- Email: [email protected]
Iron and insulin resistance: Effect of iron manipulation on insulin receptor in HEPG2 hepatocytes
30
Tuesday, April 1, 2003
Parallel Session 9: Experimental
I83
ACTIVATION CAUSES
OF PPAR ALPHA-DEPENDENT
RAPID REGRESSION
STEATOHEPATITIS
Hepatology
2
PATHWAYS
OF FIBROSING
IN MCD DIET-FED
MICE
l? Ha112, R. Kirsch2, I. Leclercq’. G. Farrell’, G. Robertson’, ‘Starr Liver Unit, Westmead Millennium Institute, University Of Sydney, Sydney, NSK Australia; 2Department Of Anatomical Pathology, Faculty Of Health Sciences, University Of Cape Town, Cape Town, South Africa
E.Ip’,
Pathogenesis of steatohepatitis involves interplay between accumulated hepatic lipid and oxidative stress that generates pro-inflammatory lipoperoxides. We have shown that activation of PPARalpha-dependent pathways clears hepatic lipid, reduces oxidative stress and prevents development of steatohepatitis induced by a methionine and choline deficient (MCD). We have now tested the hypothesis that PPARalpha activation could ameliorate established steatohepatitis. Male C57BL6 mice were fed MCD diet for 8 weeks. During the last 5 days, Wy-14, 643 (0.1% w/w), a potent PPARalpha agonist, was added to the diet; another group (controls) were maintained on the MCD diet. Liver sections were scored for steatosis and necroinflammation; Sirius red was used to stain collagen and PPARalpha-responsive gene expression was determined (northern blots or RNase protection assay). In MCD-fed mice, ALT levels were elevated and livers exhibited widespread macrovesicular steatosis, necroinflammation and extensive pericellular fibrosis. In mice given Wy-14,643 for 5 days, ALT levels were lower and livers showed minimal inflammation and less steatosis. Consistent with reduced steatosis, Wy-14, 643 increased expression of genes involved with fatty acid turnover. Wy-14, 643 treatment produced impressive regression of hepatic fibrosis, demonstrated by unweighting of extracellular collagen networks leaving only scattered fib&. In conclusion, treatment with Wy-14, 643 reverses established fibrosing steatohepatitis in mice. This PPARalpha agonist rapidly depletes the liver of lipid, decreases inflammation and appears to promote collagen degradation caused by the MCD diet.
I
84
PEROXISOME
PROLIFERATOR
(PPAR-ALPHA)
EXPRESSION
ENZYME
RESPONSE
ACTIVATED IS INDUCED
TO CLOFIBRATE
RECEPTOR-ALPHA BUT PEROXISOMAL
IS BLUNTED
IN A RAT
MODEL OF FAlTY LIVER
F. Akbiyik’, K. Cinar2, T. Ozsullu’, E. Demirpence’, R. Tunca3, R. Haziroglu3, 0. 0nder4, S. Civris4, C. Yurdaydin2, 0. Uzunalimoglu2, H. Bozkaya2. ‘Biochemistry, Hacettepe University, Faculty Of Medicine; 2Gastroenterology, Ankara University; ‘Pathology, Veterinary Faculty Of Ankara University; 41nternal Medicine, Ankara University, Ankara, Turkey Background and Aims: In this study we aimed to asses the PPAR-alpha expression pattern and mitocondrial/peroxisomal enzyme activities in response to clofibrate in a rat model of fatty liver(n). Methods: Four-week-old/male/star-Albino rats(48) were grouped (12 each group) as: 1: normal diet(ND) (6. weeks) (10 kcal% fat). 2: ND(6weeks) +clofibrate(last 2-weeks) (0.25%wt/wt). 3: high fat diet(HFD) (6. weeks) (60 kcal% fat). 4: HFD(6-weeks) +clofibrate(last 2-weeks). After 6 weeks, animals were sacrificed. Peroxisomal-acyl-CoA-oxidase(AOX), mitocondrial-acyl-CoA-dehydrogenase(ACD), catalase activities and malonaldehyde(MDA) and glutathion(GSH) levels were measured in the liver tissues by spectrophotometrically. Liver steatosis was graded by Oil-red staining. PPAR-alpha expression was immunohistochemically determined. Results: All animals fed HFD developed FL while only 2/12 animals fed HFD+clofibrate developed FL. PPAR-alpha expression was increased in
rats fed HID. Clofibrate further increased PPAR-alpha expression (labeling index for l/2/3/4: 2.5/50/10/49, p
I 85
INHIBITION
OF MICROSOMAL
TRIGLYCERIDE
PROTEIN (MTP) AND HEPATIC LIPOPROTEIN ANOTHER
IMPORTANT
MECHANISM
TRANSFER SECRETION:
