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Publications (15)36.05 Total impact

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    ABSTRACT: Retroviral vectors are widely used tools for gene therapy. However, in vivo gene transfer is only effective in dividing cells, which, in liver, requires a regenerative stimulus. Follistatin is effective in promoting liver regeneration after 90% and 70% hepatectomy in rats. We studied its efficacy on liver regeneration and retroviral-mediated gene delivery in 50% hepatectomized rats. When human recombinant follistatin was infused into the portal vein immediately after 50% hepatectomy, hepatocyte proliferation was significantly higher than in control 50% hepatectomized rats. A single injection of virus particles administered 23 h after follistatin infusion resulted in more than 20% gene transduction efficiency in hepatocytes compared to 3% in control rats. It is concluded that a single injection of follistatin induces onset of proliferation in 50% hepatectomized rats and allows efficient retroviral-mediated gene transfer to the liver.
    Biochemical and Biophysical Research Communications 04/2005; 328(4):937-43. · 2.28 Impact Factor
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    ABSTRACT: Because intrauterine transplantation of fetal hepatocytes could become an effective approach for treating severe genetic disorders of the liver, the objective of this study was to demonstrate the feasibility of in utero allotransplantation of fetal hepatocytes in a nonhuman primate model using direct intraparenchymal administration of donor cells. Fetal primary hepatocytes were isolated from 3 fetal primates (MACACA MULATTA) at 89-120 days of gestation, and cryopreserved. When a recipient was available, the cells were thawed and transduced by a beta-galactosidase-expressing retrovirus (3 cases) or labelled with a fluorescent dye (4 cases). Hepatocytes were infused directly into the fetal liver under surgical visual control. Engraftment was assessed by surgical liver biopsies taken 8-60 days following transplantation. Six recipients survived until liver biopsy, and 1 died during the surgical procedure. There was no evidence of engraftment in the 3 fetuses that received genetically marked hepatocytes. All 3 monkeys who received 20-25 x 10(6) hepatocytes from an 89-day-old donor labelled with fluorescent dye had positive liver biopsies 8-11 days following intrauterine transplantation. In utero allotransplantation of fetal hepatocytes is feasible in the nonhuman primate, and direct intraparenchymal administration enables short-term detection of persisting donor hepatocytes.
    Fetal Diagnosis and Therapy 01/2004; 19(1):92-9. · 1.90 Impact Factor
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    ABSTRACT: Engineered retroviruses are widely used vectors for cancer gene therapy approaches. However, the ability to target cells of therapeutic interest while controlling the expression of the transferred genes would improve both the efficiency and the safety of viral vectors. In this study, we investigated the ability of a retroviral amphotropic envelope displaying single-chain variable-fragment (scFv) directed against the c-Met receptor, to target the entry of recombinant retroviruses to human hepatocarcinoma cells. Four single-chain antibody fragments directed against the c-Met receptor were generated and inserted into the viral envelope protein as an N-terminal fusion. The modified envelopes were incorporated into virus particles and one of the chimeric viruses, 3D6-Env, transduced preferentially human hepatoma cells rather than proliferating human hepatocytes. In another construct, the urokinase cleavage site was inserted between the scFv moiety and the envelope. Chimeric scFv-urokinase-Env viruses transduced hepatoma cells with a similar efficiency to that of the control virus and their infectivity in human hepatocytes remained low. These results indicate that amphotropic retroviruses with engineered envelopes to display scFv directed against the c-Met receptor can efficiently and selectively deliver genes into hepatoma cells.
    Cancer Gene Therapy 12/2003; 10(11):840-9. · 2.95 Impact Factor
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    ABSTRACT: Liver regeneration after partial hepatectomy results primarily from the simple division of mature hepatocytes. However, during embryonic and fetal development or in circumstances under which postnatal hepatocytes are injured, organ regeneration is believed to occur from a compartment of epithelial liver stem or progenitor cells with biliary and hepatocytic bipotentiality. The ability to identify, isolate, and transplant epithelial liver stem cells from fetal liver would greatly facilitate the treatment of hepatic diseases currently requiring orthotopic liver transplantation. Here we report the identification and immortalization by retrovirus-mediated transfer of the simian virus 40 large T antigen gene of primate fetal epithelial liver cells with a dual hepatocytic biliary phenotype. These cells grow indefinitely in vitro and express the liver epithelial cell markers cytokeratins 8/18, the hepatocyte-specific markers albumin and alpha-fetoprotein, and the biliary-specific markers cytokeratins 7 and 19. Bipotentiality of gene expression was confirmed by clonal analysis initiated from single cells. Endogenous telomerase also is expressed constitutively. After orthotopic transplantation via the portal vein, approximately 50% of the injected cells integrated into the liver parenchyma of athymic mice without tumorigenicity. Three weeks after transplantation, cells having seeded in the liver parenchyma expressed both albumin and alpha-fetoprotein but had lost expression of cytokeratin 19. These results provide strong evidence for the existence of a bipotent epithelial liver stem cell in nonhuman primates. This unlimited source of donor cells also should enable the establishment of a model of allogenic liver cell transplantation in a large animal closely related to humans and shed light on important questions related to liver organogenesis and differentiation.
