RESUMEN
Amyotrophic lateral sclerosis (ALS) is mainly a sporadic neurodegenerative disorder characterized by loss of cortical and spinal motoneurons. Some familial ALS cases (FALS) have been linked to dominant mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Transgenic mice overexpressing a mutated form of human SOD1 with a Gly93Ala substitution develop progressive muscle wasting and paralysis as a result of spinal motoneuron loss and die at 5 to 6 months. We investigated the effects of neurotrophic factor gene delivery in this FALS model. Intramuscular injection of an adenoviral vector encoding cardiotrophin-1 (CT-1) in SOD1G93A newborn mice resulted in systemic delivery of CT-1, supplying motoneurons with a continuous source of trophic factor. CT-1 delayed the onset of motor impairment as assessed in the rotarod test. Axonal degeneration was slowed and skeletal muscle atrophy was largely reduced by CT-1 treatment. By monitoring the amplitude of the evoked motor response, we showed that the time-course of motor impairment was significantly decreased by CT-1 treatment. Thus, adenovirus-mediated gene transfer of neurotrophic factors might delay neurogenic muscular atrophy and progressive neuromuscular deficiency in ALS patients.
Asunto(s)
Citocinas/genética , Degeneración Nerviosa/terapia , Enfermedades Neuromusculares/terapia , Adenoviridae/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/terapia , Animales , Animales Recién Nacidos , Atrofia , Conducta Animal/fisiología , Peso Corporal , Citocinas/fisiología , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Inyecciones Intramusculares , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Músculo Esquelético/patología , Mutación , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/fisiopatología , Unión Neuromuscular/fisiología , Nervio Frénico/patología , Superóxido Dismutasa/genética , Análisis de Supervivencia , Factores de TiempoRESUMEN
Cardiotrophin-1 (CT-1) is a potent neurotrophic factor for motoneurons but its clinical use in motor neuron diseases is precluded by side effects on the heart and liver. We explored the possibility of targeting CT-1 to neurons by coupling with the tetanus toxin fragment TTC. Genetic fusion proteins between CT-1 or GFP and TTC were produced in Escherichia coli and assayed in vitro. In contrast to uncoupled CT-1 or GFP, TTC-coupled proteins bound with high affinity to cerebral neurons and spinal cord motoneurons and were rapidly internalized. Glia, hepatocytes, or cardiomyocytes did not show detectable binding or uptake of TTC-coupled proteins. Similar to CT-1, TTC-coupled CT-1 induced IL-6 secretion by KB cells, activated Reg-2 gene expression, and promoted motoneuron survival in a dose-dependent manner. In vivo studies will test whether TTC-coupled CT-1 might be targeted to degenerating spinal cord or brain-stem motoneurons and migrate trans-synaptically to cortical motoneurons, which are also affected in amyotrophic lateral sclerosis.
Asunto(s)
Células Cultivadas/efectos de los fármacos , Citocinas/farmacología , Enfermedad de la Neurona Motora/tratamiento farmacológico , Neuronas Motoras/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Toxina Tetánica/farmacología , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/metabolismo , Citocinas/genética , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Feto , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Proteínas Fluorescentes Verdes , Corazón/efectos de los fármacos , Corazón/fisiología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Ratones , Enfermedad de la Neurona Motora/metabolismo , Enfermedad de la Neurona Motora/fisiopatología , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Factores de Crecimiento Nervioso/genética , Fragmentos de Péptidos/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Toxina Tetánica/genéticaRESUMEN
Cardiotrophin-1 (CT-1), an IL-6-related cytokine, causes hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types, including motoneurons. Here, we analyzed systemic CT-1 effects in progressive motor neuronopathy (pmn) mice that suffer from progressive motoneuronal degeneration, muscle paralysis, and premature death. Administration of an adenoviral CT-1 vector to newborn pmn mice leads to sustained CT-1 expression in the injected muscles and bloodstream, prolonged survival of animals, and improved motor functions. CT-1-treated pmn mice showed a significantly reduced degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. The terminal innervation of skeletal muscle, grossly disturbed in untreated pmn mice, was almost completely preserved in CT-1-treated pmn mice. The remarkable neuroprotection conferred by CT-1 might become clinically relevant if CT-1 side effects, including cardiotoxicity, could be circumvented by a more targeted delivery of this cytokine to the nervous system.
Asunto(s)
Adenoviridae/genética , Citocinas/genética , Terapia Genética , Enfermedad de la Neurona Motora/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Animales , Axones/efectos de los fármacos , Células Cultivadas , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratones Mutantes , Enfermedad de la Neurona Motora/genética , Nervios Periféricos/efectos de los fármacosRESUMEN
Several neurotrophic factors (CNTF, BDNF, IGF-1) have been suggested for the treatment of motor neuron diseases. In ALS patients, however, the repeated subcutaneous injection of these factors as recombinant proteins is complicated by their toxicity or poor bioavailability. We have constructed an adenovirus vector coding for neurotrophin-3 (AdNT-3) allowing for stable and/or targeted delivery of NT-3 to motoneurons. The intramuscular administration of this vector was tested in the mouse mutant pmn (progressive motor neuronopathy). AdNT-3-treated pmn mice showed prolonged lifespan, improved neuromuscular function, reduced motor axonal degeneration and efficient reinnervation of muscle fibres. NT-3 protein and also adenovirus vectors, when injected into muscle, can be transported by motoneurons via retrograde axonal transport to their cell bodies in the spinal cord. Using ELISA and RT-PCR analyses in muscle, spinal cord and serum of AdNT-3-treated pmn mice, we have investigated the contribution of these processes to the observed therapeutic effects. Our results suggest that most if not all therapeutic benefit was due to the continuous systemic liberation of adenoviral NT-3. Therefore, viral gene therapy vectors auch as adenoviruses, AAVs, lentiviruses and new types of gene transfer not based on viral vectors that allow for efficient in vivo liberation of neurotrophic factors have potential for the future treatment of human motor neuron diseases.
Asunto(s)
Terapia Genética/métodos , Enfermedad de la Neurona Motora/terapia , Músculo Esquelético/metabolismo , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/uso terapéutico , Adenoviridae/genética , Animales , Transporte Axonal , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Electromiografía , Ensayo de Inmunoadsorción Enzimática , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Ratones , Ratones Mutantes Neurológicos , Enfermedad de la Neurona Motora/genética , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Factores de Crecimiento Nervioso/biosíntesis , Neurotrofina 3RESUMEN
Tay-Sachs disease is a severe neurodegenerative disorder due to mutations in the HEXA gene coding for the alpha-chain of the alpha-beta heterodimeric lysosomal enzyme beta-hexosaminidase A (HexA). Because no treatment is available for this disease, we have investigated the possibility of enzymatic correction of HexA-deficient cells by HEXA gene transfer. Human HEXA cDNA was subcloned into a retroviral plasmid generating to G.HEXA vector. The best Psi-CRIP producer clone of G.HEXA retroviral particles was isolated, and murine HexA-deficient fibroblasts derived from hexa -/- mice were transduced with the G.HEXA vector. Transduced cells overexpressed the alpha-chain, resulting in the synthesis of interspecific HexA (human alpha-chain/murine beta-chain) and in a total correction of HexA deficiency. The alpha-chain was secreted in the culture medium and taken up by HexA-deficient cells via mannose-6-phosphate receptor binding, allowing for the restoration of intracellular HexA activity in non-transduced cells.