FOR DRUG-INDUCED
STEATOSIS F! Letteron,
A. Sutton, A. Mansomi, B. Fromenty, U481, Hospital Beaujon, Clichy, France
D. Pessayre.
‘ZNSERM
Although several steatogenic drugs have been shown to inhibit mitochondrial fatty acid beta-oxidation, little is known on their effects on hepatic lipoprotein secretion. MTP lipidates apolipoprotein B (apo B) in the microsomal lumen to form triglyceride (TG) -rich very low density lipoproteins (VLDL), which are secreted, whereas incompletely lipidated apo B is degraded. We studied MTP activity, the lipoproteins present in the microsomal lumen and hepatic lipoprotein secretion 4 hours after a single dose of amineptine (1 mmol/kg), amiodarone (1 mmol/kg), doxycycline (0.25 mmol/kg), tetracycline (0.25 mmol/kg), tianeptine (0.5 mmol/kg) or pirprofen (2 mmol/kg) in mice. These doses acutely inhibit lipid oxidation and were also shown to cause steatosis after repeated doses (doxycycline) or a single dose (other compounds). We found that doxycycline had no effect on MTP or lipoprotein secretion. In contrast, amineptine, amiodarone, pirprofen, tetracycline and tianeptine inhibited MTP in vitro, decreased ex viva MTP activity in the hepatic homogenate (by 46, 48, 24, 70 and 60%), decreased TG in the luminal VLDL fraction of microsomes (by 89, 83, 33, 83 and 59%) and decreased the in viva hepatic secretion of TG (by 57, 51, 24, 36 and 51%) and that of Apo B (by 53, 59, 36, 45 and 85%). These results indicate that several steatogenic drugs inhibit not only mitochondrial beta-oxidation, but also MTP activity, which decreases Apo B lipidation and hepatic lipoprotein secretion. Drugs inhibiting both betaoxidation and MTP may be more steatogenic than drugs acting only on beta-oxidation or only MTl?
