Efficiency Of Retrovirus-mediated Gene Transfer Research Articles

A BMT Model Mice for Myelodysplastic Syndromes (MDS) and Transformation to AML.

To understand myelodysplastic syndromes (MDS) at the molecular level, we used a mouse bone marrow transplant (BMT) model. Several leukemogenic fusion proteins were demonstrated to induce leukemia in mouse BMT models. However, analysis of molecular basis of MDS has been hampered by the lack of mouse MDS models. Mutations of a transcription factor AML-1/Runx-1 were identified in 15–40% of MDS-RAEB and MDS/AML and 5–10% of de novo AML. To test transforming abilities of the mutant AML-1, we transduced AML-1 with different mutations (#5 and #27) into mouse bone marrow cells using our highly efficient retrovirus-mediated gene transfer, transplanted the transduced cells to irradiated mice, and found that most mice developed leukemia within 4–13 months after the transplant (table1 and figure1). The leukemic mice exhibited marked hepato-splenomegaly, and morphological abnormalities of myeloid and erythroid lineages were frequently observed. Some mice developed to leukemia after a long latency with MDS-like symptoms, either with pancytopenia or leukocytosis with anemia, thus mimicking the human disease. To clarify the differences between the leukemic mice with early onsets and those that developed leukemia after a long period of MDS-like symptoms in the mutant AML-1-induced mouse diseases, the integration sites of the transduced retroviruses were investigated using a polymerase chain reaction (PCR)-based technique bubble PCR. Our preliminary results suggested that the integration sites of the retroviruses harboring the mutant AML-1 contribute to the early onset of leukemia. In conclusion, we have developed a useful mouse in vivo model of MDS/AML that should help understand the molecular basis of MDS and progression of MDS to leukemia.penetrance and latencymutation numberpositionformpenetrancelatency#5D171Npoint mutation in RHD69.6 %4–13 months#27S291fsX300truncation type100 %4–9 months [Display omitted]

Retroviral Gene Delivery from Engineering Perspective

Although a few physical and chemical approaches have been documented to enhance the efficiency of retrovirus-mediated gene transfer, systematic analysis of the processes involved in the retroviral transduction system remains to be important for further improvement. With the aid of reasonable mathematical description of an experimental system, we can therefore predict and optimize the retroviral gene delivery on a quantitative basis of understanding.

Follistatin allows efficient retroviral-mediated gene transfer into rat liver

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.

Phagocytosis‐mediated retroviral transduction: co‐internalization of deactivated retrovirus and calcium‐alginate microspheres by macrophages

Efficiency of retrovirus-mediated gene transfer has been hampered by short retroviral half-life due to shedding of retroviral envelope proteins which is of utmost importance to the interaction between retrovirus and cell. Calcium-alginate microspheres with average size of 780 nm were prepared by reverse emulsification and characterized by scanning electron microscopy and microelectrophoresis. To obtain deactivated retroviruses produced from 293 packaging cells, retrovirus-containing media were pre-incubated at 37 degrees C for 6, 12, 18, and 24 h, respectively. Murine J774A.1 macrophages were co-treated with Ca-alginate microspheres and deactivated retroviruses encoding the enhanced green fluorescent protein (eGFP) gene. Through phagocytosis, deactivated retroviruses and Ca-alginate microspheres were co-internalized into macrophages. After retroviral transduction for 24 and 48 h, the percentages of macrophages with eGFP expression were determined by bright-field and fluorescence microscopy. After 48-h incubation with Ca-alginate microspheres and deactivated retroviruses, phagosomes turned into large vacuoles occupied almost half of the cytoplasmic space. This was probably attributed to the erosion of Ca-alginate microspheres by destructive agents within vacuolar compartments and concomitant osmotic swelling. It was reasoned that deactivated retroviruses escaped such enlarged vesicles easily and underwent reverse transcription in the cytosol. The expression of eGFP in macrophages infected by retroviruses pre-incubated for 24 h in the presence of Ca-alginate microspheres was thereby augmented up to tenfold in comparison with the cells treated with 24-h deactivated retroviruses only. Ca-alginate microspheres performed as auxiliary agents for the enhancement of retrovirus-mediated gene transfer efficiency even though retroviruses had been deactivated due to the loss of envelope proteins.

