Lentivirus-Mediated Expression of Mutant MGMTP140K Protects Human CD34+ Cells Against the Combined Toxicity of O6-Benzylguanine and 1,3-Bis(2-Chloroethyl)-Nitrosourea or Temozolomide

Lentiviral vectors are capable of efficiently transducing nondividing and slowly dividing cells, including hematopoietic stem cells, resulting in stable integration and sustained transgene expression. We constructed human immunodeficiency virus type 1-based self-inactivating lentiviral vectors to express either wild-type or an O6-benzylguanine (O6-beG)-resistant mutant form of the human O6-alkylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:[protein]-L-cysteine S-methyltransferase, EC 2.1.1.63) and transduced K562 and granulocyte colony-stimulating factor-mobilized human peripheral blood CD34+ cells. After transduction, K562 cells expressed high levels of MGMT as determined by Western blot, immunocytochemistry, and biochemical assay. A colony-forming survival assay showed significant protection against O6-beG plus 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) or temozolomide (TMZ) toxicity. Similarly, a single transduction of CD34+ cells resulted in a 13- to 14-fold increase in the level of MGMT ex...