I 86
IRON AND INSULIN RESISTANCE: MANIPULATION
EFFECT
ON INSULIN RECEPTOR
OF IRON
IN HEPGS
HEPATOCYTES F! Dongiovanni,
L. Valenti, G. Occhino, A.L. Fracanzani, M. Mattioli, S. Fargion. Dipartimento Di Medicina Interna, Universit De&i Studi, Ospedale Policlinico IRCCS, Milano, Italy
Background: Mild hepatic iron overload is a frequent clinical feature in patients with NAFLD, characterized by hepatic insulin resistance. Iron depletion improves insulin sensitivity in patients with diabetes, but the mechanisms are unclear. Aim: To define the interaction between iron and insulin sensitivity, we determined the effect of iron manipulation on insulin receptor expression in a hepatocyte cell line. Methods: HepG2 cells were cultured in RPM1 complete medium. 1125. insulin binding was evaluated 48 hours after plating in basal condition and in cells treated for 24 hours with iron (FeAmCit/holo-transfenin),
Parallel Session 9: Experimental desfenioxamine and glucose. Insulin receptor expression was evaluated by Western blotting. Results: The number of binding sites at the saturating insulin concentration of 100 nglml was 20000/tell in basal conditions. Iron depletion, obtained with desferrioxamine 100 mM, increased by two-fold (P
I
87
ALCOHOL
AND HEPATITIS C VIRUS CORE PROTEIN
ADDITIVELY
INCREASE
SYNERGISTICALLY EXPRESSION
LIPID PEROXIDATION
TRIGGER
AND
IN A TRANSGENIC
Background & Aims: Alcohol consumption accelerates the appearance of liver fibrosis and hepatocellular carcinoma in patients with chronic hepatitis C virus (HCV) infection, but the mechanisms of these interactions are unknown. HCV core protein and ethanol share common effects on lipid metabolism. Both impair microsomal triglyceride transfer protein and increase lipid peroxidation. We therefore investigated the effects of chronic ethanol consumption in HCV core protein-expressing transgenic mice. Methods & Results: Ethanol was progressively added (up to 20%) to the drinking water that was given ad libidum. In viva fatty acid oxidation (measured by [14C]CO2 exhalation after [U-14Clpalmitic acid administration) was not inhibited by ethanol consumption and/or HCV core expression. Both chronic ethanol consumption and HCV core expression decreased hepatic lipoprotein secretion (measured by triton WR1339 method) and caused steatosis, but had no additive effects on lipoprotein secretion or steatosis. However, chronic ethanol consumption and HCV core protein additively increased lipid peroxidation (measured by ethane exhalation and hepatic TBARS) and acted synergistically to increase the hepatic expression of transforming growth factor-b (TGF-b) and, to a less extent, tumor necrosis factor-a (TNI-a) (RNAse protection assay). Conclusions: HCV core protein expression and chronic alcohol consumption have no effects on in viva fatty acid oxidation and do not additively impair hepatic lipoprotein secretion. However, HCV core and alcohol additively increase hepatic lipid peroxidation and act synergistically to increase hepatic TNI-a and TGF-b expression. These effects may be involved in the activation of fibrogenesis and the development of hepatocellular carcinoma in patients cumulating alcohol abuse and HCV infection.
ISCHEMIC
31
Methods: 60 minutes &hernia was performed in mice of 6 and 60 weeks old. Liver injury was determined by AST, apoptosis by caspase 3 and TUNEL test. Necrosis was investigated 24hr after reperfusion by H&E staining. Finally, ischemic preconditioning was performed in both mice. Results: The 60 weeks old mice had significantly higher AST levels (125OOU/I_ vs 8200 U/L; piO.05) and caspase 3 activity (98 vs 67 AUF/mg p=O.O4) than young mice. In addition, old mice had significantly more TUNEL pos. hepatocytes after reperfusion (55% vs 77%; p
MOUSE MODEL
l? Letteron2, F. Carnot3, F. Zavala4, D. Pessayre2, B. Nalpas’, C. Brechot’. ‘Liver Cancer And Molecular Virology, Institut National De La Sante Et De La Recherche Medicale Unite 370, Faculte De Medecine Necker-Enfants Malades, Paris; 21NSERM U 481, Hopital Beaujon, Clichy; ‘Service D’Anatomopathologie, Hopital Europeen Georges Pompidou, Paris; 41NSERM U 25, Hopital Neckes Paris, France
INCREASED
2
HEPATIC CYTOKINE
G. Perlemuter’,
I 88
Hepatology
INJURY IN THE OLD LIVER. A NOVEL
PATHWAY OF INJURY N. Selzner’, M. Selzner2, P.-A. Clavien2. ‘Department Of Visceral Surgery; 2 Visceral Surgery, University Hospital Of Zurich, Zurich, Switzerland
Due to our aging population, liver surgery is increasingly performed in older patients. However, the effect of age on ischemic injury is unknown.
