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Recombinant Adeno-Associated Virus-Based Vectors Provide Short-Term Rather Than Long-Term Transduction of Primitive Hematopoietic Stem Cells

^a Joint Center for Radiation Therapy, Department of Radiation Oncology, Harvard Medical School, Boston, MA;
^b Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA;
^c Department of Infectious Diseases, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Key Words. Primitive stem cells • rAAV • Gene therapy • Transduction • Cobblestone-area-forming cells • Long-term repopulation

Dr. Ronald van Os, Department of Hematology, Leiden University Medical Center, Building 1, C2-R, PO Box 9600, 2300 RC Leiden, The Netherlands.

Bone marrow stem cells collected from B6-Gpi-1^a mice pretreated with 5-fluorouracil were incubated for 2 h at 37°C in the presence of the recombinant adenovirus-associated virus-based vector (rAAV) SSV9. As measured in vitro immediately following transduction, SSV9 was found to be effective in transducing the primitive cobblestone-area-forming cell (CAFC)-35 subset (60% transduction efficiency). However, this did not predict long-term expression as the presence of the transgene could not be detected six months after transplantation of 1-2 x 10⁶ transduced bone marrow stem cells into lethally irradiated recipients. CAFC analysis of bone marrow cells and Southern blot analysis of bone marrow and spleen cells were negative, and polymerase chain reaction analysis showed less than 0.1% transduction in bone marrow cells. Therefore, based on our study we conclude that rAAV transiently transduces hematopoietic stem cells but fails to integrate into the genome, leading to the loss of the reporter gene within the first six months after transplantation in vivo.