in the regulation of significantly more mRNA transcripts then did casting. Analysis of the regulated genes and pathways suggest that the mechanisms of atrophy are distinct between these models. The degradation following casting was ubiquitin-proteasome mediated, while degradation following tenotomy was lysosomal and matrix-metalloproteinase mediated, suggesting a possible role for autophagy. These data suggest that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat atrophy resulting from different conditions.
Asunto(s)
Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Tendón Calcáneo/lesiones , Tendón Calcáneo/metabolismo , Animales , Perfilación de la Expresión Génica , Miembro Posterior/lesiones , Miembro Posterior/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Atrofia Muscular/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TenotomíaRESUMEN
The perichondrium, a structure made of undifferentiated mesenchymal cells surrounding growth plate cartilage, regulates chondrocyte maturation through poorly understood mechanisms. Analyses of loss- and gain-of-function models show that Twist-1, whose expression in cartilage is restricted to perichondrium, favors chondrocyte maturation in a Runx2-dependent manner. Runx2, in turn, enhances perichondrial expression of Fgf18, a regulator of chondrocyte maturation. Accordingly, compound heterozygous embryos for Runx2 and Fgf18 deletion display the same chondrocyte maturation phenotype as Fgf18-null embryos. This study identifies a transcriptional basis for the inhibition of chondrocyte maturation by perichondrium and reveals that Runx2 fulfills antagonistic functions during chondrogenesis.
Asunto(s)
Condrogénesis/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Condrocitos/citología , Condrocitos/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Eliminación de Gen , Placa de Crecimiento/embriología , Placa de Crecimiento/metabolismo , Mesodermo/citología , Ratones , Ratones Mutantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
IGF binding protein-1 (IGFBP-1) inhibits the mitogenic actions of the IGFs. Circulating IGFBP-1 is elevated in newborns and experimental animals with fetal growth restriction (FGR). To establish a causal relationship between high circulating IGFBP-1 and FGR, we have generated transgenic mice using the mouse alpha-fetoprotein gene promoter to target overexpression of human IGFBP-1 (hIGFBP-1) in the fetal liver. These transgenic mice (AFP-BP1) expressed hIGFBP-1 mainly in the fetal hepatocytes, starting at embryonic d 14.5 (E14.5), with lower levels in the gut. The expression peaked at 1 wk postnatally (plasma concentration, 474 +/- 34 ng/ml). At birth, AFP-BP1 pups were 18% smaller [weighed 1.34 +/- 0.02 g compared with 1.62 +/- 0.04 g for wild type (WT); P < 0.05], and they did not demonstrate any postnatal catch-up growth. The placentas of the AFP-BP1 mice were larger than WT from E16.5 onwards (150 +/- 12 for AFP-BP1 vs. 100 +/- 5 mg for WT at E16.5; P < 0.05). Thus, this model of FGR is associated with a larger placenta, but without postnatal catch-up growth. Overall, these data clearly demonstrate that high concentrations of circulating IGFBP-1 are sufficient to cause FGR.
Asunto(s)
Retardo del Crecimiento Fetal/genética , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Animales , Northern Blotting , Southern Blotting , Western Blotting , Peso Corporal , ADN/metabolismo , Cartilla de ADN/química , ADN Complementario/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hepatocitos/metabolismo , Humanos , Inmunohistoquímica , Hibridación in Situ , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ligandos , Hígado/embriología , Hígado/metabolismo , Ratones , Ratones Transgénicos , Modelos Genéticos , Modelos Estadísticos , Fosforilación , Placenta/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución Tisular , Transgenes , alfa-Fetoproteínas/genéticaRESUMEN
Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Animales , Desarrollo Óseo , Diferenciación Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Relacionadas con Receptor de LDL , Operón Lac , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Osteogénesis , Osteopetrosis/etiología , Osteoprotegerina , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Wnt , beta CateninaRESUMEN
Coffin-Lowry Syndrome (CLS) is an X-linked mental retardation condition associated with skeletal abnormalities. The gene mutated in CLS, RSK2, encodes a growth factor-regulated kinase. However, the cellular and molecular bases of the skeletal abnormalities associated with CLS remain unknown. Here, we show that RSK2 is required for osteoblast differentiation and function. We identify the transcription factor ATF4 as a critical substrate of RSK2 that is required for the timely onset of osteoblast differentiation, for terminal differentiation of osteoblasts, and for osteoblast-specific gene expression. Additionally, RSK2 and ATF4 posttranscriptionally regulate the synthesis of Type I collagen, the main constituent of the bone matrix. Accordingly, Atf4-deficiency results in delayed bone formation during embryonic development and low bone mass throughout postnatal life. These findings identify ATF4 as a critical regulator of osteoblast differentiation and function, and indicate that lack of ATF4 phosphorylation by RSK2 may contribute to the skeletal phenotype of CLS.