I
89
RAPID EX VIVO PROTOCOL GENE TRANSFER TRANSDUCTION
FOR EFFICIENT
AND STABLE
IN THE LIVER VIA HEPATOCYTE BY FLAP LENTIVIRAL
VECTORS
C. Giannini”3,
S. Morosan 1,4, .I G. Tralhao’,5, J.E. Guidotti’, S. Battaglia 1,3 , K Mollier2, L. Hannoun6, D. Kremsdorf’, l? Charneau2. ‘INSERM U-370 Pasteur-Necker Institutes, Paris, France; 2Groupe De Virologie Moleculaire Et Vectorologie, Pasteur Institute, Paris, France; ‘Department Of Internal Medicine, University Of Florence, Florence, Italy; 4Faculty Of Veterinary Medicine, University Of Agronomical Science And Veterinary Medicine, Iasi, Romania; ‘Department Of Surgery 3, H. U. Coimbra, Medical Faculty, Coimbra, Portugal; 6Service De Chit-u&e Digestive, Hopital Pitie-Salpetriere, Paris, France Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and may can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture, nor transduced with integrative viral vectors and are very sensitive to trypsine dissociation before their reimplantation into the recipient. In the present study, we demonstrate that primary human and rat hepatocytes can be efficiently transduced with a flap lentiviral vector without the need for to be plating and culture. Efficient transduction of non-adherent primary hepatocytes was achieved with short period of contact with vector particles and without modifying hepatocyte viability. We also show that the presence of the DNA flap in the vector construct was essential to reach high levels of transduction. Transplanted in immunodeficient mice, the genetically modified hepatocytes integrated the mouse liver parenchyma where they stably expressed the GFP transgene for several months. Moreover, lentivirally transduced primary human hepatocytes extensively repopulated uPA/SCID mouse liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, this work opens new perspectives for the applicability of human liver directed ex viva gene therapy. In fact, the use of flap lentiviral vectors permits a high efficiency transduction in a short period of time, still maintaining optimum hepatocyte engrafting and functionality in viva.
Tuesday, April 1, 2003
Parallel Session 9: Experimental
I83
ACTIVATION CAUSES
OF PPAR ALPHA-DEPENDENT
RAPID REGRESSION
STEATOHEPATITIS
Hepatology
2
PATHWAYS
OF FIBROSING
IN MCD DIET-FED
MICE
l? Ha112, R. Kirsch2, I. Leclercq’. G. Farrell’, G. Robertson’, ‘Starr Liver Unit, Westmead Millennium Institute, University Of Sydney, Sydney, NSK Australia; 2Department Of Anatomical Pathology, Faculty Of Health Sciences, University Of Cape Town, Cape Town, South Africa
E.Ip’,
Pathogenesis of steatohepatitis involves interplay between accumulated hepatic lipid and oxidative stress that generates pro-inflammatory lipoperoxides. We have shown that activation of PPARalpha-dependent pathways clears hepatic lipid, reduces oxidative stress and prevents development of steatohepatitis induced by a methionine and choline deficient (MCD). We have now tested the hypothesis that PPARalpha activation could ameliorate established steatohepatitis. Male C57BL6 mice were fed MCD diet for 8 weeks. During the last 5 days, Wy-14, 643 (0.1% w/w), a potent PPARalpha agonist, was added to the diet; another group (controls) were maintained on the MCD diet. Liver sections were scored for steatosis and necroinflammation; Sirius red was used to stain collagen and PPARalpha-responsive gene expression was determined (northern blots or RNase protection assay). In MCD-fed mice, ALT levels were elevated and livers exhibited widespread macrovesicular steatosis, necroinflammation and extensive pericellular fibrosis. In mice given Wy-14,643 for 5 days, ALT levels were lower and livers showed minimal inflammation and less steatosis. Consistent with reduced steatosis, Wy-14, 643 increased expression of genes involved with fatty acid turnover. Wy-14, 643 treatment produced impressive regression of hepatic fibrosis, demonstrated by unweighting of extracellular collagen networks leaving only scattered fib&. In conclusion, treatment with Wy-14, 643 reverses established fibrosing steatohepatitis in mice. This PPARalpha agonist rapidly depletes the liver of lipid, decreases inflammation and appears to promote collagen degradation caused by the MCD diet.
