François Moreau-Gaudry

Publications of François Moreau-Gaudry

• Neonatal bone marrow transplantation prevents liver disease in a murine model of erythropoietic protoporphyria.

  Authors: Yann Duchartre, Nicolas Petit, Corrine Moya, Magalie Lalanne, Pierre Dubus, Hubert de Verneuil, François Moreau-Gaudry, Emmanuel Richard

  Journal of hepatology. 10/2010; 55(1):162-70.

  Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin IX (PPIX) accumulation in erythrocytes,Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin IX (PPIX) accumulation in erythrocytes, responsible for skin photosensitivity. In some EPP patients, the development of cholestatic liver injury due to PPIX accumulation can lead to hepatic failure. In adult EPP mice, bone marrow transplantation (BMT) leads to skin photosensitivity correction but fails to reverse liver damages, probably because of the irreversible nature of liver fibrosis. Our aim was to determine the time course of liver disease progression in EPP mice and to evaluate the protective effect of BMT into neonates. We studied the development of liver disease from birth in EPP mice, in relation with erythroid and hepatic PPIX accumulation. To prevent the development of liver disease, BMT was performed into newborn mice using a novel busulfan-mediated preconditioning assay. We showed that hepatic PPIX accumulates during the first 2 weeks and correlates with the onset of a progressive liver fibrosis in 12-day-old EPP mice. Transplantation of normal congenic hematopoietic stem cells into EPP neonates led to long-term donor hematopoiesis recovery. A full correction of erythroid PPIX accumulation and skin photosensitivity was obtained. Furthermore, five months after neonatal BMT, liver damage was almost completely prevented. We demonstrated for the first time that BMT could be successfully used to prevent liver disease in EPP mice and suggested that BMT would be an attractive therapeutic option to prevent severe liver dysfunction in EPP patients.

• Modeling of congenital erythropoietic porphyria by RNA interference: a new tool for preclinical gene therapy evaluation.

  Authors: Elodie Robert-Richard, Magalie Lalanne, Isabelle Lamrissi-Garcia, Véronique Guyonnet-Duperat, Emmanuel Richard, Vincent Pitard, Fréderic Mazurier, François Moreau-Gaudry, Cécile Ged, Hubert de Verneuil

  The journal of gene medicine. 08/2010; 12(8):637-46.

  Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosyntheticCongenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector-mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS-deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients. RNA interference (RNAi) has been used to develop a new human model of the disease from normal cord blood HSCs. Lentivectors were developed for this purpose. We were able to down-regulate the level of human UROS in human cell lines and primary hematopoietic cells. A 97% reduction of UROS activity led to spontaneous uroporphyrin accumulation in human erythroid bone marrow cells of transplanted immune-deficient mice, recapitulating the phenotype of cells derived from patients. A strong RNAi-induced UROS inhibition allowed us to test the efficiency of different lentiviral vectors with the aim of selecting a safer vector. Restoration of UROS activity in these small hairpin RNA-transduced CD34(+) cord blood cells by therapeutic lentivectors led to a partial correction of the phenotype in vivo. The RNAi strategy is an interesting new tool for preclinical gene therapy evaluation.

• Analysis of post-transcriptional regulations by a functional, integrated and quantitative method.

  Authors: Benoît Laloo, Delphine Simon, Véronique Veillat, Dorine Lauzel, Véronique Guyonnet-Duperat, François Moreau-Gaudry, Francis Sagliocco, Christophe Grosset

  Molecular & cellular proteomics : MCP. 06/2009;