Asunto(s)
Anomalías Múltiples/genética , Regulación Enzimológica de la Expresión Génica , Osteoblastos/metabolismo , Proteínas Quinasas S6 Ribosómicas/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Anomalías Múltiples/enzimología , Factor de Transcripción Activador 4 , Animales , Diferenciación Celular , Línea Celular , Núcleo Celular/química , Colágeno Tipo I/biosíntesis , Matriz Extracelular/química , Genes Reguladores , Discapacidad Intelectual/genética , Ratones , Ratones Mutantes , Morfogénesis , Osteocalcina/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Síndrome , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional , Cromosoma XRESUMEN
Runx2 is necessary and sufficient for osteoblast differentiation, yet its expression precedes the appearance of osteoblasts by 4 days. Here we show that Twist proteins transiently inhibit Runx2 function during skeletogenesis. Twist-1 and -2 are expressed in Runx2-expressing cells throughout the skeleton early during development, and osteoblast-specific gene expression occurs only after their expression decreases. Double heterozygotes for Twist-1 and Runx2 deletion have none of the skull abnormalities observed in Runx2(+/-) mice, a Twist-2 null background rescues the clavicle phenotype of Runx2(+/-) mice, and Twist-1 or -2 deficiency leads to premature osteoblast differentiation. Furthermore, Twist-1 overexpression inhibits osteoblast differentiation without affecting Runx2 expression. Twist proteins' antiosteogenic function is mediated by a novel domain, the Twist box, which interacts with the Runx2 DNA binding domain to inhibit its function. In vivo mutagenesis confirms the antiosteogenic function of the Twist box. Thus, relief of inhibition by Twist proteins is a mandatory event precluding osteoblast differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Factores Reguladores Miogénicos/fisiología , Proteínas Nucleares/fisiología , Osteoblastos/fisiología , Osteogénesis/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Northern Blotting/métodos , Western Blotting/métodos , Células Cultivadas , Chlorocebus aethiops , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Análisis Mutacional de ADN/métodos , Ensayo de Cambio de Movilidad Electroforética/métodos , Embrión de Mamíferos , Regulación de la Expresión Génica/fisiología , Heterocigoto , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores Reguladores Miogénicos/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Pruebas de Precipitina/métodos , Prolina/genética , Estructura Terciaria de Proteína/fisiología , ARN/análisis , Ratas , Proteínas Represoras/genética , Serina/genética , Esqueleto , Coloración y Etiquetado , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Transfección/métodos , Proteína 1 Relacionada con TwistRESUMEN
In mammals stanniocalcin (STC) is widely expressed, and in the kidney and gut it regulates serum calcium levels by promoting phosphate reabsorption. To shed further light on its functional significance in mammals we have created several lines of mice that express a human STC (hSTC) transgene. Three lines expressed the hSTC transgene, but only two lines exhibited high expression and contained circulating hSTC, and in these animals there was a reduction in postnatal growth (30-50%) that persisted after weaning. Moreover, even wild-type pups exhibited a growth retardation phenotype when nursed by a transgenic foster mother, and this implies that hSTC overexpression deleteriously affects maternal behavior and/or lactation. The reproductive potential of female transgenic mice was also compromised, as evidenced by significantly smaller litter sizes, but transgenic male fertility was unchanged even though the transgene was most highly expressed in testes. Interestingly, transgene-derived serum hSTC increased significantly after puberty and was severalfold higher in females than in males, suggesting a gender-specific mechanism for maintaining elevated circulating levels of STC. Blood analysis revealed that both transgenic lines had elevated phosphate and decreased alkaline phosphatase levels, indicative of altered kidney and bone metabolism. These studies provide the first evidence that STC is involved in growth and reproduction and reaffirm its role in mineral homeostasis.