I
84
PEROXISOME
PROLIFERATOR
(PPAR-ALPHA)
EXPRESSION
ENZYME
RESPONSE
ACTIVATED IS INDUCED
TO CLOFIBRATE
RECEPTOR-ALPHA BUT PEROXISOMAL
IS BLUNTED
IN A RAT
MODEL OF FAlTY LIVER
F. Akbiyik’, K. Cinar2, T. Ozsullu’, E. Demirpence’, R. Tunca3, R. Haziroglu3, 0. 0nder4, S. Civris4, C. Yurdaydin2, 0. Uzunalimoglu2, H. Bozkaya2. ‘Biochemistry, Hacettepe University, Faculty Of Medicine; 2Gastroenterology, Ankara University; ‘Pathology, Veterinary Faculty Of Ankara University; 41nternal Medicine, Ankara University, Ankara, Turkey Background and Aims: In this study we aimed to asses the PPAR-alpha expression pattern and mitocondrial/peroxisomal enzyme activities in response to clofibrate in a rat model of fatty liver(n). Methods: Four-week-old/male/star-Albino rats(48) were grouped (12 each group) as: 1: normal diet(ND) (6. weeks) (10 kcal% fat). 2: ND(6weeks) +clofibrate(last 2-weeks) (0.25%wt/wt). 3: high fat diet(HFD) (6. weeks) (60 kcal% fat). 4: HFD(6-weeks) +clofibrate(last 2-weeks). After 6 weeks, animals were sacrificed. Peroxisomal-acyl-CoA-oxidase(AOX), mitocondrial-acyl-CoA-dehydrogenase(ACD), catalase activities and malonaldehyde(MDA) and glutathion(GSH) levels were measured in the liver tissues by spectrophotometrically. Liver steatosis was graded by Oil-red staining. PPAR-alpha expression was immunohistochemically determined. Results: All animals fed HFD developed FL while only 2/12 animals fed HFD+clofibrate developed FL. PPAR-alpha expression was increased in
rats fed HID. Clofibrate further increased PPAR-alpha expression (labeling index for l/2/3/4: 2.5/50/10/49, p
I 85
INHIBITION
OF MICROSOMAL
TRIGLYCERIDE
PROTEIN (MTP) AND HEPATIC LIPOPROTEIN ANOTHER
IMPORTANT
MECHANISM
TRANSFER SECRETION:
FOR DRUG-INDUCED
STEATOSIS F! Letteron,
A. Sutton, A. Mansomi, B. Fromenty, U481, Hospital Beaujon, Clichy, France
D. Pessayre.
‘ZNSERM
Although several steatogenic drugs have been shown to inhibit mitochondrial fatty acid beta-oxidation, little is known on their effects on hepatic lipoprotein secretion. MTP lipidates apolipoprotein B (apo B) in the microsomal lumen to form triglyceride (TG) -rich very low density lipoproteins (VLDL), which are secreted, whereas incompletely lipidated apo B is degraded. We studied MTP activity, the lipoproteins present in the microsomal lumen and hepatic lipoprotein secretion 4 hours after a single dose of amineptine (1 mmol/kg), amiodarone (1 mmol/kg), doxycycline (0.25 mmol/kg), tetracycline (0.25 mmol/kg), tianeptine (0.5 mmol/kg) or pirprofen (2 mmol/kg) in mice. These doses acutely inhibit lipid oxidation and were also shown to cause steatosis after repeated doses (doxycycline) or a single dose (other compounds). We found that doxycycline had no effect on MTP or lipoprotein secretion. In contrast, amineptine, amiodarone, pirprofen, tetracycline and tianeptine inhibited MTP in vitro, decreased ex viva MTP activity in the hepatic homogenate (by 46, 48, 24, 70 and 60%), decreased TG in the luminal VLDL fraction of microsomes (by 89, 83, 33, 83 and 59%) and decreased the in viva hepatic secretion of TG (by 57, 51, 24, 36 and 51%) and that of Apo B (by 53, 59, 36, 45 and 85%). These results indicate that several steatogenic drugs inhibit not only mitochondrial beta-oxidation, but also MTP activity, which decreases Apo B lipidation and hepatic lipoprotein secretion. Drugs inhibiting both betaoxidation and MTP may be more steatogenic than drugs acting only on beta-oxidation or only MTl?
