title: Genetic modification of mouse bone marrow by lentiviral vector-mediated delivery of hypoxanthine-Guanine phosphoribosyltransferase short hairpin RNA confers chemoprotection against 6-thioguanine cytotoxicity.
abstract: We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection.
authors.affiliation: Department of Medicine, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA 90095, USA.
authors.forename: K
authors.initials: K
authors.lastname: Hacke
authors.forename: J A
authors.initials: JA
authors.lastname: Treger
authors.forename: B T
authors.initials: BT
authors.lastname: Bogan
authors.forename: R H
authors.initials: RH
authors.lastname: Schiestl
authors.forename: N
authors.initials: N
authors.lastname: Kasahara
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000201:enzymology*; D016026:Bone Marrow Transplantation; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D013866:Thioguanine / Q000494:pharmacology*
chemical_list: D034741:RNA, Small Interfering; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine