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.
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.
Kikuchi
Jiro
J
Division of Cell and Molecular Medicine, The Center for Molecular Medicine, Jichi Medical School, Tochigi-ken 329-0498, Japan.
Mimuro
Jun
J
Ogata
Kyoichi
K
Tabata
Toshiaki
T
Ueda
Yasuji
Y
Ishiwata
Akira
A
Kimura
Kouzoh
K
Kimura
Konzoh
K
Takano
Katsuhiro
K
Madoiwa
Seiji
S
Mizukami
Hiroaki
H
Hanazono
Yutaka
Y
Kume
Akihiro
A
Hasegawa
Mamoru
M
Ozawa
Keiya
K
Sakata
Yoichi
Y
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*
D018952:Antigens, CD34; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D005169:Factor VIII