title: Chemoprotection of murine hematopoietic cells by combined gene transfer of cytidine deaminase (CDD) and multidrug resistance 1 gene (MDR1).
abstract: BACKGROUND
  Hematologic toxicity represents a major side effect of cytotoxic chemotherapy frequently preventing adequately dosed chemotherapy application and impeding therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells represents a potential strategy to overcome this problem. To apply this concept in the context of acute myeloid leukemia and myelodysplasia, we have investigated the overexpression of the multidrug resistance 1 (MDR1) and the cytidine deaminase (CDD) gene conferring resistance to anthracyclines and cytarabine (Ara-C), the two most important drugs in the treatment of these diseases.
  
  
  METHODS
  State-of-the-art, third generation, self-inactivating (SIN) lentiviral vectors were utilized to overexpress a human CDD-cDNA and a codon-optimized human MDR1-cDNA corrected for cryptic splice sites from a spleen focus forming virus derived internal promoter. Studies were performed in myeloid 32D cells as well as primary lineage marker negative (lin(-)) murine bone marrow cells and flow cytometric analysis of suspension cultures and clonogenic analysis of vector transduced cells following cytotoxic drug challenge were utilized as read outs.
  
  
  RESULTS
  Efficient chemoprotection of CDD and MDR1 transduced hematopoietic 32D as well as primary lin(-) cells was proven in the context of Ara-C and anthracycline application. Both, CTX-R transduced 32D as well as primary hematopoietic cells displayed marked resistance at concentrations 5-20 times the LD50 of non-transduced control cells. Moreover, simultaneous CDD/MDR1 gene transfer resulted in similar protection levels even when combined Ara-C anthracycline treatment was applied. Furthermore, significant enrichment of transduced cells was observed upon cytotoxic drug administration.
  
  
  CONCLUSIONS
  Our data demonstrate efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following Ara-C and/or anthracycline application, arguing for the efficacy as well as feasibility of our approach and warranting further evaluation of this concept.
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brennig
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.|JRG Translational Hematology of Congenital Diseases, REBIRTH Cluster-of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Lachmann
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Theresa
authors.initials: T
authors.lastname: Buchegger
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Hetzel
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.|Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany. moritz.thomas@mh-hannover.de.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany. moritz.thomas@mh-hannover.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Moritz
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D018943:Anthracyclines / Q000008:administration & dosage; D003561:Cytarabine / Q000008:administration & dosage; D003564:Cytidine Deaminase / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000187:drug effects*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D051379:Mice; D009190:Myelodysplastic Syndromes / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D022821:RNA Splice Sites / Q000235:genetics
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018943:Anthracyclines; D018076:DNA, Complementary; D022821:RNA Splice Sites; D003561:Cytarabine; D003564:Cytidine Deaminase