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Blood, 15 September 2007, Vol. 110, No. 6, pp. 1779-1787.
Prepublished online as a Blood First Edition Paper on May 11, 2007; DOI 10.1182/blood-2006-11-053710.
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Submitted November 2, 2006
Accepted April 11, 2007
Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced intensity selection for MGMT P140K transgene expression
Claudia R Ball, Ingo H Pilz, Manfred Schmidt, Sylvia Fessler, David A Williams, Christof von Kalle*, and Hanno Glimm
Department of Translational Oncology, The National Center for Tumor Diseases (NCT) and the German Cancer Research Center, Heidelberg, Germany
Faculty of Biology, Albert-Ludwigs-University, Freiburg, Germany
Institute of Molecular Medicine and Cell Research, Albert-Ludgwigs-University, Freiburg, Germany
Experimental Hematology Molecular and Gene Therapy Program, Cincinnati Children's Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States
Department of Internal Medicine I, Albert-Ludwigs-University, Freiburg, Germany
* Corresponding author; email: christof.kalle{at}nct-heidelberg.de.
Efficient in vivo selection increases survival of gene corrected hematopoietic stem cells (HSC) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSC lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K) expressing HSC and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6 BG and BCNU did not impair long-term differentiation and proliferation of MGMT P140K expressing stem cell clones in serially transplanted mice and did not lead to clonal exhaustion. Interestingly, not all gene modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.
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