Stem cell gene therapy remains an elusive therapeutic goal. Furthermore, without strategies to select for low levels of genetically altered stem cells expressing the gene of interest in vivo, it may be difficult if not impossible to impact hematopoiesis. The investigators have analyzed mutant forms of MGMT that provide resistance to the MGMT inhibitor, 06 benzylguanine (BG) and confer over 5 fold resistance to BG + BCNU in vitro and over 300 fold survival and hematopoietic reconstitution advantage in vivo following therapy with BG and BCNU. In the first Specific Aim, the investigators will evaluate human lentiviral vectors because of their enhanced ability to transduce human repopulating stem cells. Lentiviral backbone modifications will be made to increase transduction and expression, and mutant MGMT will be integrated to evaluate transduction into human CD34 and NOD/SCID repopulating human cells.
In Aim 2, a dual gene vector will be evaluated containing the X linked Chronic Granulomatous Disease gene, gp91phox and MGMT. Transduction of murine stem cells from mice defective in gp9lphox followed by transplantation and non-myeloablative treatment with BG + BCNU will establish whether the MGMT gene can be used to select for a therapeutic gene in vivo.
The third Aim i s admittedly novel: the use of the MGMT gene to select for marrow-derived stem cells in nonhematopoietic organs such as liver, brain and lung. Since the preliminary studies by others indicate the plastic differentiation capacity of hematopoietic stem cells, we hypothesize that selective enrichment through a drug resistance strategy offers a potentially potent approach to tissue replacement. Establishing that a BG-resistant mutant MGMT gene can act as a therapeutic drug selection gene to protect stem cells and to enhance tissue engraftment in vivo has far reaching implications for stem cell gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA073062-09
Application #
7060488
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Wolpert, Mary K
Project Start
1997-08-20
Project End
2008-05-31
Budget Start
2006-06-01
Budget End
2008-05-31
Support Year
9
Fiscal Year
2006
Total Cost
$289,970
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
Roth, Justin C; Alberti, Michael O; Ismail, Mourad et al. (2015) MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors. Virus Res 196:170-80
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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