title: Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.
abstract: BACKGROUND
  Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII).
  
  
  METHODS
  CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied.
  
  
  RESULTS
  We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 10(6) CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells.
  
  
  CONCLUSIONS
  Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients.
authors.affiliation: Division of Cell and Molecular Medicine, The Center for Molecular Medicine, Jichi Medical School, Tochigi-ken 329-0498, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Kikuchi
authors.forename: Jun
authors.initials: J
authors.lastname: Mimuro
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Ogata
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Tabata
authors.forename: Yasuji
authors.initials: Y
authors.lastname: Ueda
authors.forename: Akira
authors.initials: A
authors.lastname: Ishiwata
authors.forename: Kouzoh
authors.initials: K
authors.lastname: Kimura
authors.forename: Konzoh
authors.initials: K
authors.lastname: Kimura
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Takano
authors.forename: Seiji
authors.initials: S
authors.lastname: Madoiwa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mizukami
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hanazono
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kume
authors.forename: Mamoru
authors.initials: M
authors.lastname: Hasegawa
authors.forename: Keiya
authors.initials: K
authors.lastname: Ozawa
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Sakata
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis*; D001854:Bone Marrow Cells / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000235:genetics*; D005312:Fetal Blood / Q000378:metabolism*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015302:Simian Immunodeficiency Virus / Q000378:metabolism*; D013997:Time Factors; D019076:Transgenes / Q000235:genetics*
chemical_list: D018952:Antigens, CD34; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D005169:Factor VIII