title: A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines.
abstract: BACKGROUND
  Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells.
  
  
  METHODS
  We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O(6)-methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency.
  
  
  RESULTS
  EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34(+) cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O(6)-benzylguanine (BG)/N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation.
  
  
  CONCLUSIONS
  This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells.
authors.affiliation: INSERM E0217, Université Victor Segalen Bordeaux 2, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Richard
authors.forename: Fabien
authors.initials: F
authors.lastname: Géronimi
authors.forename: Magalie
authors.initials: M
authors.lastname: Lalanne
authors.forename: Cécile
authors.initials: C
authors.lastname: Ged
authors.forename: Isabelle
authors.initials: I
authors.lastname: Redonnet-Vernhet
authors.forename: Isabelle
authors.initials: I
authors.lastname: Lamrissi-Garcia
authors.forename: Stanton L
authors.initials: SL
authors.lastname: Gerson
authors.forename: Hubert
authors.initials: H
authors.lastname: de Verneuil
authors.forename: François
authors.initials: F
authors.lastname: Moreau-Gaudry
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D000970:Antineoplastic Agents / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D005294:Ferrochelatase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D017121:Porphyria, Hepatoerythropoietic / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes / Q000276:immunology; D013997:Time Factors; D019076:Transgenes
chemical_list: D018952:Antigens, CD34; D000970:Antineoplastic Agents; D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005294:Ferrochelatase; D002330:Carmustine