I 86
IRON AND INSULIN RESISTANCE: MANIPULATION
EFFECT
ON INSULIN RECEPTOR
OF IRON
IN HEPGS
HEPATOCYTES F! Dongiovanni,
L. Valenti, G. Occhino, A.L. Fracanzani, M. Mattioli, S. Fargion. Dipartimento Di Medicina Interna, Universit De&i Studi, Ospedale Policlinico IRCCS, Milano, Italy
Background: Mild hepatic iron overload is a frequent clinical feature in patients with NAFLD, characterized by hepatic insulin resistance. Iron depletion improves insulin sensitivity in patients with diabetes, but the mechanisms are unclear. Aim: To define the interaction between iron and insulin sensitivity, we determined the effect of iron manipulation on insulin receptor expression in a hepatocyte cell line. Methods: HepG2 cells were cultured in RPM1 complete medium. 1125. insulin binding was evaluated 48 hours after plating in basal condition and in cells treated for 24 hours with iron (FeAmCit/holo-transfenin),
Parallel Session 9: Experimental desfenioxamine and glucose. Insulin receptor expression was evaluated by Western blotting. Results: The number of binding sites at the saturating insulin concentration of 100 nglml was 20000/tell in basal conditions. Iron depletion, obtained with desferrioxamine 100 mM, increased by two-fold (P
I
87
ALCOHOL
AND HEPATITIS C VIRUS CORE PROTEIN
ADDITIVELY
INCREASE
SYNERGISTICALLY EXPRESSION
LIPID PEROXIDATION
TRIGGER
AND
IN A TRANSGENIC
Background & Aims: Alcohol consumption accelerates the appearance of liver fibrosis and hepatocellular carcinoma in patients with chronic hepatitis C virus (HCV) infection, but the mechanisms of these interactions are unknown. HCV core protein and ethanol share common effects on lipid metabolism. Both impair microsomal triglyceride transfer protein and increase lipid peroxidation. We therefore investigated the effects of chronic ethanol consumption in HCV core protein-expressing transgenic mice. Methods & Results: Ethanol was progressively added (up to 20%) to the drinking water that was given ad libidum. In viva fatty acid oxidation (measured by [14C]CO2 exhalation after [U-14Clpalmitic acid administration) was not inhibited by ethanol consumption and/or HCV core expression. Both chronic ethanol consumption and HCV core expression decreased hepatic lipoprotein secretion (measured by triton WR1339 method) and caused steatosis, but had no additive effects on lipoprotein secretion or steatosis. However, chronic ethanol consumption and HCV core protein additively increased lipid peroxidation (measured by ethane exhalation and hepatic TBARS) and acted synergistically to increase the hepatic expression of transforming growth factor-b (TGF-b) and, to a less extent, tumor necrosis factor-a (TNI-a) (RNAse protection assay). Conclusions: HCV core protein expression and chronic alcohol consumption have no effects on in viva fatty acid oxidation and do not additively impair hepatic lipoprotein secretion. However, HCV core and alcohol additively increase hepatic lipid peroxidation and act synergistically to increase hepatic TNI-a and TGF-b expression. These effects may be involved in the activation of fibrogenesis and the development of hepatocellular carcinoma in patients cumulating alcohol abuse and HCV infection.