    Proceedings of the National Academy of Sciences 04/2002; 99(6):3639-44. · 9.81 Impact Factor
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    ABSTRACT: The transplantation of isolated hepatocytes in large animals, including nonhuman primates, must be evaluated before clinical trials are performed. However, in the absence of large transgenic animals and large-animal (as opposed to small-animal) models of genetic deficiencies, it is difficult to evaluate the fate of transplanted hepatocytes, their localization, survival, and function within the parenchyma of the host liver. In this work, we aimed to develop a technique for delivering hepatocytes to the liver of a nonhuman primate and to evaluate their localization and functionality in the short term. A 20% hepatectomy was performed in 34 cynomolgus monkeys (Macaca fascicularis) and hepatocytes were isolated. Hepatocytes were labeled in vitro with a recombinant retrovirus expressing the beta-galactosidase gene and returned to the liver by infusion through a portal catheter left in place. Liver biopsies were performed 4 and 7 d after transplantation. Twenty-four monkeys underwent surgery to define the necessary technical adjustments and to optimize conditions. Six monkeys died. The whole protocol, including the transplantation of genetically marked hepatocytes and procurement of liver biopsies, was performed in the remaining 10 monkeys. In eight monkeys, transplanted hepatocytes expressing the beta-galactosidase gene were widely distributed in the portal tracts, sinusoids, and hepatocyte plates of the host liver 4 and 7 d after transplantation. We have developed an experimental nonhuman primate model for the evaluation of hepatocyte transplantation. We demonstrated the engraftment and functioning of transplanted hepatocytes in the host liver 4 and 7 d after transplantation.
    Transplantation 10/2001; 72(5):811-8. · 3.78 Impact Factor
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    ABSTRACT: The main impediment to effective ex vivo liver gene therapy of metabolic diseases is the lack of experimental work on large animals to resolve such important issues as effective gene delivery, cell-processing techniques, and the development of appropriate vectors. We have used a nonhuman primate, as a preclinical model, to analyze the limiting steps of this approach using recombinant retroviruses. Seven monkeys (Macaca fascicularis) underwent the complete protocol: their left liver lobe was resected, a catheter was placed in the inferior mesenteric vein and connected to an infusion chamber, and the hepatocytes were isolated, cultured, and transduced with a retroviral vector containing the beta-galactosidase gene. The hepatocytes were harvested and returned to the host via the infusion chamber. Biopsies were taken 4-40 days later. No animal was killed in the course of the experiments. They all tolerated the procedure well. We have developed and defined conditions that permit the proliferation and transduction of up to 90% of the plated hepatocytes. A significant proportion of genetically modified cells, representing up to 3% of the liver mass, were safely delivered to the liver via the chamber. Polymerase chain reaction analysis detected integrated viral DNA sequences and quantitative analysis of the in situ beta-Gal-expressing hepatocytes indicated that a significant amount of transduced hepatocytes, up to 2%, had become integrated into the liver and were functional. These results represent substantial advances in the development of the ex vivo approach and suggest that this approach is of clinical relevance for liver-directed gene therapy.
    Human Gene Therapy 02/2001; 12(2):169-79. · 4.02 Impact Factor
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    ABSTRACT: We are developing cell therapy approaches on non-human primates as a preclinical model for the treatment of hepatic metabolic diseases. In foetuses, the tissues, including liver, are in expansion, which should facilitate hepatocytes engraftment, and the immune system becomes fully mature only after birth. We have set out conditions for isolation of fetal hepatocytes from macaca mulatta at the end of the 2nd trimester of gestation (90-100 days), their cryopreservation and retroviral transduction. Two different routes of administration of hepatocytes were evaluated: the umbilical vein which was deleterious for the foetuses, and the intraparenchymatous injection which was well tolerated by the animals. Administration of hepatocytes into the hepatic parenchyma resulted in microchimerism and allogenic cells were visualized 9 days after transplantation. Another approach has been to immortalize simian foetal hepatocytes using a retroviral vector expressing SV40 Large T flanked by lox sites. A cell line has been established for 2 years, which is not tumorigenic when injected subcutaneously into nude mice and display characteristics of bipotent hepatoblasts, precursors of hepatocytes and biliary cells. After orthotopic transplantation into nude mice via the portal vein, these cells expressed albumin until the sacrifice of the animals (17 days). The next steps will be to define conditions for transplantation of retrovirally transduced fetal primary and/or immortalized hepatocytes into young foetuses (60 days of gestation) and post-natally.
