Highly Efficient Lentiviral Vector-Mediated Transduction of Nondividing, Fully Reimplantable Primary Hepatocytes
Authors: Tuan Huy Nguyen, José Oberholzer, Jacques Birraux, Pietro Majno, Philippe Morel, Didier Trono
Journal: Molecular Therapy (2002)

Gene therapy is an attractive approach for the treatment of liver disease. We demonstrate that a so-called third-generation human immunodeficiency virus (HIV)-derived vector system can govern the efficient delivery, integration, and stable expression of a transgene into primary human hepatocytes in the complete absence of cell division. We also show that rodent hepatocytes exhibit a significant degree of resistance to HIV vector-mediated transduction, a phenotype that is particularly pronounced in murine hepatocytes and that results from a block in the immediate-early phase of infection. We finally describe a methodology, that allows very high rates of transduction through minimal in vitro manipulation, in which hepatocytes are kept in suspension and reimplanted within a few hours of harvest with a fully preserved engraftment potential. These results have immediate implications for the treatment of liver diseases by the transplantation of genetically modified hepatocytes, an approach that could be applied to a number of hereditary and acquired hepatic disorders. Gene therapy is an attractive approach for the treatment of liver disease. We demonstrate that a so-called third-generation human immunodeficiency virus (HIV)-derived vector system can govern the efficient delivery, integration, and stable expression of a transgene into primary human hepatocytes in the complete absence of cell division. We also show that rodent hepatocytes exhibit a significant degree of resistance to HIV vector-mediated transduction, a phenotype that is particularly pronounced in murine hepatocytes and that results from a block in the immediate-early phase of infection. We finally describe a methodology, that allows very high rates of transduction through minimal in vitro manipulation, in which hepatocytes are kept in suspension and reimplanted within a few hours of harvest with a fully preserved engraftment potential. These results have immediate implications for the treatment of liver diseases by the transplantation of genetically modified hepatocytes, an approach that could be applied to a number of hereditary and acquired hepatic disorders. IntroductionThe liver is an important target for gene therapy because numerous inherited diseases affect the metabolic functions of this organ or its ability to secrete systemically active proteins. Acquired hepatic diseases could also benefit from genetic approaches if, for instance, liver cells could be made refractory to the replication of hepatitis B or C viruses. The proliferative capacity of hepatocytes further indicates that both in vivo and ex vivo approaches might be of value in this organ [1Mulligan R. The basic science of gene therapy.Science. 1993; 260: 926-932Crossref PubMed Scopus (1520) Google Scholar]. Hepatocytes are indeed amongst a handful of differentiated cell types that can, by still unclear mechanisms involving cytokines and growth factors, regain their mitotic potential upon parenchymal loss and divide until the original liver mass is restored [2Michalopoulos G. DeFrances M. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2864) Google Scholar]. Therefore, the stable introduction of a therapeutic gene into hepatocytes, be it in vivo or by ex vivo manipulation, can have long-lasting effects provided that a suitable gene delivery system is used. Adenoviral vectors, although extremely efficient in the liver, remain episomal and non-replicating and are therefore progressively lost over time [3Li Q. Kay M. Finegold M. Stratford-Perricaudet L. Woo S. Assessment of recombinant adenoviral vectors for hepatic gene therapy.Hum. Gene Ther. 1993; 4: 403-409Crossref PubMed Scopus (295) Google Scholar]. Adeno-associated virus vectors do not have this drawback, but their limited cloning capacity and uneasy production constitute important disadvantages [4Snyder R. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.Nat. Genet. 1997; 16: 270-276Crossref PubMed Scopus (496) Google Scholar]. Vectors derived from oncoretroviruses such as murine leukemia virus (MLV) integrate their cargo into the chromosome of target cells, but only if those divide shortly after infection [5Miller D. Adam M. Miller A. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.Mol. Cell. Biol. 1990; 10: 4239-4242Crossref PubMed Scopus (1271) Google Scholar]. This is a serious shortcoming for liver-based gene therapy, because adult hepatocytes hardly proliferate in vivo unless significant regeneration is induced, for example, by partial hepatectomy, and respond poorly to growth factors in vitro [6Webber E.M. Godowski P.J. Fausto N. In vivo response of hepatocytes to growth factors requires an initial priming stimulus.Hepatology. 