title: Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.
abstract: Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias.
authors.affiliation: Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran; Department of Histology and Embryology, Faculty of Science, Urmia University, Urmia, Iran. Electronic address: m.sam@urmia.ac.ir.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Sam
authors.affiliation: Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran; Department of Histology and Embryology, Faculty of Science, Urmia University, Urmia, Iran.
authors.forename: Azadeh Sadat
authors.initials: AS
authors.lastname: Azadbakhsh
authors.affiliation: Department of Histology and Embryology, Faculty of Science, Urmia University, Urmia, Iran.
authors.forename: Farrah
authors.initials: F
authors.lastname: Farokhi
authors.affiliation: Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran.
authors.forename: Kobra
authors.initials: K
authors.lastname: Rezazadeh
authors.affiliation: Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran.
authors.forename: Sohrab
authors.initials: S
authors.lastname: Sam
authors.affiliation: Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Zomorodipour
authors.affiliation: Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
authors.forename: Aliakbar
authors.initials: A
authors.lastname: Haddad-Mashadrizeh
authors.affiliation: Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
authors.forename: Nowruz
authors.initials: N
authors.lastname: Delirezh
authors.affiliation: Department of Immunology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran.
authors.forename: Aram
authors.initials: A
authors.lastname: Mokarizadeh
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006467:Hemophilia A / Q000235:genetics / Q000628:therapy; D006801:Humans; D007438:Introns / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D014162:Transfection / Q000379:methods; D055544:beta-Globins / Q000235:genetics
chemical_list: D055544:beta-Globins; D005164:Factor IX; D012194:RNA-Directed DNA Polymerase
keywords: Bone-marrow mesenchymal stem cells; Cell-based therapy; Gene therapy; Hematopoietic lineage; Hemophilia B; Human factor IX