title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression.
abstract: Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs 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 HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation 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.
authors.affiliation: National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany.
authors.forename: Claudia R
authors.initials: CR
authors.lastname: Ball
authors.forename: Ingo H
authors.initials: IH
authors.lastname: Pilz
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Sylvia
authors.initials: S
authors.lastname: Fessler
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000187:drug effects / Q000201:enzymology*; D016026:Bone Marrow Transplantation; D002330:Carmustine / Q000494:pharmacology; D002454:Cell Differentiation; D015316:Genetic Therapy / Q000379:methods; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006410:Hematopoiesis / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D022423:Myeloid Cells / Q000187:drug effects / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D020079:Terminal Repeat Sequences / Q000502:physiology; D014161:Transduction, Genetic; D014184:Transplantation, Homologous
chemical_list: D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine