Incorporation of drug resistance genes into gene vectors has 2 important roles in stem cell gene therapy: increasing the proportion of gene-corrected cells in vivo (ie, in vivo selection) and marrow protection to permit higher or more tightly spaced doses of chemotherapy in the treatment of malignant diseases. We studied in a clinically relevant canine model of gene therapy the P140K mutant of the drug resistance gene methylguanine methyltransferase (MGMT), which encodes a DNA-repair enzyme that confers resistance to the combination of the MGMT inhibitor O 6 -benzylguanine (O 6 BG) and nitrosourea drugs such as carmustine and methylating agents such as temozolomide. Two dogs received MGMT(P140K)-transduced autologous CD34 ؉ -selected cells. After stable engraftment, gene marking in granulocytes was between 3% and 16% in the 2 animals, respectively. Repeated administration of O 6 BG and temozolomide resulted in a multilineage increase in genemodified repopulating cells with marking levels of greater than 98% in granulocytes.MGMT(P140K) overexpression prevented the substantial myelosuppression normally associated with this drug combination. Importantly, hematopoiesis remained polyclonal throughout the course of the study. Extrahematopoietic toxicity was minimal, and no signs of myelodysplasia or leukemia were detected. These large-animal data support the evaluation of MGMT(P140K) in con IntroductionGene transfer to hematopoietic stem cells holds significant promise for the treatment of genetic diseases, AIDS, and cancer. The use of drug resistance genes in stem cell gene therapy has 2 important clinical applications. First, drug selection of genetically corrected cells in vivo increases the proportion of corrected cells in gene therapy protocols for genetic diseases affecting the hematopoietic system. This strategy could circumvent the relatively low gene transfer levels obtained after nontoxic reduced-intensity conditioning regimens. Second, this strategy could also be applied to protect the bone marrow in the context of chemotherapy for solid tumors. Chemoprotection could permit dose-intensified cancer chemotherapy regimens, averting the dose-limiting myelosuppression normally associated with intensified chemotherapy regimens. [1][2][3][4] Rescue with unmanipulated autologous stem cells has been widely used to this end, but this strategy allows for only a limited number of high-dose chemotherapy cycles and is still limited by short periods of significant myelosuppression in the period immediately after transplantation. In contrast, a genetically protected bone marrow could make possible tightly spaced dose-intense chemotherapy regimens over extended time periods in the complete absence of any myelosuppression.A particularly attractive drug resistance gene for use in gene therapy is the DNA-repair enzyme methylguanine methyltransferase (MGMT). 3,4 Overexpression of this enzyme renders primary hematopoietic cells resistant to nitrosoureas such as carmustine (BCNU) [3][4][5][6] and to methylating agents, such as t...

Efficient gene transfer to hematopoietic stem cells by Moloney murine leukemia virus-derived retroviral vectors benefits from ex vivo culture and cytokine support. Both also increase the risks of apoptosis and differentiation among cells targeted for transduction. In an effort to maximize the retention of stem cell properties in target cells, we developed a transduction protocol with a focus on minimizing graft manipulation, cytokine stimulation, and ex vivo exposure duration. Based on their wide host range and ability to transduce quiescent cells, human immunodeficiency virus (HIV)-derived lentivirus vectors are ideally suited for this purpose. Our present studies in a murine model show that whole bone marrow cells are readily transduced after a 1-hour vector exposure in the presence of stem cell factor and CH296 fibronectin fragment. Using this rapid transduction protocol, we achieved long-term, multilineage reconstitution of murine recipients with up to 25% GFP-expressing cells in primary and secondary recipients. Our results demonstrate the unique ability of HIV-derived vectors to transduce hematopoietic stem cells in the absence of enrichment, under minimal cytokine stimulation, and following brief exposures.