Expression of Recombinant Coagulation Factor IX in Human Amniotic Membrane-derived Mesenchymal Stem Cells: A New Strategy to Gene Therapy of Hemophilia B

Background: hemophilia b is an x-linked hereditary disorder of blood coagulation system which is caused by factor ix (fix) deficiency. factor ix is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. replacement of factor ix with plasma-derived or recombinant factor ix is the conventional treatment for hemophilia b to raise the factor ix level to therapeutic range. recently, gene therapy has been regarded as a promising approach to treat hemophilia b. this study was aimed to express the factor ix in human amniotic membrane-derived mesenchymal stem cells (ham-mscs). materials and methods: human amniotic membrane-derived mesenchymal stem cells were isolated and characterized from amnion membrane. factor ix from commercially available plasmid was sub-cloned into pcdna3.1 vector. recombinant pcdna3.1-fix construct was confirmed by pcr, enzymatic digestion and dna sequencing. mesenchymal stem cells were transfected with the recombinant vector. expression of factor ix was determined by rt-pcr, elisa and its biological activity assay was performed using aptt. results: isolated ham-mscs expressed specific mesenchymal stem cells markers and were able to differentiate to osteocytes and adipocytes lineages. ham-mscs expressed hrfix at mrna and protein level. the maximum amount of hrfix was 120 ng/ml at 72 hrs after ham-mscs transfection. this hrfix was biologically active (11% activity), formed fibrin clot in aptt test and caused more than two fold decrease in clotting time. conclusion: the ham-mscs expressing factor ix would be useful for gene therapy of hemophilia b. however further studies are required to prove these finding.