729. Receptor Trafficking Affects the Efficiency of Retrovirus Transduction

We have constructed a model system to study the role of receptor trafficking on the efficiency of retrovirus-mediated gene transfer. We constructed a panel of HeLa cell lines to stably express one of three interleuken 2 receptor alpha chain (TAC) chimeras (TAC, TAC-CD16, TAC-DKQTLL), each of which is internalized via a distinct intracellular trafficking pathway. Targeted retroviruses to bind these receptors were also constructed by fusing a single-chain antibody variable fragment against TAC (ScFv anti-TAC) to the N-terminus of the murine leukemia virus (MLV) amphotropic and ecotropic envelope proteins. We found that retroviruses pseudotyped with the ScFv anti-TAC amphotropic fusion protein, but not those pseudotyped with the ecotropic fusion protein, were able to transduce HeLa cells that expressed the chimeric TAC receptors. Gene transfer was significantly less efficient in HeLa cells that expressed TAC-DKQTLL, a receptor that is rapidly internalized and transported to the lysosomes, than in HeLa cells that expressed TAC, TAC-CD16, or in the parent cell line that did not express any of the chimeric TAC receptors. Gene transfer was most efficient to HeLa cells that expressed TAC, a receptor with a long residence time on the cell surface (half-life > 40 hours). Our findings suggest that the efficiency of retrovirus transduction is a function of the intracellular trafficking itinerary of cell-surface retrovirus-binding proteins. The implications of our findings to the development of improved gene transfer technologies for the purposes of human gene therapy will be discussed.

Portal branch ligation induces efficient retrovirus‐mediated gene delivery in rat liver

The in vivo transduction of hepatocytes with conventional retrovirus vectors requires the induction of cell division and this can currently only be achieved by invasive surgery or by inducing severe liver damage. We hypothesised that partial portal branch ligation (PBL) could induce hepatocyte proliferation and efficient gene transfer in the rat. We ligated the portal branch serving 70% of the liver and measured the kinetics of liver mass restoration and cell proliferation and the distribution of dividing hepatocytes after administration of 5-bromo-2'-deoxyuridine. The efficiency of retrovirus-mediated gene transfer after PBL was tested by use of beta-galactosidase-expressing recombinant retroviruses. The viruses were administered in a single injection via the portal vein at different times after PBL and the livers of transduced animals were analysed 4 days later. We found that the number of cycling hepatocytes remained stable between 24 and 44 h after PBL (approximately 12.5%). Although there was a high level of inter-animal variability, hepatocyte proliferation was always initiated in the same lobe of the liver. In animals that had undergone PBL, 19% of hepatocytes were transduced 28 h after the administration of a single high-titre injection of retroviruses, mainly around the portal spaces. PBL can mediate the efficient transduction of hepatocytes in vivo after a single intravenous injection of recombinant retroviruses. This approach is feasible in humans.

Factors influencing immune response after in vivo retrovirus-mediated gene transfer to the liver.

Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo triggers an immune response directed against transduced hepatocytes. This effect may be due either to spreading of retroviral vectors in the blood stream with subsequent infection of antigen presenting cells (APCs) or to cross-presentation of the transgene product present as a contaminant in the viral stock. In order to decrease immune response, we evaluated the effect of asanguineous perfusion of the liver as well as purification of the viral stock on long-term transduction of hepatocytes using the nls-lacZ marker gene. Animals were divided in four groups. In group 1, the viral supernatant was perfused in the regenerating liver after complete vascular exclusion of the organ. In group 2, using the same strategy, animals received retroviral supernatant that was passed through a beta-galactosidase affinity column to reduce beta-galactosidase contamination. In two control groups (respectively groups 3 and 4) the corresponding viral supernatants were delivered via peripheral injection. In group 1, 23.1% of animals had no immune response 2 months after gene delivery vs. 33.4% in group 2, 4.3% in control group 3, and 0% in control group 4. Statistical analysis of the results demonstrated that only the difference between groups 2 and 3 was statistically significant. This indicated that both asanguineous perfusion together with passage through an affinity column were required to decrease significantly immune response. Our present results suggest that both supernatant contamination and viral spreading contribute to immune response after retrovirus-mediated gene delivery to the liver.

In-vivo retroviral gene transfer to the liver is cancelled by an immune response against the corrected cells. Can it be avoided?