    Journal de la Société de Biologie 02/2001; 195(1):57-63.
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    ABSTRACT: The aim of this study was to assess liver regeneration after partial portal ligation. 70% partial portal occlusion was obtained by ligation of the left portal vein branch. Total liver weight ratio were measured 96 hours after partial portal occlusion and in sham operated animals. The kinetics of hepatocytes division was evaluated by measuring the incorporation of 5-bromo-21-deoxyuridine into replicating cells at various time points by immunohistochemistry. Partial portal occlusion did not alter the total liver weight 96 hours after surgery. It resulted in atrophy of the ligated lobes and hypertrophy of the lobes with preserved portal flow. Hypertrophy was associated to an increase of the percentage of replicating hepatocytes. The replication rate was maximum at 28 hours with a peak at 12.5% and was prolonged beyond the 48th hour. Partial portal occlusion results in major and prolonged regeneration process in the liver lobes with preserved portal flow.
    Annales de Chirurgie 03/2000; 125(2):144-8. · 0.35 Impact Factor
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    ABSTRACT: Effect of portal branch ligation on rat liver regeneration.Goal: The aim of this study was to assess liver regeneration after partial portal ligation.Methods: 70 % partial portal occlusion was obtained by ligation of the left portal vein branch. Total liver weight ratio were measured 96 hours after partial portal occlusion and in sham operated animals. The kinetics of hepatocytes division was evaluated by measuring the incorporation of 5-bromo-21-deoxyuridine into replicating cells at various time points by immunohistochemistry.Results: Partial portal occlusion did not alter the total liver weight 96 hours after surgery. It resulted in atrophy of the ligated lobes and hypertrophy of the lobes with preserved portal flow. Hypertrophy was associated to an increase of the percentage of replicating hepatocytes. The replication rate was maximun at 28 hours with a peak at 12.5 % and was prolonged beyond the 48th hour.Conclusions: Partial portal occlusion results in major and prolonged regeneration process in the liver lobes with preserved portal flow.
    Annales de Chirurgie 02/2000; 125(2):144-148. · 0.35 Impact Factor
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    ABSTRACT: Effect of portal branch ligation on rat liver regeneration.Goal: The aim of this study was to assess liver regeneration after partial portal ligation.Methods: 70 % partial portal occlusion was obtained by ligation of the left portal vein branch. Total liver weight ratio were measured 96 hours after partial portal occlusion and in sham operated animals. The kinetics of hepatocytes division was evaluated by measuring the incorporation of 5-bromo-21-deoxyuridine into replicating cells at various time points by immunohistochemistry.Results: Partial portal occlusion did not alter the total liver weight 96 hours after surgery. It resulted in atrophy of the ligated lobes and hypertrophy of the lobes with preserved portal flow. Hypertrophy was associated to an increase of the percentage of replicating hepatocytes. The replication rate was maximun at 28 hours with a peak at 12.5 % and was prolonged beyond the 48th hour.Conclusions: Partial portal occlusion results in major and prolonged regeneration process in the liver lobes with preserved portal flow.
    Annales De Chirurgie - ANN CHIR. 01/2000; 125(2):144-148.
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    ABSTRACT: In utero allotransplantation of fetal hepatocytes into a preimmune fetus could be used in early treatment of many inherited hepatic metabolic diseases. This study was designed to assess the tolerance to hepatocyte transplantation and to test the feasability and toxicity of such an injection in a primate model. Fetal hepatocytes were obtained from two 120-day-old Macaca mulatta fetuses and cryopreserved. They were thawed, cultured in vitro, and transduced with a recombinant retrovirus expressing beta-galactosidase. Transduction efficiency was 75-85%. Three unrelated fetuses (90, 100, and 104 days old) were each given 1-2 x 10(7) transduced cells via the umbilical vein. This caused vasospasm and severe bradycardia. Two fetuses died in the 48 hours after transplantation; the third survived and was killed at the end of gestation. No evidence of the infused cells was found. Three fetuses (90 days old) were, therefore, given 3-4 10(7) hepatocytes by direct intrahepatic injection. All the fetuses survived without side effect. Donor cells were not apparent from histochemical staining and PCR reactions. There was no evidence of inflammatory reaction. These findings indicate that the protocole could be improved by increasing the number of transplanted cells and using specific hepatic promoters in the retroviral vectors to achieve an effective postnatal chimerism.