1994; 19: 489-497Crossref PubMed Scopus (201) Google Scholar, 7Runge D.M. Epidermal growth factor- and hepatocyte growth factorreceptor activity in serum-free cultures of human hepatocytes.J. Hepatol. 1999; 30: 265-274Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Gomez-Lechon M.J. Effects of hepatocyte growth factor on the growth and metabolism of human hepatocytes in primary culture.Hepatology. 1995; 21: 1248-1254Crossref PubMed Google Scholar]. The advent of vectors derived from human immunodeficiency virus (HIV) and other lentiviruses could, in principle, allow us to overcome these obstacles, as lentiviral vectors have a large cloning capacity, are easy to generate, and can govern the efficient delivery, integration and long-term expression of transgenes into nondividing cells both in vitro and in vivo [9Naldini L. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (3978) Google Scholar].The therapeutic potential of lentiviral vectors was first demonstrated in the central nervous system. The injection of a lentiviral vector encoding the relevant therapeutic protein achieved a long-term benefit in animal models of type VII mucopolysaccharidosis and Parkinson's disease [10Bosch A. Perret E. Desmaris N. Trono D. Heard J. Reversal of pathology in the entire brain of mucopolysaccharidosis type VII mice after lentivirus-mediated gene transfer.Hum. Gene Ther. 2000; 11: 1139-1150Crossref PubMed Scopus (123) Google Scholar, 11Kordower J. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease.Science. 2000; 290: 767-773Crossref PubMed Scopus (1138) Google Scholar]. The liver, like the brain, is composed of nonproliferating, highly differentiated, and metabolically active cells, so that by analogy, it is tempting to predict that lentiviral vectors will represent tools of choice in this organ as well. However, recent reports have suggested that hepatocytes are less sensitive than neurons to lentivirus-mediated transduction. Although the issue is still controversial, it appears that mouse or rat hepatocytes can be transduced in vivo but with an efficiency that is relatively low and well inferior to that of other cell types found in the liver [12Kafri T. Blomer U. Peterson D.A. Gage F.H. Verma I.M. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.Nat. Genet. 1997; 17: 314-317Crossref PubMed Scopus (543) Google Scholar, 13Park F. Ohashi K. Chiu W. Naldini L. Kay M. Efficient lentiviral transduction of liver requires cell cycling in vivo.Nat. Genet. 2000; 24: 49-52Crossref PubMed Scopus (204) Google Scholar, 14Pfeifer, A., et al.2001. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging. Mol. Ther.3: 319–322,Google Scholar].Ex vivo methods are considered for the genetic treatment of liver-based disorders. In a prototypic approach, hepatocytes are isolated, placed in culture for 3–4 days in the presence of growth factors, genetically modified, for instance, with an oncoretroviral vector, and reinjected into the portal vein. Stimulated by results obtained in an animal model of familial hypercholesterolemia [15Chowdhury J.R. Long-term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR-deficient rabbits.Science. 1991; 254: 1802-1805Crossref PubMed Scopus (323) Google Scholar], Grossman et al. [16Grossman M. Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia.Nat. Genet. 1994; 6: 335-341Crossref PubMed Scopus (495) Google Scholar] conducted an ex vivo gene therapy trial in five patients with a homozygous mutation in the gene coding for the low-density lipoprotein receptor (LDLR). While the feasability and safety of this approach were demonstrated, two pitfalls limited its clinical outcome. On one hand, the efficiency of gene transfer was limited; on the other hand, two-thirds of the hepatocytes were lost during their in vitro culture. Still, the small decrease in serum cholesterol levels observed in some patients argued for the potential value of the approach, provided that greater numbers of hepatocytes could be genetically modified and reimplanted [17Grossman M. A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolemia.Nat. Med. 1995; 1: 1148-1154Crossref PubMed Scopus (456) Google Scholar].In the present work, we investigated whether HIV-derived lentiviral vectors could help overcome these obstacles, and more generally, represent interesting tools for the gene therapy of liver disorders. We first examined the susceptibility to HIV-derived vectors of human hepatocytes cultured as previously performed in the context of an ex vivo gene therapy