In vivo selection for human and murine hematopoietic cells transduced with a therapeutic MGMT lentiviral vector that inhibits HIV replication

We have developed an HIV-based lentiviral vector, VRX496, which efficiently transduces human CD34(+) progenitors and CD4(+) T lymphocytes. VRX496 contains an antisense sequence against the HIV envelope and is currently being evaluated for safety in a clinical trial for treatment of HIV. Selective outgrowth of transduced hematopoietic cells in vivo is anticipated to increase the therapeutic efficacy of this treatment by maximizing the persistence of virus-resistant cells in the body. Although HIV resistance is selective, additional selection may aid in treatment efficacy due to the vast quantity of target cells. Therefore, we engineered VRX496 to express the P140K MGMT gene to drive potent drug-mediated in vivo selection for transduced hematopoietic long-term repopulating cells. Suboptimally transduced T cell cultures treated with O-6-benzylguanine and BCNU were selected from 3 to 100%, and after selection cultures did not support HIV replication. Primary CD34(+) progenitors derived from G-CSF-mobilized peripheral blood were transduced at 27 to 35% efficiency. Approximate sixfold selection was observed for transduced CD34(+) progenitors, colony-forming units, and long-term culture-initiating cells. Multilineage in vivo selection was demonstrated for transduced murine hematopoietic cells in human CD34(+)-derived hematopoietic cells in NOD-SCID mice. These results establish efficient ex vivo and in vivo selection for hematopoietic cells transduced with lentiviral vectors and support the potential therapeutic benefit of this strategy in human gene therapy.