title: Chemoselection of allogeneic HSC after murine neonatal transplantation without myeloablation or post-transplant immunosuppression.
abstract: The feasibility of allogeneic transplantation, without myeloablation or post-transplant immunosuppression, was tested using in vivo chemoselection of allogeneic hematopoietic stem cells (HSCs) after transduction with a novel tricistronic lentiviral vector (MGMT(P140K)-2A-GFP-IRES-TK (MAGIT)). This vector contains P140K-O(6)-methylguanine-methyltransferase (MGMT(P140K)), HSV-thymidine kinase (TK(HSV)), and enhanced green fluorescent protein (eGFP) enabling (i) in vivo chemoselection of HSC by conferring resistance to benzylguanine (BG), an inhibitor of endogenous MGMT, and to chloroethylating agents such as 1,3-bis(2-chloroethyl)nitrosourea (BCNU) and, (ii) depletion of proliferating cells such as malignant clones or transduced donor T cells mediating graft versus host disease (GVHD), by expression of the suicide gene TK(HSV) and Ganciclovir (GCV) administration. Non-myeloablative transplantation of transduced, syngeneic, lineage-depleted (Lin(-)) BM in neonates resulted in 0.67% GFP(+) mononuclear cells in peripheral blood. BG/BCNU chemoselection, 4 and 8 weeks post-transplant, produced 50-fold donor cell enrichment. Transplantation and chemoselection of major histocompatibility complex (MHC)-mismatched MAGIT-transduced Lin(-) BM also produced similar expansion for >40 weeks. The efficacy of this allotransplant approach was validated in Hbb(th3) heterozygous mice by correction of β-thalassemia intermedia, without toxicity or GVHD. Negative selection, by administration of GCV resulted in donor cell depletion without graft ablation, as re-expansion of donor cells was achieved with BG/BCNU treatment. These studies show promise for developing non-ablative allotransplant approaches using in vivo positive/negative selection.
authors.affiliation: Department of Medicine, University of California-San Francisco, San Francisco, California, USA.
authors.forename: Rustom
authors.initials: R
authors.lastname: Falahati
authors.forename: Jianqing
authors.initials: J
authors.lastname: Zhang
authors.forename: Linda
authors.initials: L
authors.lastname: Flebbe-Rehwaldt
authors.forename: Yimin
authors.initials: Y
authors.lastname: Shi
authors.forename: Stanton L
authors.initials: SL
authors.lastname: Gerson
authors.forename: Karin Ml
authors.initials: KM
authors.lastname: Gaensler
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000998:Antiviral Agents / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D049109:Cell Proliferation; D002469:Cell Separation; D048430:Cell Shape; D002470:Cell Survival / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000235:genetics; D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology; D006082:Graft Enhancement, Immunologic; D006084:Graft Rejection / Q000276:immunology / Q000517:prevention & control*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D013937:Thymidine Kinase / Q000235:genetics; D014161:Transduction, Genetic; D014184:Transplantation, Homologous; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D017086:beta-Thalassemia / Q000628:therapy*
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D025521:Tumor Suppressor Proteins; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D013937:Thymidine Kinase; D045643:DNA Repair Enzymes; D015774:Ganciclovir; D002330:Carmustine