Recombinant adeno-associated virus-based vectors provide short-term ratherthan long-term transduction of primitive hematopoietic stem cells

Bone marrow stem cells collected from B6-Gpi-1(a) mice pretreated with 5-fl uorouracil were incubated for 2 h at 37 degrees C in the presence of the re combinant adenovirus-associated virus-based vector (rAAV) SSV9, As measured in vitro immediately following transduction, SSV9 was found to be effectiv e in transducing the primitive cobblestone-area-forming cell (CAFC)-35 subs et (60% transduction efficiency). However, this did not predict long-term e xpression as the presence of the transgene could not be detected six months after transplantation of 1-2 x 10(6) transduced bone marrow stem cells int o lethally irradiated recipients. CAFC analysis of bone marrow cells and So uthern blot analysis of hone marrow and spleen cells were negative,and poly merase chain reaction analysis showed less than 0.1% transduction in bone m arrow cells. Therefore, based on our study we conclude that rAAV transientl y transduces hematopoietic stem cells but fails to integrate into the genom e, leading to the loss of the reporter gene within the first six months aft er transplantation in vivo.