Highly efficient retroviral-mediated gene transfer into hepatocytes in vivo has been previously reported in rats, but some reports described transient expression of the transgene that may be related to induction of an immune response against the transgene product. To devise a surgical approach to circumvent this drawback, two-thirds partial hepatectomy was performed in Wistar male rats to induce the hepatocyte division required to achieve retrovirus integration. Delivery of amphotrophic retroviral vectors (RVV) encoding Escherichia coli beta-galactosidase was performed 24 h after partial hepatectomy. In a first group (n = 11), gene delivery was performed by peripheral injection of 2 ml retrovirus-containing medium. For the second group (n = 11), asanguineous perfusion of the regenerating liver after complete vascular exclusion was carried out with 20 ml viral solution. Liver biopsies were performed sequentially in each group. In the first group, beta-galactosidase was expressed at day 7 in 7 +/- 6.3% of hepatocytes and the labeled hepatocytes had disappeared in less than 4 weeks. Polymerase chain reaction experiments demonstrated the elimination of the transduced cells and the appearance of antibodies against beta-galactosidase. Of the 11 rats in the second group, 8 were still able to express beta-galactosidase more than 6 weeks after asanguineous perfusion with no detectable antibody response. Asanguineous perfusion of the regenerating liver with RVV after complete vascular exclusion enabled long-term expression in rats and avoided the immune response present after peripheral delivery in most animals. These results suggest that the immune reaction is secondary to viral infection of antigen presenting cells. Asanguineous perfusion could thus be a way to perform gene therapy for inherited liver diseases without immunosuppressive therapy.

Clinical-scale selection of anti-CD3/CD28-activated T cells after transduction with a retroviral vector expressing herpes simplex virus thymidine kinase and truncated nerve growth factor receptor.

Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.

Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes

Cationic polymers, such as polybrene and protamine sulfate, are typically used to increase the efficiency of retrovirus-mediated gene transfer, however, the mechanism of their enhancement of transduction has remained unclear. As retrovirus transduction is fundamentally limited by the slow diffusion of virus to the target cell surface, we investigated the ability of polybrene to modulate this initial transport step. We compared the ability of both envelope (gp70) and capsid (p30) protein based assays to quantitate virus adsorption and found that p30 based assays were more reliable due to their ability to distinguish virus binding from free gp70 binding. Using the p30 based assay, we established that polybrene concentrations, which yielded 10-fold increases in transduction also, yielded a significant increase in virus adsorption rates on murine fibroblasts. Surprisingly, this enhancement, and adsorption in general, were receptor and envelope independent, as adsorption occurred equivalently on receptor positive and negative Chinese hamster ovary cells, as well as with envelope positive and negative virus particles. These findings suggest that the currently accepted physical model for early steps in retrovirus transduction may need to be reformulated to accommodate an initial adsorption step whose driving force does not include the retrovirus concentration, and the reclassification of currently designated ‘receptor’ molecules as fusion triggers. The implication of these findings with respect to the development of targeted retrovirus-mediated gene therapy protocols is discussed.

Amelioration of retrovirus-mediated gene transfer into hepatocellular carcinoma cells

Recombinant retroviruses are by far the most frequently used vehicle in clinical gene therapy. No serious side-effects have been reported so far in clinical gene therapy trials using recombinant retroviral systems. Low titers of recombinant retroviruses, however, have limited the usefulness of recombinant retroviruses. To improve the efficiency of retrovirus-mediated gene transfer, we previously introduced the polyomavirus early region into amphotropic PA317 cells and established a modified retroviral packaging cell line, PAMP51. We demonstrate here that recombinant retroviruses produced by PAMP51-derived retroviral producing cells have approximately 10-fold higher titers compared with those produced by conventional PA317-derived retroviral producing cells. Importantly, recombinant retroviruses produced by PAMP-derived retroviral producing cells could infect hepatocellular carcinoma cells much more efficiently and could induce much stronger expression of a lacZ reporter gene in HCC cells compared with those produced by PA317-derived ones. These results indicate that recombinant retroviruses prepared from PAMP51-derived retroviral producing cells are much more useful than those prepared from PA317-derived ones and that the use of PAMP51 retroviral packaging cells may open up new avenues for the treatment of various types of cancer including hepatocellular carcinoma.

Fas/CD95 pathway induces mouse liver regeneration and allows for highly efficient retrovirus-mediated gene transfer

Stable gene transfer into hepatocytes has been proposed to compensate for genetic deficiencies that affect liver function, or to deliver diffusible factors into the circulation. This strategy can be achieved using retroviral vectors; however, cell division must occur. We describe a simple and reproductive method that enables the induction of hepatocyte replication in a controlled fashion, thus allowing an efficient in vivo retroviral liver transduction that is applicable to mouse models of human genetic disorders. The approach is based on liver susceptibility to apoptosis via the Fas/CD95 pathway. We show that, 4 days following a single Fas agonist antibody (JO2) injection, hepatocyte replication occurs, the intensity of which is correlated with the level of the induced hepatic cytolysis. This treatment enables in vivo liver transduction, and its efficiency also correlates with the level of hepatic cytolysis. When recombinant retroviral vectors were infused intravenously during the period of hepatocyte replication, 15.4% ± 1.7% of the hepatocytes were transduced, reaching up to 32.5%. (H EPATOLOGY 2001;33:10-15.)

Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin

The low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have impeded studies to optimize the retroviral transduction of stem cells from adult sources. To address this problem, first a cytokine combination was defined that would both maximize the kinetics of adult BM CD34+CD38− cell mitogenesis and minimize the period of prestimulation required for the transduction of these cells by a MSCV-GFP/neor virus in tissue culture dishes in the absence of fibronectin. Three days of stimulation with flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and sufficient to obtain 83% ± 2% GFP+ CD34+CD38− cells, 75% ± 10% G418-resistant clonogenic progenitors, and 50% ± 20% transduced long-term culture-initiating cells as recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this prestimulation protocol, repeated daily exposure to new virus (3×) did not alter the proportion of transduced cells over that obtained with a single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-β2M−/−) mice were also efficiently transduced (10%-20% GFP+ human lymphoid and myeloid cells present 6-8 weeks after transplant) using a 6-day prestimulation and infection protocol. A clinically useful efficiency of retrovirus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activation into cycle and concomitant increased expression of virus receptor transcripts before virus exposure.

Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin

AbstractThe low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have impeded studies to optimize the retroviral transduction of stem cells from adult sources. To address this problem, first a cytokine combination was defined that would both maximize the kinetics of adult BM CD34+CD38− cell mitogenesis and minimize the period of prestimulation required for the transduction of these cells by a MSCV-GFP/neor virus in tissue culture dishes in the absence of fibronectin. Three days of stimulation with flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and sufficient to obtain 83% ± 2% GFP+ CD34+CD38− cells, 75% ± 10% G418-resistant clonogenic progenitors, and 50% ± 20% transduced long-term culture-initiating cells as recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this prestimulation protocol, repeated daily exposure to new virus (3×) did not alter the proportion of transduced cells over that obtained with a single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-β2M−/−) mice were also efficiently transduced (10%-20% GFP+ human lymphoid and myeloid cells present 6-8 weeks after transplant) using a 6-day prestimulation and infection protocol. A clinically useful efficiency of retrovirus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activation into cycle and concomitant increased expression of virus receptor transcripts before virus exposure.

Stimulation of mouse bone marrow cells with kit ligand, FLT3 ligand, and thrombopoietin leads to efficient retrovirus-mediated gene transfer to stem cells, whereas interleukin 3 and interleukin 11 reduce transduction of short- and long-term repopulating cells.

The effects of cytokine stimulation during retroviral transduction on in vivo reconstitution of mouse hematopoietic stem cells was tested in a murine competitive repopulation assay with alpha-thalassemia as a marker to distinguish donor and recipient red blood cells (RBCs) and the enhanced green fluorescent protein (EGFP) as a marker for gene transfer. After transplantation, EGFP was detected in up to 90% of circulating RBCs, platelets, and leukocytes, and in primitive progenitors in bone marrow (BM), spleen, and thymus of individual transplanted mice for observation periods of more than 6 months. Large quantitative differences in reconstitution were observed after transplantation with graded numbers (1000-30, 000) of EGFP(+) cells preconditioned with various combinations of Kit ligand (KL), FLT-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), and IL-11. Relative to nonmanipulated BM cells, repopulation of EGFP(+) cells was maintained by KL/FL/TPO stimulation, but approximately 30-fold reduced after KL/FL/TPO/IL-3, or KL/FL/IL-3/IL-11. These differences were not caused by changes in the ability of immature hematopoietic cells to home to the BM, which was only moderately reduced. In conclusion, these quantitative transplantation studies of mice demonstrate the importance of optimal ex vivo cytokine stimulation for gene transfer to stem cells with retention of their in vivo hematopoietic potential, and also emphasize that overall in vitro transduction frequency does not predict gene transfer to repopulating stem cells.