  In the past ten years, transcriptome and proteome analyses have provided valuable data on global gene expression and cell functional networks. However, when integrated, these analyses revealedIn the past ten years, transcriptome and proteome analyses have provided valuable data on global gene expression and cell functional networks. However, when integrated, these analyses revealed partial correlations between mRNA expression levels and protein abundance thus suggesting that post-transcriptional regulations may be in part responsible for this discrepancy. In the present work, we report the development of a functional, integrated and quantitative method to measure post-transcriptional regulations that we named FunREG. This method enables (i) quantitative measure of post-transcriptional regulations mediated by selected 3' untranslated regions and exogenous siRNA or miRNAs, and (ii) comparison of these regulatory processes in physiologically relevant systems (e.g. cancer vs. primary untransformed cells). We applied FunREG to the study of liver cancer and we demonstrate for the first time the differential regulatory mechanisms controlling gene expression at a post-transcriptional level in normal and tumoral hepatic cells. As an example, translation efficiency mediated by Heparin Binding-Epidermal Growth Factor 3'UTR was increased three fold in liver cancer cells compared to normal hepatocytes, whereas stability of an mRNA containing a portion of Cyclin D1 3'UTR was increased more than two fold in HepG2 cells compared to normal hepatocytes. Consequently, we believe that the method presented herein may become an important tool in fundamental and medical research. This approach is convenient and easy to perform, accessible to any investigator and should be adaptable to a large number of cell type, functional and chemical screens as well as genome-scale analyses. Finally, FunREG may represent a helpful tool to reconcile transcriptome and proteome data.

• Erythropoietic porphyrias: animal models and update in gene-based therapies.

  Authors: Emmanuel Richard, Elodie Robert-Richard, Cécile Ged, François Moreau-Gaudry, Hubert de Verneuil

  Current gene therapy. 07/2008; 8(3):176-86.

  The inherited porphyrias are inborn errors of haem biosynthesis, each resulting from the deficient activity of a specific enzyme of the haem biosynthetic pathway. Porphyrias are divided intoThe inherited porphyrias are inborn errors of haem biosynthesis, each resulting from the deficient activity of a specific enzyme of the haem biosynthetic pathway. Porphyrias are divided into erythropoietic and hepatic according to the predominant porphyrin-accumulating tissue. Three different erythropoietic porphyrias (EP) have been described: erythropoietic protoporphyria (EPP, MIM 177000) the most frequent, congenital erythropoietic porphyria (CEP, MIM 263700), and the very rare hepatoerythropoietic porphyria (HEP, MIM 176100). Bone marrow transplantation is considered as the only curative treatment for severe cases of erythropoietic porphyria (especially CEP), if donors are available. Some EPP patients who undergo liver failure may require hepatic transplantation. Murine models of EPP and CEP have been developed and mimic most of the human disease features. These models allow a better understanding of the pathophysiological mechanisms involved in EP as well as the development of new therapeutic strategies. The restoration of deficient enzymatic activity in the bone marrow compartment following gene therapy has been extensively studied. Murine oncoretroviral, and recently, lentiviral vectors have been successfully used to transduce hematopoietic stem cells, allowing full metabolic and phenotypic correction of both EPP and CEP mice. In CEP, a selective survival advantage of corrected cells was demonstrated in mice, reinforcing the arguments for a gene therapy approach in the human disease. These successful results form the basis for gene therapy clinical trials in severe forms of erythropoietic porphyrias.

• Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells.

  Authors: Elodie Robert-Richard, François Moreau-Gaudry, Magalie Lalanne, Isabelle Lamrissi-Garcia, Muriel Cario-André, Véronique Guyonnet-Dupérat, Laurence Taine, Cécile Ged, Hubert de Verneuil

  American journal of human genetics. 02/2008; 82(1):113-24.

  Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severeAchieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut248) mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.

• Imatinib and nilotinib induce apoptosis of chronic myeloid leukemia cells through a Bim-dependant pathway modulated by cytokines.

  Authors: Francis Belloc, François Moreau-Gaudry, Maialen Uhalde, Laurie Cazalis, Marie Jeanneteau, Francis Lacombe, Vincent Praloran, François-Xavier Mahon

  Cancer biology & therapy. 07/2007; 6(6):912-9.

  It is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated byIt is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated by extracellular factors. Apoptosis induced by imatinib and nilotinib was determined in BCR-ABL expressing cell lines and primary CML CD34+ cells. Both molecules induced apoptosis of BCR-ABL expressing cells. This apoptosis was inhibited by protein synthesis inhibition in both K562 and CML CD34+ cells. In K562, 80% inhibition of the BCR-ABL auto-phosphorylation by either imatinib or nilotinib induced a two fold increase in Bim-EL expression and induction of apoptosis in 48 h. Bim accumulation preceded apoptosis induction which was completely abolished by depletion in Bim using shRNA. However, the anti-proliferative effect of imatinib was preserved in Bim-depleted cells. When K562 cells were cultured in a cytokine containing medium, the pro-apoptotic effect of nilotinib was decreased by 68% and this was related to a decrease in Bim-EL dephosphorylation and accumulation. Similarly, the presence of a combination of cytokines inhibited 88% of NIL- and 39% of IMA-induced apoptosis in primary CML CD34+ cells. In conclusion, both nilotinib and imatinib induce apoptosis through Bim accumulation independently of cell cycle arrest. However, the pro-apoptotic effect of both molecules can be attenuated by the presence of cytokines and growth factors, particularly concerning nilotinib. Thus BCR-ABL inhibition restores the cytokine dependence but is not sufficient to induce apoptosis when other signaling pathways are activated.

