In VivoSelection of Human Hematopoietic Cells in a Xenograft Model Using Combined Pharmacologic and Genetic Manipulations

Abstract

Strategies that increase the ability of human hematopoietic stem and progenitor cells to repair alkylator-induced DNA damage may prevent the severe hematopoietic toxicity in patients with cancer undergoing high-dose alkylator therapy. In the context of genetic diseases, this approach may allow for selection of small numbers of cells that would not otherwise have a favorable growth advantage. No studies have tested this approach in vivo using human hematopoietic stem and progenitor cells. Human CD34(+) cells were transduced with a bicistronic oncoretrovirus vector that coexpresses a mutant form of O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and the enhanced green fluorescent protein (EGFP) and transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Mice were either not treated or treated with O(6)-benzylguanine (6BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). At 8-weeks postinjection, a 2- to 8-fold increase in the percentage of human CD45(+)EGFP(+) cells in 6BG/BCNU-treated versus nontreated mice was observed in the bone marrow and was associated with increased MGMT(P140K)-repair activity. Functionally, 6BG/BCNU-treated mice demonstrated multilineage differentiation in vivo, although some skewing in the maturation of myeloid and B cells was observed in mice transplanted with granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood compared to umbilical cord blood. Expansion of human cells in 6BG/BCNU-treated mice was observed in the majority of mice previously transplanted with transduced umbilical cord blood cells. In addition, a significant increase in the number of EGFP(+) progenitor colonies in treated versus nontreated mice were observed in highly engrafted mice indicating that selection and maintenance of human progenitor cells can be accomplished by expression of MGMT(P140K) and treatment with 6BG/BCNU.

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O6-methylguanine-DNA methyltransferaseP140K

“…37 The most intense studied genes for chemoprotective gene therapy are MDR1 [6][7][8][9][10] and MGMT. [24][25][26] As drug combinations are common in most chemotherapeutic regimens, gene therapy with a combination of several genes is particularly promising and would increase the therapeutic index of combination chemotherapy.…”

“…20 Thus, gene transfer of MGMT P140K into hematopoietic stem cells opened a way to better exploit the therapeutic potential of combined treatment with O 6 -BG and alkylating agents. [21][22][23] This approach was already applied successfully in several in vivo investigations with mice 24,25 and in a canine model demonstrating that retroviral overexpression of MGMT P140K in blood stem cells prevented myelosuppression associated with combination of O 6 -BG and the alkylating agent temozolomide (TMZ). 26 As in clinical protocols usually drug combinations are used, simultaneous expression of two chemoprotective proteins with different mechanisms of action would be advantageous.…”

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

“…This approach provides selective protection of hematopoietic cells at the same time sensitizing MGMT-expressing tumor cells to chemotherapy. [35][36][37][38][39] Such use of mutant MGMT vectors is currently underway in several cancer chemotherapy trials (S Gerson, personal communication; L Wagner, personal communication). In the current study, the detection of vector for prolonged periods of time in the majority of subjects suggest expression of wild-type MGMT protein was not immunogenic when administered in this setting and may serve as a baseline for monitoring future gene therapy studies using the mutant protein.…”

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

“…In addition to potential use in generating chemoresistance, we and others have found that the combined use of 6-BG and MGMT mutants leads to effective in vivo selection of transduced stem cells, including human HSC in xenograft models [ 35,37,62,64 ]. This approach could have additional therapeutic applications in genetic diseases, as demonstrated in mouse models of thalassemia [ 65 ]; protoporphyria [ 66 ]; HIV therapy [ 67,68 ]; and graft modulation following allogeneic transplant.…”