RESUMEN
STUDY DESIGN: Experimental study. OBJECTIVE: Several neuro-degenerative disorders such as Alzheimer's dementia, Parkinson's disease and amyotrophic lateral sclerosis (ALS) are associated with genetic mutations, and replacing or disrupting defective sequences might offer therapeutic benefits. Single gene delivery has so far failed to achieve significant clinical improvements in humans, leading to the advent of co-expression of multiple therapeutic genes. Co-transfection using two or more individual constructs might inadvertently result in disproportionate delivery of the products into the cells. To prevent this, and in order to rule out interference among the many promoters with varying strength, expressing multiple proteins in equimolar amounts can be achieved by linking open reading frames under the control of only one promoter. SETTING: Kazan, Russian Federation. METHODS: Here we describe a strategy for adeno-viral co-expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) interconnected through picorna-viral 2A-amino-acid sequence in transfected human umbilical cord blood mono-nuclear cells (hUCB-MCs). RESULTS: Presence of both growth factors, as well as absence of immune response to 2A-antigen, was demonstrated after 28-52 days. Following injection of hUCB-MCs into ALS transgenic mice, co-expression of VEGF and FGF2, as well as viable xeno-transplanted cells, were observed in the spinal cord after 1 month. CONCLUSION: These results suggest that recombinant adeno-virus containing 2A-sequences could serve as a promising alternative in regenerative medicine for the delivery of therapeutic molecules to treat neurodegenerative diseases, such as ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/terapia , Células Sanguíneas/metabolismo , Células Sanguíneas/trasplante , Cisteína Endopeptidasas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Virales/metabolismo , Adenoviridae/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Cisteína Endopeptidasas/genética , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Sangre Fetal/citología , Factor 2 de Crecimiento de Fibroblastos/genética , Vectores Genéticos/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutación/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutasa-1/genética , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas Virales/genéticaRESUMEN
STUDY DESIGN: Experimental study. OBJECTIVE: To evaluate the treatment of spinal cord injury with glial cell-derived neurotrophic factor (GDNF) delivered using an adenoviral vector (AdV-GDNF group) in comparison with treatment performed using human umbilical cord blood mononuclear cells (UCB-MCs)-transduced with an adenoviral vector carrying the GDNF gene (UCB-MCs+AdV-GDNF group) in rat. SETTING: Kazan, Russian Federation. METHODS: We examined the efficacy of AdV-GDNF and UCB-MCs+AdV-GDNF therapy by conducting behavioral tests on the animals and morphometric studies on the spinal cord, performing immunofluorescence analyses on glial cells, investigating the survival and migration potential of UCB-MCs, and evaluating the expression of the recombinant GDNF gene. RESULTS: At the 30th postoperative day, equal positive locomotor recovery was observed after both direct and cell-based GDNF therapy. However, after UCB-MCs-mediated GDNF therapy, the area of preserved tissue and the number of spared myelinated fibers were higher than those measured after direct GDNF gene therapy. Moreover, we observed distinct changes in the populations of glial cells; expression patterns of the specific markers for astrocytes (GFAP, S100B and AQP4), oligodendrocytes (PDGFαR and Cx47) and Schwann cells (P0) differed in various areas of the spinal cord of rats treated with AdV-GDNF and UCB-MCs+AdV-GDNF. CONCLUSION: The differences detected in the AdV-GDNF and UCB-MCs+AdV-GDNF groups could be partially explained by the action of UCB-MCs. We discuss the insufficiency and the advantages of these two methods of GDNF gene delivery into the spinal cord after traumatic injury.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia Genética , Factor Neurotrófico Derivado de la Línea Celular Glial/uso terapéutico , Recuperación de la Función , Traumatismos de la Médula Espinal/terapia , Animales , Acuaporina 4/metabolismo , Conexinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/fisiología , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Locomoción/fisiología , Masculino , Proteína P0 de la Mielina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Traumatismos de la Médula Espinal/genética , Estadísticas no ParamétricasRESUMEN