protocol [17Grossman M. A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolemia.Nat. Med. 1995; 1: 1148-1154Crossref PubMed Scopus (456) Google Scholar], except that we omitted growth factors. We then defined optimal parameters for transduction of these cultured human hepatocytes and compared their susceptibility with that of hepatocytes from various species. We observed that nondividing human hepatocytes are highly sensitive to HIV-derived vectors, while rodent hepatocytes, in particular of murine origin, show a significant degree of resistance. Finally, we developed a new ex vivo gene transfer protocol that allows very high rates of transduction of uncultured hepatocytes with a full preservation of their reimplantation capacity. These data pave the way to clinically relevant and effective protocols for the ex vivo gene therapy of liver diseases.ResultsEfficient Transduction of Primary Human Hepatocytes in the Absence of Cell ProliferationWe purified human hepatocytes from liver biopsies and placed them in culture on Primaria dishes in serum-free medium in the absence of growth factors. In this experimental setting, the cells maintained their differentiation status throughout the time of culture as demonstrated by the albumin content (Fig. 1A). Furthermore, bromodeoxyuridine (BrdU) incorporation indicated that less than 2% of the hepatocytes entered mitosis (Fig. 1B), consistent with the absence of expression of proliferating cell nuclear antigen (PCNA), a marker for the G1 and G2/S/M phases of the cell cycle (data not shown). We produced vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles containing an HIV-derived lentiviral vector encoding green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (CLL-GFP-CLL) from a third-generation stable packaging cell line [18Klages, N., Zufferey, R., and Trono, D.2000. A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol. Ther.2: 170–176,Google Scholar], and we infected hepatocytes on day 2 post-plating through a 16-hour incubation with this virus at a multiplicity of infection (MOI) of 30 HeLa transducing units (HTU) per cell. Cells expressed detectable levels of the transgene within 36 hours, and by 4 days, up to 80% of the hepatocytes were strongly GFP-positive as demonstrated by fluorescence-activated cell sorting (FACS) analysis (Fig. 1C, upper right panel). The induction of GFP expression was completely blocked when infection was conducted in the presence of nevirapine, an inhibitor of reverse transcription, demonstrating that it did not result from pseudotransduction (Fig. 1C, lower left panel). The large fraction of transduced cells in a population that exhibited a very low proliferation index indicated that gene transfer occurred in the absence of cell cycling. Confirming this point, a comparable murine retroviral vector could only transduce the background of dividing cells in the culture, even at a high MOI (Fig. 1C, lower right panel). Furthermore, when hepatocytes plated 2 days earlier were infected with the lentiviral vector in the presence of BrdU, robust levels of transduction were observed without detectable BrdU incorporation, which is in sharp contrast with results obtained in similarly treated HeLa cells (Fig. 1D). Finally, the cell cycle inhibitor aphidicolin did not reduce the efficiency of hepatocyte transduction (data not shown).To demonstrate the integration of the lentiviral vector, we conducted an Alu-HIV PCR analysis as described [14Pfeifer, A., et al.2001. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging. Mol. Ther.3: 319–322,Google Scholar, 19Chun T. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.Proc. Natl. Acad. Sci. USA. 1997; 94: 13193-13197Crossref PubMed Scopus (1542) Google Scholar]. A first round of PCR was performed using one primer complementary to Alu sequences and one specific for the HIV long terminal repeat (LTR). As Alu regions are repetitive sequences scattered throughout the human genome and vectors randomly integrate at multiple sites, this first reaction generated products with variable sizes (Fig. 2, PCR1). The second round of PCR, using nested primers within the HIV-1 LTR, generated the expected 140-bp product from transduced but not from control hepatocytes (Fig. 2, PCR1+PCR2). Genomic DNA from transduced cells subjected only to PCR2 amplification did not yield any signal, confirming the validity of the Alu-HIV PCR technique to detect integrated proviruses [14Pfeifer, A., et al.2001. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging. Mol. Ther.3: 319–322,Google Scholar, 19Chun T. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.Proc. Natl. Acad. Sci. USA. 1997; 94: 13193-13197Crossref PubMed Scopus (1542) Google Scholar] (Fig. 2, PCR2).FIG. 2Detection of integrated vector provirus. Genomic DNA isolated from primary human hepatocytes, either mock-treated or transduced with CLL-GFP-CLL vector at the indicated MOI was subjected to nested Alu-HIV PCR. Amplification products were resolved on 1% agarose gel containing ethidium bromide and detected by UV transillumination. PCR1 and PCR2 correspond respectively to the first and second rounds of PCR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine more quantitatively the efficacy of lentivector-mediated gene transfer into human primary hepatocytes, we exposed these cells to increasing amounts of infectious particles, from 0 to 100 HTU per hepatocyte. At low MOIs, human hepatocytes were as susceptible as HeLa cells, which served as control targets because they are highly permissive to lentiviral transduction (Fig. 3A). For both cell types, the linear range of the transduction/vector dose curve was for MOIs between 0.1 and 1 (r = 0.99). As much as 80% of human hepatocytes were transduced at an MOI of 10. However, in contrast to HeLa cells, transduction never reached 100% in the liver cells. The addition of polybrene, a polycation that promotes virion attachment, did not increase transduction efficiency (data not shown).FIG. 3Dose-response of human hepatocytes to lentiviral transduction. (A) Human hepatocytes and Hela cells were exposed during 16 hours to increasing MOI (equals the number of HTU per cell) of HIV-derived vector and GFP-positive cells were quantified by FACS. The values represent the mean of three measurements with standard deviation. (B) FACS analysis of human hepatocytes transduced on day 2 by a 16-hour exposure to 5 × 103 and 5 × 104 cpm of RT activity equivalent of GFP vectors with cPPT-CTS in sense (TRIP-GFP) or antisense (TRIPinv-GFP) orientations. Percentage of GFP-positive cells and mean fluorescence intensity (MFI) are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Accessory Genes and cPPT-CTSThe third-generation packaging system used in these experiments comprises only the gag, pol, and rev genes of HIV-1 [18Klages, N., Zufferey, R., and Trono, D.2000. A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol. Ther.2: 170–176,Google Scholar]. However, transduction efficiency was unchanged when a first-generation producer system, including tat, vif, vpr, vpu, and nef, was substituted (data not shown). We next determined whether lentiviral vectors containing a central polypurine tract (cPPT) and central terminal sequence (CTS) had an increased ability to transduce hepatocytes. This cis-acting element can stimulate the efficiency of an as yet incompletely defined early step of infection, possibly nuclear import [20Follenzi A. Ailles L. Bakovic S. Geuna M. Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences.Nat. Genet. 2000; 25: 217-222Crossref PubMed Scopus (780) Google Scholar, 21Zennou V. HIV-1 genome nuclear import is mediated by a central DNA flap.Cell. 2000; 10: 173-185Abstract Full Text Full Text PDF Scopus (700) Google Scholar]. We exposed human hepatocytes to equal amounts of particles (as assessed by reverse transcriptase (RT) content) containing GFP-expressing vectors with the cPPT-CTS in either sense or antisense orientations (TRIP-GFP or TRIPinv-GFP, respectively). At a nonsaturating dose of viral particles, the percentage of transduced cells and their mean fluorescence index were increased by the cPPT-CTS (Fig. 3B). However, the increase in the relative infectivity of the particles was of equal magnitude in hepatocytes and in HeLa cells (not illustrated). Furthermore, increasing the amounts of cPPT-CTS-containing lentivector up to 300 HTU per hepatocyte did not raise the maximal transduction efficiency above the 80% plateau observed in the absence of cPPT-CTS (data not shown).Lower Susceptibility of Rodent Hepatocytes to Lentiviral Vector-Mediated Gene TransferRodents can serve as animal models for numerous human liver diseases and were previously used to study the efficiency of lentivector-mediated gene transfer in this organ [12Kafri T. Blomer U. Peterson D.A. Gage F.H. Verma I.M. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.Nat. Genet. 1997; 17: 314-317Crossref PubMed Scopus (543) Google Scholar, 13Park F. Ohashi K. Chiu W. Naldini L. Kay M. Efficient lentiviral transduction of liver requires cell cycling in vivo.Nat. Genet. 2000; 24: 49-52Crossref PubMed Scopus (204) Google Scholar, 14Pfeifer, A., et al.2001. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging. Mol. Ther.3: 319–322,Google Scholar]. Therefore, we compared the susceptibility of rodent and human hepatocytes to lentiviral vector-mediated transduction. We cultured freshly isolated rat and mouse hepatocytes for 2 days in the same conditions as their human counterparts before exposing them to increasing amounts of the CLL-GFP-CLL vector particles. At an MOI of 1 and below, both types of rodent hepatocytes were strikingly less sensitive to transduction than human hepatocytes (Fig. 4A). This resistance could be partly overcome by increasing the MOI. However, at HTU/cell ratios of 20 and above, levels of transduction reached a plateau at 30% and 50% for mouse and rat hepatocytes, respectively (Fig. 4B). Mouse hepatocytes were particularly resistant to genetic modification immediately after harvest, compared with human and rat cells (Fig. 4C).FIG. 4Decreased sensitivity of rodent hepatocytes to lentiviral transduction. (A) Human and rodent hepatocytes were transduced on day 2 post-plating with a GFP-expressing HIV-derived vector at a low MOI to stay in the linear range of the assay and GFP positive cells were scored by FACS. (B) Same experiment with increasingly high MOIs. (C) Same experiment, transducing human and rodent hepatocytes at an MOI of 30 HTU/cell at different times after plating. (D) PCR analysis of reverse transcription. Lysates of hepatocytes infected at different MOIs (0, untransduced; 1, MOI = 1; 10, MOI = 10) prepared at the indicated times post-infection were amplified by PCR with LTR-, GFP- and β-actin-specific primers. LTR5/LTR6 primers amplify a 140-bp fragment from strong stop DNA (early reverse transcript) and GFP primers a 417-bp fragment from elongated reverse transcription products. As a control, human cells were infected at an MOI of 10 in the presence of nevirapine (N).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the level of the block observed in rodent cells, we monitored by PCR the synthesis of viral reverse transcripts in human and murine hepatocytes plated and infected on the day of harvest (Fig. 4D). Both cell types exhibited readily detectable levels of minus-strand strong-stop DNA, the earliest product of reverse transcription, 2 hours post-infection (Fig. 4D, top). Nevirapine treatment had no effect on this particular DNA species, possibly because it is already contained in virions [22Lori F. Viral DNA carried by human immunodeficiency virus type 1 virions.J. Virol. 1992; 66: 5067-5074Crossref PubMed Google Scholar, 23Trono D. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses.J. Virol. 1992; 66: 4893-4900PubMed Google Scholar]. Human hepatocytes further accumulated GFP-specific elongated reverse transcripts in a nevirapine-sensitive manner 5 hours post-infection, confirming that these latter products reflected de novo reverse transcription (Fig. 4D, bottom). In contrast, murine hepatocytes did not harbor significant levels of this DNA, even 24 hours post-infection. The finding of early but not late viral DNA species in mouse hepatocytes indicates that these targets can internalize the lentiviral vector but support subsequent steps of the infectious process very inefficiently.A Clinically Relevant Protocol for the ex Vivo Transduction of HepatocytesThe transplantation of genetically modified hepatocytes is considered for the treatment of several liver-based and systemic disorders. For this type of approach to be successful, however, both the efficacy of gene transfer and the preservation of the cells before reimplantation are crucial. As a first step towards defining a clinically suitable ex vivo transduction protocol, we seeded human hepatocytes at different cell densities and infected them at an MOI of 30 on day 2 post-plating. We harvested and analyzed the cells by FACS 4 days later to determine the percent of viable (% of cells gated in R1; Fig. 5A) and GFP-positive cells. Cell survival and gene transfer were optimal when hepatocytes were seeded between 2 and 5 × 105 cells per well (data not shown). The time of exposure between cells and viral particles was then reduced to 4 hours instead of the usual overnight incubation. Although the relative efficacy of gene transfer was lower, up to 50% and 75% of human hepatocytes were still transduced at MOIs of 30 and 100, respectively (Fig. 5B). Hypothesizing that hepatocytes exposed to large amounts of viral particles while in culture were placed under a particularly important stress, we added vitamin E, an anti-oxidant, to the transduction medium. This resulted in a significant increase in both hepatocyte survival and transduction efficiency. Under these conditions, and after a 4-hour exposure to the vector at an MOI of 30, more than 65% of the cells survived and up to 78% were stably transduced (Fig. 5B). These data prompted us to ask whether gene transfer required any plating of the cells. For this, we incubated human hepatocytes with the vector for 4 hours while still in suspension, immediately