Delivery of a Retroviral Vector Expressing Human β-Glucuronidase to the Liver and Spleen Decreases Lysosomal Storage in Mucopolysaccharidosis VII Mice

Mucopolysaccharidosis VII (MPS VII) is caused by beta-glucuronidase (beta-gluc) deficiency and results in lysosomal storage due to the inability to degrade glycosaminoglycans. Transfer of a beta-gluc gene into the liver reduces hepatic pathology as well as storage in other organs via uptake of secreted protein. A Moloney murine leukemia-based retroviral vector expressing the human beta-gluc cDNA was injected intravascularly into MPS VII mice during hepatocyte replication, which was induced with im injection of an adenoviral vector that transiently expressed hepatocyte growth factor (Ad.CMV. HGF). This procedure resulted in transduction of approximately 1% of hepatocytes, 1% of normal liver enzyme activity, and a reduction in lysosomal storage in the liver at 3.5 months. Surprisingly, controls that received retroviral vector without HGF had transduction of nonparenchymal cells in the liver, significant levels of enzyme and RNA in the liver at 2 but not 3.5 months, and reduced lysosomal storage at 3.5 months. Transduction was also achieved in the replicating cells of the spleen, where lysosomal storage was reduced. An approach using a retroviral vector without a growth factor might temporarily reduce lysosomal storage in the liver and spleen in humans. Addition of HGF might be used to augment and prolong gene transfer.

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

In vivo retrovirus-mediated gene transfer into lamb liver.

Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo has been previously reported in the rat. Before considering human applications of these techniques in the treatment of inherited liver diseases, it was necessary to document its efficiency in a large animal model. Lamb was choosen because the liver was similar to human liver regarding size and anatomy. To induce hepatocyte division which is necessary for infection with retroviral particles, animals were subjected to a left hepatectomy. Kinetics of liver regeneration were assessed on sequential liver biopsies after partial hepatectomy in order to provide an evaluation of the peak of maximal liver regeneration in a first animal group. Recombinant retroviruses encoding a reporter gene (E. coli beta galactosidase) were then perfused through the portal vein of the regenerating liver in a second animal group. The more intense liver regeneration occurred from one to 6 days after partial hepatectomy, with the highest thymidine kinase rate and MIB-1 antibody staining on the second day. The proportion of genetically modified lamb hepatocytes expressing the reporter gene was less than 1%, despite the use of higher titers of retroviral particles than those described in previous reports. The results obtained in rodent livers with this in vivo gene transfer methodology cannot currently be scaled up in a large ruminant model. The efficacy of vectors has to be tested in other large mammals before planning gene therapy trials for the treatment of inherited liver diseases.

Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells.

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic approach for a number of diseases. To overcome the difficulty in generating specific CTLs, we established stable artificial antigen-presenting cells (AAPCs) that can be used to stimulate T cells of any patient of a given human leukocyte antigen (HLA) type. Mouse fibroblasts were retrovirally transduced with a single HLA-peptide complex along with the human accessory molecules B7.1, ICAM-1, and LFA-3. These AAPCs consistently elicit strong stimulation and expansion of HLA-restricted CTLs. Owing to the high efficiency of retrovirus-mediated gene transfer, stable AAPCs can be readily engineered for any HLA molecule and any specific peptide.

In situ Perfusion of the Liver Enhances the Efficiency of Retrovirus-Mediated Gene Transfer to Hepatocytes

To increase the efficiency of retrovirus-mediated gene transfer targeting an individual’s liver in vivo, the liver was perfused in situ with the retrovirus vector during hepatic cold ischemia. Four weeks prior to gene transfer, the spleen was transpositioned to the left subcutaneous position to develop a portosplenic shunt, which was performed in order to prevent intestinal congestion during hepatic ischemia. Traditional retrovirus vectors (1 × 10⁵ CFU/ml) which encode genes for the Escherichia coli β-galactosidase (LacZ) were used in this study. Twenty-four hours after partial hepatectomy (70%), the remnant liver was surgically isolated, perfused with 1 ml of vector solution through the portal vein, and kept in contact with the vector for 30 min under cold ischemia (group 1). Hepatic ischemia could thus be performed without any intestinal congestion, due to the preestablished portosystemic shunt. In group 2, the liver was perfused with 1 ml of vector solution through the portal vein without in situ perfusion of the liver. Animals were sacrificed 1, 3, 7 and 28 days after gene transfer. In X-gal staining, the transferred LacZ was detected positive in 10–15% of the hepatocytes only in group 1, 3 days after gene transfer. Graft histology and a liver function test showed no difference between both groups 24 h after gene transfer. In conclusion, in situ perfusion of the liver greatly enhanced the efficacy of retrovirus-mediated gene transfer, targeting an individual’s liver in vivo.