402. Comparison of Pseudotyped rAAV Vectors for Transduction of Primary Hematopoietic Stem and Progenitor Cells Assayed In Vitro and In Vivo

We evaluated a panel of AAV serotypes for their ability to transduce hematopoietic stem and progenitor cells of human and murine origin. rAAV-2 genomes encoding either dsRed1 or firefly luciferase under the control of the hybrid CBA promoter were packaged into capsids derived from AAV-1, AAV-5, AAV-7, AAV-8 and AAV-9 and purified by successive rounds of cesium chloride gradient centrifugation. Hematopoietic cells were transduced overnight at low cytokine concentrations, following which cells were assayed in vitro for transgene expression or transplanted into sublethally irradiated NOD/SCID mice. For murine marrow cells, AAV-7 and AAV-showed the highest levels of expression as compared with the other serotypes. For analysis of human cells, hematopoietic stem/progenitor cells were purified from cord blood, cytokine-primed peripheral blood and bone marrow samples obtained using IRB approved protocols. CD34 cells were immunomagnetically purified and transduced with rAAV-dsRed. Flow cytometric analyses revealed that AAV-7 consistently showed the highest level of transgene expression in vitro in CD34 cells derived from cord blood and bone marrow with levels ranging from 36-64% 24 hours after transduction. This was followed by AAV-8 and AAV-1, at 10-43% expression at 24 hours post-transduction, while the other serotypes exhibited lower levels of transgene expression. We next compared transduction of the CD34+CD3//Q-population, which is highly enriched for stem cells. CD34+CD3//Q-cells were flow sorted to purity prior to transduction with the different serotypes. Notably, this primitive population yielded higher levels of transduction than the more heterogeneous CD34 population, with approximately 60-70% of CD34+CD3//Q-cells from cord blood and cytokine primed peripheral blood showing dsRed expression 24 hours after transduction with AAV-7. As with CD34 cells, transduction with AAV-8 and AAV-1 were the next most efficient. We have previously demonstrated that although rAAV-2 transduction in vitro results in poor expression at early time points, possibly due to defined limitations in uncoating, nuclear entry and second strand synthesis, long term studies and in vivo assays demonstrate that rAAV-2 efficiently transduces primitive human hematopoietic stem cells capable of serial engraftment in immune deficient mice. Since in vitro assays may not reflect true stem cell activity, we initiated in vivo transplantation studies of stem cells transduced with different AAV serotypes to determine the relative long term engraftment and gene expression potentials of AAV genomes packaged in the different serotype capsids. Initial results following live imaging of luciferase-transduced recipients confirm both the early onset of gene expression with AAV-7 and AAV-8 and the hierarchy of transgene expression seen with the serotypes in vitro. Overall, our results suggest that rAAV genomes pseudotyped in AAV-7 and AAV-8 capsids are very promising for gene transfer into hematopoietic stem cells.