• Proteasome inhibition specifically sensitizes leukemic cells to anthracyclin-induced apoptosis through the accumulation of Bim and Bax pro-apoptotic proteins.

  Authors: Arnaud Pigneux, François-Xavier Mahon, François Moreau-Gaudry, Maialene Uhalde, Hubert de Verneuil, Francis Lacombe, Josy Reiffers, Noel Milpied, Vincent Praloran, Francis Belloc

  Cancer biology & therapy. 05/2007; 6(4):603-11.

  Proteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicinProteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicin (IDA) alone or together with a low concentration of MG132 or bortezomib. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than the additive effect of both drugs administered separately (p < 0.01). Isobologram analysis showed that both MG132 and bortezomib interacted synergistically with IDA to induce apoptosis of THP1 cells. Western blot analysis of Bax and Bim show an acumulation of these pro-apoptotic proteins in THP1 treated cells. This increase in Bim preceded the induction of apoptosis and participated in idarubicin-induced apoptosis. Proteasome inhibition also potentiated IDA-induced apoptosis in primary blast cells from 22 AML patients while no such effect was found on normal lymphocytes, PHA-stimulated lymphocytes, normal cord blood CD34+ cells or bone marrow normal myeloid cells. These data show that MG132 and bortezomib specifically sensitize leukemic cells to IDA through an increase in BIM and Bax pro-apoptotic Bcl-2 family proteins.

• Murine retroviral but not human cellular promoters induce in vivo erythroid-specific deregulation that can be partially prevented by insulators.

  Authors: Elodie Robert-Richard, Emmanuel Richard, Punam Malik, Cécile Ged, Hubert de Verneuil, François Moreau-Gaudry

  Molecular therapy : the journal of the American Society of Gene Therapy. 02/2007; 15(1):173-82.

  We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT)We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT) selective gene in a constitutive way. We report that transduction of murine hematopoietic stem cells (HSCs) with a human phosphoglycerate kinase promoter-based vector at low multiplicity of infection (MOI) does not result in a selective in vivo expansion in the presence of alkylating agents. In contrast, by replacing this cellular promoter with the powerful retroviral-derived myeloproliferative sarcoma virus enhancer, negative control region-deleted, dl587rev primer-binding site substituted promoter, the vector allowed efficient chemoprotection of transduced HSCs at low MOI. However, this promoter interacted with the erythroid HS40/ankyrin enhancer/promoter driving green fluorescent protein, leading to an unexpected loss of erythroid specificity. A partial restoration of tissue-specific expression was obtained by interposition of insulator sequences between the expression units. Alternatively, we found that the strong human cellular elongation factor1-alpha promoter allows similar chemoprotection but without any deregulation of the erythroid-specific promoter in the absence of insulators. These data demonstrate that the level of in vivo deregulation induced by a promoter is not correlated with its transcriptional activity.

• 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

  The journal of gene medicine. 10/2003; 5(9):737-47.

  BACKGROUND: Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to theBACKGROUND: 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.

• A SIN lentiviral vector containing PIGA cDNA allows long-term phenotypic correction of CD34+-derived cells from patients with paroxysmal nocturnal hemoglobinuria.

  Authors: David Robert, François-Xavier Mahon, Emmanuel Richard, Gabriel Etienne, Hubert de Verneuil, François Moreau-Gaudry

  Molecular therapy : the journal of the American Society of Gene Therapy. 04/2003; 7(3):304-16.

  Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell (HSC) disorder in which an acquired somatic mutation of the X-linked PIGA gene results in a deficiency in GPI-anchored surfaceParoxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell (HSC) disorder in which an acquired somatic mutation of the X-linked PIGA gene results in a deficiency in GPI-anchored surface proteins. Clinically, PNH is dominated by a chronic hemolytic anemia, often associated with recurrent nocturnal exacerbations, neutropenia, thrombocytopenia, and thrombotic tendency. Allogenic bone marrow transplantation is the only potentially curative treatment for severe forms of PNH but is associated with a high treatment-related morbidity and mortality. HSC gene therapy could provide a new therapeutic option, especially when an HLA-matched donor is not available. To develop an efficient gene transfer approach, we have designed a new SIN lentiviral vector (TEPW) that contains the PIGA cDNA driven by the human elongation factor 1 alpha promoter, the central DNA flap of HIV-1, and the WPRE cassette. TEPW transduction led to a complete surface expression of the GPI anchor and CD59 in PIGA-deficient cell lines without any selection procedure. Moreover, efficient gene transfer was achieved in bone marrow and mobilized peripheral blood CD34(+) cells derived from two patients with severe PNH disease. This expression was stable during erythroid, myeloid, and megakaryocytic liquid culture differentiation. CD59 surface cell expression was fully restored during 5 weeks of long-term culture.

• MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models.

  Authors: François-Xavier Mahon, Francis Belloc, Valérie Lagarde, Claudine Chollet, François Moreau-Gaudry, Josy Reiffers, John M Goldman, Junia V Melo

  Blood. 04/2003; 101(6):2368-73.

  Inappropriate expression of the multidrug resistance (MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previouslyInappropriate expression of the multidrug resistance (MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp. Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1 gene overexpression, grew continuously in the presence of 1 microM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture. Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC(50)) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 microM for K562/DOX, K562/DOX plus verapamil, and K562, respectively. Retroviral-mediated transfection of the BCR-ABL(+) AR230 cell line with the MDR1 gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil. The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined. We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon.

• Highly efficient lentiviral gene transfer in CD34+ and CD34+/38-/lin- cells from mobilized peripheral blood after cytokine prestimulation.

  Authors: Fabien Géronimi, Emmanuel Richard, Isabelle Redonnet-Vernhet, Isabelle Lamrissi-Garcia, Magalie Lalanne, Cécile Ged, François Moreau-Gaudry, Hubert de Verneuil

  Stem cells (Dayton, Ohio). 01/2003; 21(4):472-80.

  Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34(+) cells and the stem/progenitor-cell-enrichedBecause mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34(+) cells and the stem/progenitor-cell-enriched CD34(+)/38(-)/lin(-) cell subset isolated from mPB. In this study, we used an optimized third-generation self-inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor lalpha promoter. This lentivector was first used to compare multiplicity of infection (MOI)-dependent gene transfer efficiency in cord blood (CB) versus mPB CD34(+)-derived cells, colony-forming cells (CFCs), and long-term culture-initiating cells (LTC-ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans-duction. Effects of a cytokine prestimulation period (18 hours) with interleukin-3, stem cell factor, Flt-3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC-ICs derived from mPB CD34(+) cells. Transduction levels in those conditions demonstrated a two- and fourfold increase in CFCs and LTC-ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34(+)/38(-)/lin(-) cells isolated from mPB, with up to >85% of colonies derived from LTC-ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.

• [Successful gene therapy of mice with congenital erythropoietic porphyria.]

  Authors: Hubert de Verneuil, Elodie Robert-Richard, Cécile Ged, Frédéric Mazurier, Emmanuel Richard, François Moreau-Gaudry

  Médecine sciences : M/S. 24(6-7):615-20.

  Porphyrias are a group of disorders due to a genetic deficiency in one of the heme biosynthetic pathway enzymes. Congenital erythropoietic porphyria (CEP) is the most severe type characterized by aPorphyrias are a group of disorders due to a genetic deficiency in one of the heme biosynthetic pathway enzymes. Congenital erythropoietic porphyria (CEP) is the most severe type characterized by a deficiency in uroporphyrinogen III synthase (UROS) activity. Bone marrow transplantation represents a curative treatment for patients, as long as human leucocyte antigen-compatible donor is available. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Urosmut 248 mice resulted in a complete and long-term enzymatic, metabolic and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate for the first time that the cure of this mouse model of CEP at moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified HSCs. double dagger.