title: Efficient in vivo regulation of cytidine deaminase expression in the haematopoietic system using a doxycycline-inducible lentiviral vector system.
abstract: Regulated transgene expression may reduce transgene-specific and genotoxic risks associated with gene therapy. To prove this concept, we have investigated the suitability of doxycycline (Dox)-inducible human cytidine deaminase (hCDD) overexpression from lentiviral vectors to mediate effective myeloprotection while circumventing the lymphotoxicity observed with constitutive CDD activity. Rapid Dox-mediated transgene induction associated with a 6-17-fold increase in drug resistance was observed in 32D and primary murine bone marrow (BM) cells. Moreover, robust Dox-regulated transgene expression in the entire haematopoietic system was demonstrated for primary and secondary recipients of hCDD-transduced R26-M2rtTA transgenic BM cells. Furthermore, mice were significantly protected from myelosuppressive chemotherapy as evidenced by accelerated recovery of granulocytes (1.9±0.6 vs 1.3±0.3, P=0.034) and platelets (883±194 vs 584±160 10(3) per μl, P=0.011). Minimal transgene expression in the non-induced state and no overt cellular toxicities including lymphotoxicity were detected. Thus, using a relevant murine transplant model our data provide conclusive evidence that drug-resistance transgenes can be expressed in a regulated fashion in the lymphohaematopoietic system, and that Dox-inducible systems may be used to reduce myelotoxic side effect of anticancer chemotherapy or to avoid side effects of high constitutive transgene expression.
authors.affiliation: REBIRTH Cluster-of-Excellence, Research Group Reprogramming, Hannover Medical School, Hannover, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Lachmann
authors.forename: S
authors.initials: S
authors.lastname: Brennig
authors.forename: N
authors.initials: N
authors.lastname: Pfaff
authors.forename: H
authors.initials: H
authors.lastname: Schermeier
authors.forename: J
authors.initials: J
authors.lastname: Dahlmann
authors.forename: R
authors.initials: R
authors.lastname: Phaltane
authors.forename: I
authors.initials: I
authors.lastname: Gruh
authors.forename: U
authors.initials: U
authors.lastname: Modlich
authors.forename: A
authors.initials: A
authors.lastname: Schambach
authors.forename: C
authors.initials: C
authors.lastname: Baum
authors.forename: T
authors.initials: T
authors.lastname: Moritz
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D016026:Bone Marrow Transplantation; D002460:Cell Line; D002478:Cells, Cultured; D003561:Cytarabine / Q000494:pharmacology; D003564:Cytidine Deaminase / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006413:Hematopoietic System / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods; D019076:Transgenes / Q000235:genetics
chemical_list: D003561:Cytarabine; D049452:Green Fluorescent Proteins; D003564:Cytidine Deaminase; D004318:Doxycycline