ISCHEMIC
31
Methods: 60 minutes &hernia was performed in mice of 6 and 60 weeks old. Liver injury was determined by AST, apoptosis by caspase 3 and TUNEL test. Necrosis was investigated 24hr after reperfusion by H&E staining. Finally, ischemic preconditioning was performed in both mice. Results: The 60 weeks old mice had significantly higher AST levels (125OOU/I_ vs 8200 U/L; piO.05) and caspase 3 activity (98 vs 67 AUF/mg p=O.O4) than young mice. In addition, old mice had significantly more TUNEL pos. hepatocytes after reperfusion (55% vs 77%; p
MOUSE MODEL
l? Letteron2, F. Carnot3, F. Zavala4, D. Pessayre2, B. Nalpas’, C. Brechot’. ‘Liver Cancer And Molecular Virology, Institut National De La Sante Et De La Recherche Medicale Unite 370, Faculte De Medecine Necker-Enfants Malades, Paris; 21NSERM U 481, Hopital Beaujon, Clichy; ‘Service D’Anatomopathologie, Hopital Europeen Georges Pompidou, Paris; 41NSERM U 25, Hopital Neckes Paris, France
INCREASED
2
HEPATIC CYTOKINE
G. Perlemuter’,
I 88
Hepatology
INJURY IN THE OLD LIVER. A NOVEL
PATHWAY OF INJURY N. Selzner’, M. Selzner2, P.-A. Clavien2. ‘Department Of Visceral Surgery; 2 Visceral Surgery, University Hospital Of Zurich, Zurich, Switzerland
Due to our aging population, liver surgery is increasingly performed in older patients. However, the effect of age on ischemic injury is unknown.
I
89
RAPID EX VIVO PROTOCOL GENE TRANSFER TRANSDUCTION
FOR EFFICIENT
AND STABLE
IN THE LIVER VIA HEPATOCYTE BY FLAP LENTIVIRAL
VECTORS
C. Giannini”3,
S. Morosan 1,4, .I G. Tralhao’,5, J.E. Guidotti’, S. Battaglia 1,3 , K Mollier2, L. Hannoun6, D. Kremsdorf’, l? Charneau2. ‘INSERM U-370 Pasteur-Necker Institutes, Paris, France; 2Groupe De Virologie Moleculaire Et Vectorologie, Pasteur Institute, Paris, France; ‘Department Of Internal Medicine, University Of Florence, Florence, Italy; 4Faculty Of Veterinary Medicine, University Of Agronomical Science And Veterinary Medicine, Iasi, Romania; ‘Department Of Surgery 3, H. U. Coimbra, Medical Faculty, Coimbra, Portugal; 6Service De Chit-u&e Digestive, Hopital Pitie-Salpetriere, Paris, France Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and may can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture, nor transduced with integrative viral vectors and are very sensitive to trypsine dissociation before their reimplantation into the recipient. In the present study, we demonstrate that primary human and rat hepatocytes can be efficiently transduced with a flap lentiviral vector without the need for to be plating and culture. Efficient transduction of non-adherent primary hepatocytes was achieved with short period of contact with vector particles and without modifying hepatocyte viability. We also show that the presence of the DNA flap in the vector construct was essential to reach high levels of transduction. Transplanted in immunodeficient mice, the genetically modified hepatocytes integrated the mouse liver parenchyma where they stably expressed the GFP transgene for several months. Moreover, lentivirally transduced primary human hepatocytes extensively repopulated uPA/SCID mouse liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, this work opens new perspectives for the applicability of human liver directed ex viva gene therapy. In fact, the use of flap lentiviral vectors permits a high efficiency transduction in a short period of time, still maintaining optimum hepatocyte engrafting and functionality in viva.