after their purification. To avoid adherence, we kept the cells during that time in regular plastic culture dishes instead of Primaria plates, with gentle shaking at hourly intervals, before extensive washing and plating. In this setting as well, vitamin E significantly increased both cell viability and transduction efficiency. In the presence of this anti-oxidant, up to 50% and close to 60% of freshly isolated hepatocytes were transduced in suspension at MOIs of 10 and 30, respectively (Fig. 5C). Notably, these cells were really infected while in suspension and not after their subsequent plating, because the supernatant of the last wash before plating contained only minimal amounts of residual infectivity (Fig. 5D). These data demonstrate that efficient transduction of human hepatocytes does not require any plating and culture and, therefore, suggest that such cells could be reimplanted to a patient immediately after their genetic modification.FIG. 5Optimized ex vivo liver gene therapy protocol. (A) Representative FACS analysis of bulk population of hepatocytes placed in culture and trypsinized; viable cells are gated in R1. (B) Vitamin E allows for an accelerated transduction protocol. Human hepatocytes were transduced on day 2 post-plating through a 4-hour exposure to increasing amounts of vectors in the presence (lozenges) or absence (squares) of vitamin E. Hepatocytes were then washed, cultured in regular hepatocyte medium, and analyzed by FACS to determine the percent of viable (closed lozenge and square) and GFP-positive cells (open lozenge and open square). (C) Transduction of freshly isolated human hepatocytes in suspension. Hepatocytes were transduced in suspension immediately after purification, by a 4-hour exposure to vector at indicated MOIs in the presence (open lozenge) or absence (closed square) of vitamin E. Cells were then washed, plated, and cultured without vitamin E. The percentage of GFP-positive and viable cells were determined by FACS 5 days later. (D) Transduction of human primary hepatocytes with residual vector from the wash medium of hepatocytes transduced in suspension. Hepatocytes were either infected in suspension for 4 hours (left and middle panel) or plated immediately after harvest. The latter hepatocytes were then incubated for 16 hours with the medium from the last wash of the suspension-transduced hepatocytes (right panel). We determined the percentage of GFP-positive cells by FACS 5 days later.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To probe this issue further, we used the rat model, as hepatocytes from this species seemed almost as trans-ducible as their human counterparts provided that high MOIs were used (Fig. 4C). We transduced freshly isolated rat hepatocytes in suspension immediately after harvest with the GFP-expressing vector at an MOI of 10 for 4 hours in the presence or absence of vitamin E. The anti-oxidant had the same stimulating effect as in human cells (Fig. 6A). After washing, we injected twenty million hepatocytes transduced in the presence of vitamin E (gene transfer efficiency, 31%) into the spleen of two rats that had been subjected 24 hours earlier to a two-thirds partial hepatectomy in order to create an environment optimal for cell transplantation. We sacrificed one of the two animals 2 weeks later, whereas the other was subjected to an additional hemi-hepatectomy and was euthanatized 3 weeks later. GFP-positive cells were detected in the liver of both rats 2 weeks post-transplantation, representing 0.4% and 1% of all hepatocytes, respectively. Transgene-positive hepatocytes were distributed throughout the liver parenchyma, albeit predominantly in the periportal area (Figs. 6B and 6C). They were present as single cells or in pairs, although one big cluster containing more than 100 cells was present in the liver of one of the two animals (data not shown). In the animal that underwent a second partial hepatectomy, the percentage of GFP-positive hepatocytes was 1% at both the 2- and 5-week examinations. At this later time-point, numerous clusters of 3 to 10 GFP-positive cells were detected, showing that the transduced hepatocytes had responded to the proliferative stimulus triggered by the hepatectomy performed 2 weeks after their implantation (Fig. 6B).FIG. 6Transplantation of lentivector-transduced hepatocytes. (A) Vitamin Emediated stimulation of rat hepatocyte transduction. Freshly isolated rat hepatocytes were transduced in suspension at an MOI of 10 for 4 hours with (open bar) or without (black bar) vitamin E, placed in culture and analyzed 5 days later for expression of the GFP transgenethen washed. Combined results of two experiments are shown, with error bar indicative of mean ± SD (B) Pr