Contributions
Abstract: EP1677
Type: ePoster
Abstract Category: Therapy - disease modifying - 27 Neuroprotection and Repair
Oligodendrocyte progenitor cells (OPCs) are capable of remyelinating injured axons caused by multiple sclerosis (MS). Demyelinated axons secrete PDGF-AA to direct chemotactic migration of PDGFRα-expressing OPCs. Still, this mechanism is not sufficient to achieve robust remyelination given that inflammation and demyelination activate fibrous astrocytes and microglia. Fibrous astrocytes and microglia generate a glial scar, which obstructs OPCs to contact with the axon thereby, blocking remyelination. Here, we demonstrate that by modifying the expression levels of PDGFRα in OPCs migration velocity can be altered. We transfected vectors based on CRISPR-Cas9 system targeting PDGFRα gene promoter to alter protein expression. CRISPR-Cas9 stimulates genome editing by creating a double strand break (DSB) in a precise locus, thereby inducing DNA repair by either non-homologous end joining (NHEJ) or homology directed repair (HDR). Transfection of two different constructs directed to PDGFRα promoter into mouse derived OPCs resulted in efficient expression of GFP reporter protein. We did not observe significant ultrastructural differences between transfected and non-transfected cells in transmission electron microscopy (TEM). By means of immunogold TEM, we observed GFP bound to the plasma membrane and to intermediate filaments as well as, inside the nucleus. We next sought to assess the effect of our approach on OPC migration efficiency by chemotaxis assays. Transfected cells exhibited a significant reduction in terms of migration velocity. This effect was more evident in OPCs transfected with PDGFRα1 promoter-Cas9, suggesting that regulation of PDGFRα-mediated signalling via CRISPR-Cas9 is a promising tool for the treatment of demyelinating lesions. Our current efforts are focused on transfecting a linear DNA sequence containing a strong promoter flanked by homologous arms together with the PDGFRα promoter-Cas9 plasmids. We expect that these genomic editions might increase OPC chemotactic migration efficiency and eventually, modified cells could be engrafted into lysolecithin injected mice to assess remyelination efficiency.
Disclosure: This research was supported by grants from the Spanish Network for Cell Therapy (ISCIII2012-RED-19-016) and the Spanish Ministry of Economy and Competitiveness (SAF2012-33683).
Jose Manuel Garcia Verdugo: Nothing to disclose
Vicente Herranz Perez: Nothing to disclose
Maria Jose Ulloa Navas: Nothing to disclose
Maria del Carmen Gonzalez Calixto: Nothing to disclose
Abstract: EP1677
Type: ePoster
Abstract Category: Therapy - disease modifying - 27 Neuroprotection and Repair
Oligodendrocyte progenitor cells (OPCs) are capable of remyelinating injured axons caused by multiple sclerosis (MS). Demyelinated axons secrete PDGF-AA to direct chemotactic migration of PDGFRα-expressing OPCs. Still, this mechanism is not sufficient to achieve robust remyelination given that inflammation and demyelination activate fibrous astrocytes and microglia. Fibrous astrocytes and microglia generate a glial scar, which obstructs OPCs to contact with the axon thereby, blocking remyelination. Here, we demonstrate that by modifying the expression levels of PDGFRα in OPCs migration velocity can be altered. We transfected vectors based on CRISPR-Cas9 system targeting PDGFRα gene promoter to alter protein expression. CRISPR-Cas9 stimulates genome editing by creating a double strand break (DSB) in a precise locus, thereby inducing DNA repair by either non-homologous end joining (NHEJ) or homology directed repair (HDR). Transfection of two different constructs directed to PDGFRα promoter into mouse derived OPCs resulted in efficient expression of GFP reporter protein. We did not observe significant ultrastructural differences between transfected and non-transfected cells in transmission electron microscopy (TEM). By means of immunogold TEM, we observed GFP bound to the plasma membrane and to intermediate filaments as well as, inside the nucleus. We next sought to assess the effect of our approach on OPC migration efficiency by chemotaxis assays. Transfected cells exhibited a significant reduction in terms of migration velocity. This effect was more evident in OPCs transfected with PDGFRα1 promoter-Cas9, suggesting that regulation of PDGFRα-mediated signalling via CRISPR-Cas9 is a promising tool for the treatment of demyelinating lesions. Our current efforts are focused on transfecting a linear DNA sequence containing a strong promoter flanked by homologous arms together with the PDGFRα promoter-Cas9 plasmids. We expect that these genomic editions might increase OPC chemotactic migration efficiency and eventually, modified cells could be engrafted into lysolecithin injected mice to assess remyelination efficiency.
Disclosure: This research was supported by grants from the Spanish Network for Cell Therapy (ISCIII2012-RED-19-016) and the Spanish Ministry of Economy and Competitiveness (SAF2012-33683).
Jose Manuel Garcia Verdugo: Nothing to disclose
Vicente Herranz Perez: Nothing to disclose
Maria Jose Ulloa Navas: Nothing to disclose
Maria del Carmen Gonzalez Calixto: Nothing to disclose