Cytotoxic immune response blunts long-term transgene expression after efficient retroviral-mediated hepatic gene transfer in rat (original) (raw)
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Journal of Hepatology, 2004
Background/Aims: Gene therapy for inherited liver diseases requires permanent expression of the therapeutic gene. However, in vivo liver transduction with retroviral vectors triggers an immune elimination of transduced hepatocytes. Here we investigated whether immune response could be prevented by treatment with compounds known to induce tolerance in organ transplantation: CTLA4Ig and LF-15-0195. Methods: CTLA4Ig was administered either via i.p. injection of the drug or by i.m. injection of recombinant adenoviruses encoding CTLA4Ig. LF-15-0195 was administered i.p. All animals were subjected to partial hepatectomy and received b-galactosidase retroviral vectors intravenously. Appearance of anti-b-galactosidase antibodies was monitored and the number of positive hepatocytes was assessed at day 7 and at sacrifice. Results: No b-galactosidase antibodies were detected as long as CTLA4Ig was detectable in serum. Short-term treatment with CTLA4Ig induced tolerance in a significant proportion of animals only at high dose (1 mg/kg). Administration of CTLA4Ig adenovectors resulted in prolonged secretion of CTLA4Ig and permanent absence of anti-b-galactosidase antibodies. LF-15-0915 administration achieved tolerance in some animals. Conclusions: In conclusion, manipulation of the immune system at the time of virus delivery using clinically relevant tolerance-inducing protocols is a promising approach to achieve long term expression after retrovirus-mediated gene transfer to the liver.
Retrovirus-mediated transduction of adult hepatocytes
Proceedings of the National Academy of Sciences, 1988
Retrovirus-mediated gene transfer was used to develop a method for introducing genes into primary cultures of adult rat hepatocytes. Subconfluent monolayers of hepatocytes, cultured in hormonally defined media on different matrix substrata, were infected with helper-free stocks of a replication-defective retrovirus that constitutively expresses high levels of fi-galactosidase. Retrovirus-mediated transduction was measured by two methods: (a) an in situ cytochemical stain that specifically detects the expression of viral expressed (3-galactosidase, and (ii) Southern blot analysis, which mea-
Retrovirus Vectors: Comparison of VSV-G and Amphotropic Vectors for Hepatic Gene Transfer
1995
Recombinant retrovirus vectors are widely used for gene transfer studies. The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo. A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector. Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli �-galactosidase-producing pseudotype and amphotropic vector. High rates of �-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer. Subsequent studies demonstrated ...
Biochemical and Biophysical Research Communications, 1996
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 -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 for in vivo liver gene therapy.
Proceedings of The National Academy of Sciences, 1987
The liver is an important target for potential gene therapy because of the critical role it plays in intermediary metabolism and synthesis of serum proteins. We report the use of retroviral vectors for transfer of recombinant genes into primary mouse hepatocytes. Hepatocytes were grown in a defined serum-free medium and expressed liver-specific functions for up to 14 days. Hepatocytes were transformed to Geneticin (G418) resistance by infection with recombinant retroviruses carrying the Tn5 neomycin-resistance gene. The G418-resistant cells exhibited characteristic hepatocyte morphology and continued to express liver-specific gene function. A retrovirus that expresses neomycin resistance driven by a herpes simplex thymidine kinase promoter produced the most efficient transformation compared with viruses using the retroviral long terminal repeat promoter or the simian virus 40 early-region promoter. These experiments indicate that primary hepatocytes can be successfully cultured and transformed with recombinant genes using retroviral vectors. These results provide a model for future somatic gene replacement therapy in which functional genes can be introduced into hepatocytes by viral-mediated gene transfer.
Gene expression in implanted rat hepatocytes following retroviral-mediated gene transfer
Somatic Cell and Molecular Genetics, 1989
An hepatocyte transplantation-gene transfer protocol has been developed whereby liver cells containing an expressing NeoRgene can be successfully implanted in vivo. Adult primary cultures of rat hepatocytes, after infection with the retroviral vector N2, were grown on a floating solid support (coated with purified collagen IV) in a serum-free hormonally defined medium designed for hepatocytes that also contained G418. Under these conditions, normal adult hepatocytes expressing the Neo R gene could be grown to high density. The solid supports holding the gene-engineered hepatocytes were then implanted into adult rats into subcutaneous and intraperitoneal sites. After one to two weeks, the supports were removed and shown to still contain the gene-engineered hepatocytes expressing the Neo R gene. These results suggest that cells from solid organs, such as the liver, are potential targets for gene transfer and expression studies in vivo.
Expression of Retrovirally Transduced Genes in Primary Cultures of Adult Rat Hepatocytes
Proceedings of The National Academy of Sciences, 1987
Differentiated primary rat hepatocyte cultures have been infected with retroviral vectors expressing human hypoxanthine/guanine phosphoribosyltransferase or the transposon Tn5 neomycin-resistance gene. Expression of the markers was detected only after infection of the cells during a short period of cell replication and transient dedifferentiation from days 1 to 5 of culture. Provirus integrated during that period remains fully expressed during the entire subsequent stationary period of culture up to at least 25 days. Selection with the neomycin analogue G418 of cells infected with the neomycin vector led to the appearance of cells with hepatocyte morphology in which newly synthesized albumin was detectable by immunoprecipitation, indicating successful infection of hepatocytes.
2009
Background: Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein. Major Findings: AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic b-galactosidase (b-gal) was performed in immune competent mice, followed by a secondary b-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in ,2% of hepatocytes almost completely protected from inflammatory T cell responses against b-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, ,10% of hepatocytes continued to express b-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8 + T cell responses to b-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer. Conclusions: These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.
Journal of virology, 1996
Recombinant retrovirus vectors are widely used for gene transfer studies. The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo. A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector. Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli beta-galactosidase-producing pseudotype and amphotropic vector. High rates of beta-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer. Subsequent studies demonst...