Paridar M, Amirizadeh N, Habibi Roudkenar M, Amiri F, Abolghasemi H, Jalili M A. Expression of Recombinant Coagulation Factor IX in Human Amniotic Membrane-derived Mesenchymal Stem Cells: A New Strategy to Gene Therapy of Hemophilia B. Iranian Journal of Blood and Cancer 2014; 6 (3) :133-141
URL:
http://ijbc.ir/article-1-462-en.html
Abstract: (11767 Views)
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.
Key words: Hemophilia B, amnion membrane, mesenchymal stem cell, factor IX, gene therapy.
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Original Article |
Subject:
Pediatric Hematology & Oncology Received: 2013/11/16 | Accepted: 2014/03/4 | Published: 2014/04/8