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.

Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells.

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fech(m1Pas)/Fech(m1Pas) murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Acerca de un caso de Agaricus muscarius / About a case of Agaricus muscarius

Caso

Ferrochelatase cDNA delivered by adenoviral vector corrects biochemical defect in protoporphyric cells.

Protoporphyria is generally an autosomal dominant disease characterized genetically by mutations in the ferrochelatase gene. The interaction between the wild-type and mutant ferrochelatase protein is unknown. The aim of this study was to evaluate the ability to correct the enzymatic and biochemical defects in cells from patients with protoporphyria, using a replication-defective human adenovirus for gene transfer. Overexpression of ferrochelatase was accomplished by construction of a vector in which expression of the wild-type ferrochelatase cDNA was driven by the constitutive cytomegalovirus (CMV) promoter, introduction and packaging of the cDNA into human adenovirus dl309, and transduction of normal and protoporphyric fibroblasts. Fibroblasts from controls and patients were infected with the ferrochelatase adenovirus or a control adenovirus and assayed for ferrochelatase activity and the accumulation of protoporphyrin upon challenge with the precursor delta-aminolevulinic acid (ALA). At a multiplicity of infection (moi) of 10, greater than 85% of both the wild-type and protoporphyric fibroblasts were infected. The recombinant adenovirus increased the ferrochelatase protein content and activity in the wild-type and protoporphyric fibroblasts with equal efficiency. Therefore, the presence of the mutant ferrochelatase protein did not inhibit the ferrochelatase activity expressed by the transgene.