To overcome myelosuppression, a major dose limiting toxicity of many chemotherapeutic agents, this application proposes to use gene therapy to overexpress a drug resistance gene in hematopoietic progenitors. The overall goal is to reduce myelosuppression after repeated doses of conventional or high dose chemotherapy. The drugs targeted are alkylating agents which are associated with cumulative myelosuppression, persistent marrow damage and delayed secondary leukemias, most likely due to direct toxicity to early hematopoietic progenitors. This group of agents includes the chloroethylating agent,, BCNU, and the methylating agent, temozolomide, [TMZ] which exert their cytotoxic effect by attack of the O6 position of guanine. The applicant has shown that O6 -DNA alkyltransferase [AGT], encoded by MGMT, repairs O6 - alkylguanine lesions and is responsible for tumor resistance to these agents. The applicant has also shown that the AGT inhibitor, O6 - benzylguanine, [BG], markedly sensitizes tumors to BCNU although its use is associated with dose-limiting myelosuppression. To protect hematopoietic cells from the combination of BG & BCNU or BG & TMZ, the applicant proposes to use a mutant MGMT, glycine 156 to alanine, [delta-MGMT] which is resistant to BG inactivation. The investigators propose that expression of delta-MGMT from retroviral vectors in murine and human hematopoietic progenitors will selectively prevent myelosuppression after therapy with BG & BCNU and BG & TMZ. Evaluation of both BCNU and TMZ is important because their mechanism of action and potential therapeutic utility and toxicities are different. The following specific aims will be studied: 1. Define the ability of delta-MGMT to transduce human CD34 cells, generating resistance to the combination of BG and BCNU or TMZ; 2. Optimize delta-MGMT transduction into early hematopoietic progenitor long term culture initiating cells [LTC-IC] and determine whether these cells become resistant to the combination of BG & BCNU or TMZ; 3. Evaluate murine models of hematopoietic drug resistance following retroviral delta-MGMT transduction and transplantation; and 4. Determine the ability of transduced murine cells to competitively repopulate the marrow of mice by repeated drug exposure. These studies will optimize gene transfer strategies to protect hematopoietic cells from the acute and long term effects of chemotherapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA073062-01A1
Application #
2410868
Study Section
Special Emphasis Panel (ZRG2-ET-1 (03))
Project Start
1997-08-20
Project End
2001-05-31
Budget Start
1997-08-20
Budget End
1998-05-31
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Alberti, Michael O; Roth, Justin C; Ismail, Mourad et al. (2012) Derivation of a myeloid cell-binding adenovirus for gene therapy of inflammation. PLoS One 7:e37812
Lin, Yuan; Cheung, Perrin; Roth, Justin C et al. (2011) Imaging stem cell-derived persistent foci after in vivo selection of lentiviral MGMT-P140K transduced murine bone marrow cells. Mol Ther 19:1342-52
Wang, Fangjing; Dennis, James E; Awadallah, Amad et al. (2009) Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging. Physiol Genomics 37:23-34
Lee, Zhenghong; Dennis, James E; Gerson, Stanton L (2008) Imaging stem cell implant for cellular-based therapies. Exp Biol Med (Maywood) 233:930-40
Reese, Jane S; Roth, Justin C; Gerson, Stanton L (2008) Bone marrow-derived cells exhibiting lung epithelial cell characteristics are enriched in vivo using methylguanine DNA methyltransferase-mediated drug resistance. Stem Cells 26:675-81
Lin, Yuan; Molter, Joe; Lee, Zhenghong et al. (2008) Bioluminescence imaging of hematopoietic stem cell repopulation in murine models. Methods Mol Biol 430:295-306
Love, Zachary; Wang, Fangjing; Dennis, James et al. (2007) Imaging of mesenchymal stem cell transplant by bioluminescence and PET. J Nucl Med 48:2011-20
Fontes, Aparecida Maria; Davis, Brian M; Encell, Lance P et al. (2006) Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells. Mol Cancer Ther 5:121-8
An, Feng-Qi; Folarin, Hope Merlene; Compitello, Nicole et al. (2006) Long-term-infected telomerase-immortalized endothelial cells: a model for Kaposi's sarcoma-associated herpesvirus latency in vitro and in vivo. J Virol 80:4833-46

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