Retroviral transduction of mutant O6-methylguanine-DNA methyltransferase as a potent selection factor in hematopoietic stem cells in mouse models.

摘要

Hematopoietic gene therapy is limited by poor retroviral transduction of quiescent hematopoietic stem cells, resulting in a low proportion of genetically modified progeny in bone marrow and peripheral blood. In vivo selection for cells transduced with bicistronic vectors expressing an appropriate selectable marker and a therapeutic gene has been proposed to improve the efficacy of hematopoietic gene therapy. One gene which mediates in vivo selection is O 6-methylguanine-DNA methyltransferase (MGMT). MGMT encodes the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) which repairs alkylation damage from the O6 position of guanine. Unfortunately, in vivo selection for MGMT-transduced cells is compromised by endogenous MGMT expressed in untransduced stem cells, limiting the strength of selection.;O6-benzylguanine (BG) is an inactivator of AGT that potentiates alkylating agent toxicity. BG has been shown to sensitize unmodified hematopoietic cells to alkylating agents (e.g. BCNU and temozolomide) resulting in greater cytotoxicity than from alkylating agents alone. A mutant form of AGT (DeltaAGT) containing a glycine to alanine substitution at amino acid 156 has 240-fold greater resistance to inactivation by BG. I hypothesize that G156A MGMT (DeltaMGMT) gene transduced hematopoietic cells will have increased resistance to BG plus alkylating agent cytotoxicity. I further hypothesize that the selective protection of DeltaMGMT transduced cells and BG sensitization of unmodified cells to the stem cell toxin BCNU will result in strong in vivo selection for hematopoietic progenitor and long term repopulating cells retrovirally transduced with DeltaMGMT. BG & BCNU selection may be stronger than other in vivo selection strategies utilizing the multidrug resistance gene-1 and dihydrofolate reductase genes, in which selection is not mediated by stem cell toxins. Furthermore, in vivo selection could be improved by optimizing mutant MGMT gene transfer using retroviral vectors which strongly express in hematopoietic cells and mutant AGT proteins with maximal activity, drug resistance and protein stability. I have compared a number of retroviral vectors and MGMT mutants for these properties. The studies described here demonstrate that BG & BCNU exerts selection pressure for DeltaMGMT transduced long term repopulating cells greater than that observed in other selection strategies.