A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines.
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.
Richard
Emmanuel
E
INSERM E0217, Université Victor Segalen Bordeaux 2, France.
Géronimi
Fabien
F
Lalanne
Magalie
M
Ged
Cécile
C
Redonnet-Vernhet
Isabelle
I
Lamrissi-Garcia
Isabelle
I
Gerson
Stanton L
SL
de Verneuil
Hubert
H
Moreau-Gaudry
François
F
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
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