    The Journal of Maternal-Fetal Medicine 01/1998; 7(6):296-303.
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    ABSTRACT: Fetal hepatocytes are an attractive target for in utero cellular transplantation. Their use could provide a very efficient way for implanting normal or transduced cells into the livers of affected fetuses. Marking cells with recombinant retroviruses is a powerful tool for evaluating the chimerism of grafted animals. The technique relies on the ex vivo transduction efficiency of the engrafted cells. We have isolated fetal primary hepatocytes from nonhuman primates. The cells were cultured and transduced with a retroviral vector carrying the Escherichia coli beta-galactosidase gene. Optimal gene transfer efficiency was obtained 48-60 hr after plating and was as high as 90%. Cryopreservation had little effect on cell viability and infectivity: The viability of thawed hepatocytes remained high (75-85%) and the infection efficiency was identical to that of freshly isolated cells. Efficient ex vivo retroviral gene transfer into fetal hepatocytes provides an appropriate system for testing allogenic grafting and for modifying immunogenicity of engrafted cells. These results open up new perspectives for cell transplantation through cell banking.
    Human Gene Therapy 03/1997; 8(3):267-74. · 4.02 Impact Factor
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    ABSTRACT: Familial hypercholesterolemia (FH) is an inherited disease caused by a defect in the gene encoding the Low Density Lipoprotein receptor (LDL-R). The ex vivo hepatic gene therapy which restore the expression of the normal protein in hepatocytes should correct the disease. Improved transduction efficiency and long lasting expression of the transduced gene remain the main goals of gene therapy research. We developed an efficient and reliable method for in vivo transduction of human, mouse and primate primary hepatocytes. A retroviral vector bearing the LDL-R cDNA driven by the liver-type pyruvate kinase promoter allows high and tissue specific expression of the gene in primary hepatocytes. A second vector with a housekeeping promoter corrects the LDL-R deficiency in fibroblasts from a FH patient. Ex vivo preclinical studies in non-human primates will provide new insight in transduced cells biology after reimplantation.
    Comptes rendus des séances de la Société de biologie et de ses filiales 02/1996; 190(1):53-65.
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    ABSTRACT: Hepatocyte Growth Factor (HGF) is the more potent mitogen of mature hepatocytes. We have examined the effect of human HGF expression by a recombinant retroviral cell line (MFG-LacZ) on retroviral transduction of primary mouse and human hepatocytes. The HGF in the supernatant of MFG-LacZ cell line was correctly processed and biologically active. Transduction of mouse and human hepatocytes with the supernatant of transfected cells was increased 5-fold, as determined by β-galactosidase activity. The production of HGF was stable and did not interfere with the viral titers of the producer cells. This study provides evidence that expression of HGF within a retrovirus-producer cell line increases the transduction rate of primary hepatocytes. Since the number of corrected cells is a limiting step for phenotypic correction of liver deficiencies, our approach should improve hepatic gene therapy efficiency. Furthermore this cell line should be useful forin vivoliver gene therapy.
    Biochemical and Biophysical Research Communications 01/1996; 222(3):726-731. · 2.28 Impact Factor
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    ABSTRACT: The critical physiological function of the long terminal repeat in Moloney murine leukemia virus (MoMLV) gene expression has been thoroughly explored and shown to include binding sites for ubiquitous and tissue-specific transcription factors, such as the glucocorticoid responsive element (GRE) and the LVb sequence recognized by phorbol 12-myristate 13-acetate (TPA)-induced factors. The present study was undertaken to determine the effect of different activators, known to enhance expression of MoMLV, on their ability to modulate retroviral transcripts in psi CRIP producing cell lines. Improvement of recombinant retrovirus production by two psi CRIP producer cells was tested by using dexamethasone, TPA and sodium butyrate (Na-But) alone or in combination. We demonstrate that 5 mM Na-But, or 5 mM Na-But plus 1 microM dexamethasone significantly enhanced viral production. These compounds could induce a 10-fold increase in viral production. There was also a good correlation between the increased viral production and the titer measured after transduction of NIH 3T3. This improvement is of general interest, since a major goal for gene therapy is the production of high retroviral titer for increased transduction efficiency.
    Gene Therapy 11/1995; 2(8):547-51. · 4.32 Impact Factor