Efficient gene transfer into human cord blood CD34(+) cells and the CD34(+)CD38(-) subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

Recombinant adeno-associated viral (rAAV) vectors have been evaluated for t heir ability to transduce primitive hematopoietic cells. Early studies docu mented rAAV-mediated gene expression during progenitor derived colony forma tion in vitro, but studies examining genome integration and longterm gene e xpression in hematopoietic cells have yielded conflicting results. Such stu dies were performed with crude vector preparations. Using improved methodol ogy, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/10(7) particles) and adenovirus. Transd uction of CD34(+) cells from umbilical cord blood was evaluated with a bici stronic rAAV vector encoding the green fluorescent protein (GFP) and a trim etrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isola ted, quiescent CD34(+) cells were resistant to transduction (less than 4%), but transduction increased to 23 +/- 2% after 2 days of cytokine stimulati on and was further augmented by addition of tumor necrosis factor alpha (51 +/- 4%) at a multiplicity of infection of 10(6). rAAV-mediated gene expres sion was transient in that progenitor derived colony formation was inhibite d by trimetrexate. Primitive CD34(+) and CD34(+), CD38(-) subsets were sequ entially transduced with a rAAV vector encoding the murine ecotropic recept or followed by transduction with an ecotropic retroviral Vector encoding GF P and DHFR. Under optimal conditions 41 +/- 7% of CD34+ progenitors and 21 +/- 6% of CD34(+), CD38(-) progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hemat opoietic cells efficiently but gene expression appears to be transient.