Myeloablation is not required to select and maintain expression of the drug-resistance gene, mutant MGMT, in primary and secondary recipients

Abstract

Gene transduction of hematopoietic progenitors capable of reconstituting both primary and secondary recipients is an important milestone in preclinical development of gene therapy. Myeloablation conditioning prior to infusion of transduced stem cells causes significant host morbidity. In contrast, drug-resistance gene transfer utilizes judicious in vivo selection of transduced stem cells over time, reaching only the level of transduction and expression required. The O(6)-benzylguanine (BG)-resistant mutant O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a potent selection gene for transduced cells. Using two different mutant MGMTs, G156A and P140K, that vary in BG resistance by a factor of 1:20, we asked whether long-term repopulating and secondary mouse-repopulating cells could be transduced, transplanted, and selected for in the nonmyeloablated recipient and whether the mutant MGMT would continue to be expressed in secondary recipient repopulating cells. We found that under stringent drug-selection competition, cells expressing the more BG-resistant variant, P140K-MGMT, were enriched over G156A-MGMT-expressing progenitors. In addition, the MFG retroviral vector transmitted the mutant MGMT gene to long-term repopulating cells that, after selective enrichment in the nonmyeloablated primary recipient, repopulated secondary mice and continued to express the transgene. Thus, MFG mutant MGMT vectors transduce repopulating hematopoietic stem cells that may be used both for chemotherapeutic drug resistance and to enrich for second therapeutic genes.

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

“…In addition, the use of O6-benzylguanine (BG)-resistant mutant O6-methylguanine-DNA methyltransferase (MGMT) or similar multi-drug resistance genes can be used for positive selection of transduced BM cells in the absence of myeloablation. 37 Notably, our technique demonstrates partial phenotypic correction in the absence of any cell or gene selective pressure and is achievable with clinically relevant numbers of autologous cells in immunocompetent individuals.…”

Live and let die: In vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection

“…The major limitation of the use of 6-BG in pre-clinical models and in human trials has been severe, dose-related bone marrow toxicity, likely due to further depletion of low endogenous MGMT levels in hematopoietic cells [50][51][52][53][54][55][56][57]. Our group and others have evaluated the efficacy of chemoprotection using mutant forms of MGMT that are highly resistant to pseudosubstrate-mediated depletion and retain repair activity [ 31,33,35,37,[58][59][60][61][62][63]. Thus, this combined genetic and pharmacologic approach allows sensitization of tumors to alkylating agents and simultaneous reduction in bone marrow toxicity [ 4,13,40 ].…”

“…In murine studies, multiple groups have demonstrated that MGMT transduced HSC are able to reconstitute secondary recipients after a chemoselective regimen has been applied [ 35,37,38,60,65,118 ]. No instances of abnormal hematopoiesis or increased rates of hematological malignancy have been reported.…”

Hematopoietic stem cell gene therapy with drug resistance genes: an update

“…134,138,201 In the b-thalassemic murine model, mice receiving gamma-globin/MGMT dual genetransduced BM following a nonmyeloablative conditioning regimen, temozolomide and BG treatment, had increased red cell gamma-globin, and consequently increased donor cell and hemoglobin levels in the recipient mice. 202 In the absence of drug selection, donor cells contributed to hematopoiesis marginally, and there was no improvement in hemoglobin levels.…”