Using rat model of spinal cord contusion injury at TVIII, we compared the effectiveness of immediate single transplantation of human mononuclear umbilical cord blood cells transfected with pBud-VEGF-FGF2 plasmid and immediate direct injection of the same plasmid into the lesion area. The results suggest that the delivery of therapeutic genes vegf and fgf2 in cells is more effective than direct injection of plasmid DNA with the same genes (judging from the number of myelinated fibers). Better tissue preservation and motor function recovery in experiments with direct injection of plasmid pBud-VEGF-FGF2 suggest that direct gene therapy seems to be an effective additional procedure to the method of gene delivery with transfected stem and progenitor cells.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Vectores Genéticos/genética , Plásmidos/genética , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapia , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Masculino , Ratas , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive death of cerebral and spinal motorneurons. Using behavioral tests we studied the efficiency of gene-cell therapy in SOD1 G93A transgenic mice receiving xenotransplantation of human umbilical cord blood mononuclear cells genetically modified with adenoviral vectors encoding vascular endothelial growth factor (VEGF) and reporter green fluorescent protein (EGFP) genes. The cells were transplanted to mice on week 27 of life (preclinical stage of the disease). Behavioral tests (open field, grip strength test) showed that transplantation of umbilical cord blood mononuclear cells expressing VEGF significantly improved the parameters of motor and explorative activity, grip strength, and animal survival. Thus, gene-cell therapy based on genetically modified mononuclear cells expressing VEGF can be efficient for the treatment of amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/terapia , Superóxido Dismutasa/metabolismo , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia Genética/métodos , Humanos , Ratones , Ratones Transgénicos , Superóxido Dismutasa/genética , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Effects of immediate single transplantation of human umbilical cord blood mononuclear cells (UCB-MC) transfected with recombinant vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) genes into the area of injury were studied on the model of rat spinal cord dosed contusion at TVIII level. UCB-MC transfected with EGFP-N2 plasmid were transplanted into the rats of the control group under similar conditions. The presence of EGFP- labeled cells were traced in white matter during 21 days after transplantation at a distance no less than 10 mm in rostral and caudal directions from the nearest point of the injection. By 30 days after the transplantation of UCB-MC transfected with pBud-VEGF-FGF2 plasmid, the cross-sectional area of sparing grey matter increased by more than 60% at a distance of 3 mm from the epicenter of injury. By that time, in the animals of this group, the number of perivascular cells expressing beta receptor of platelet-derived growth factor (PDGFbetaR) was increased by an average of 30% in the outer zones of white matter 1.5 cm from the injury epicenter. Delivery of the therapeutic genes VEGF and FGF2 to the damaged region and their expression in cell carriers stimulates vascularization and post-traumatic spinal cord regeneration.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Regulación de la Expresión Génica , Neovascularización Fisiológica , Traumatismos de la Médula Espinal/terapia , Regeneración de la Medula Espinal , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Ratas , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Using the model of the rat spinal cord dosed contusion injury at T8 level, cross sectional area of the pathological cavities was measured and the number of myelinated nerve fibers was calculated in the outer zones of white matter after immediate single injection in the damaged area of human umbilical cord blood mononuclear cells (UCB-MC) transfected with plasmid with vegf and fgf2 genes. UCB-MC transfected with pEGFP-N2 plasmid with egfp gene of enhanced green fluorescent protein were injected into the rats of control group under similar conditions. By Day 30 after the injection of UCB-MC transfected with vegf and fgf2 genes, total cross-sectional area of the cavities in outer zones of white matter at a distance of 3 mm caudally from the epicenter of the injury was reduced more than twice as compared with that found in control group. Number of myelinated nerve fibers in the same zones of white matter at the same distance from the epicentre in rostral and caudal directions, was increased by 20% on the average as compared with control, and at a distance of 5 mm in rostral direction--by 40 to 70%. Thus, the delivery to the injury region of the therapeutic genes vegf and fgf2 reduced cavitation, restrained the processes of secondary degeneration and supported the number of myelinated fibers in the injured spinal cord.