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- Title
A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines.
- Authors
Emmanuel Richard; Fabien Géronimi; Magalie Lalanne; Cécile Ged; Isabelle Redonnet-Vernhet; Isabelle Lamrissi-Garcia; Stanton L Gerson; Hubert de Verneuil; François Moreau-Gaudry
- Abstract
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. We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O6-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. 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 O6-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. 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. Copyright © 2003 John Wiley & Sons, Ltd.
- Subjects
ERYTHROPOIETIC failure; HEMATOPOIETIC system; HEMATOPOIESIS; BLOOD diseases; STEM cells
- Publication
Journal of Gene Medicine, 2003, Vol 5, Issue 9, p737
- ISSN
1099-498X
- Publication type
Article
- DOI
10.1002/jgm.407