Mixed hematopoietic molecular chimerism results in permanent transgene expression from retrovirally transduced hepatocytes in mice

BACKGROUND: Cytotoxic immune elimination of transduced hepatocytes may limit gene therapy for inherited liver diseases. Using beta-galactosidase as a marker gene, we studied whether creation of mixed beta-galactosidase molecular hematopoietic chimerism could induce tolerance to beta-galactosidase-transduced hepatocytes. METHODS: Molecular hematopoietic chimerism was established in irradiated recipient mice by transplantation of either a mixture of wild-type and beta-galactosidase-transgenic bone marrow or autologous bone marrow stem cells that were transduced with beta-galactosidase lentiviral vectors. After transplantation, mice were hepatectomized and injected with beta-galactosidase recombinant retroviruses to transduce regenerating hepatocytes. We monitored the presence of beta-galactosidase-expressing hepatocytes as well as the appearance of anti-beta-galactosidase antibodies during the time. RESULTS: In control animals, anti-beta-galactosidase antibodies and cytotoxic T-lymphocyte (CTL) response developed as early as 3 weeks after gene transfer. Transduced hepatocytes disappeared concomitantly. In bone marrow transplanted mice, tolerance could be observed in a significant proportion of animals. Tolerance resulted in permanent liver transgene expression and was absent unless a chimerism above 1% was achieved, demonstrating a threshold effect. CONCLUSIONS: Creation of a molecular hematopoietic chimerism can result in transgene tolerance and evade immune rejection of retrovirally transduced hepatocytes. This strategy may be useful for hepatic inherited diseases in which the transgene product behaves as a non-self protein.     

Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver

Adenovirus-mediated gene transfer has become an important tool with which to introduce genetic material into cells. Available data emphasize efficient adenoviral transduction of parenchymal liver cells (i.e., hepatocytes) in both in vitro and in vivo model systems, typically in normal cells. The aim of this study was to evaluate gene transfer to nonparenchymal (and parenchymal) cells of the normal and injured rat liver. Hepatocytes, stellate cells, and endothelial cells were isolated by standard methods. Liver injury was induced by bile duct ligation or carbon tetrachloride administration. Cells were transduced in vitro with an adenovirus encoding beta-galactosidase (Ad.beta-gal) over a range of viral titers, and transduced cells were identified by detection of X-gal. In vivo transduction efficiency was studied in normal and injured livers using cell isolation techniques. Nonparenchymal cells were transduced with greater frequency than hepatocytes at all adenoviral titers tested, both in vitro and in vivo. After liver injury, adenoviral transduction was reduced for all liver cell types compared with that for cells from normal livers (at all virus titers). Notably, transduction efficiency remained greater in nonparenchymal cells than in hepatocytes after liver injury. This work implies that, to achieve comparable gene expression in the injured liver, higher adenoviral titers may be required, an important consideration as gene therapy in disease states is considered.     

Therapeutic potential of hepatocyte transplantation

Liver repopulation with transplanted cells offers unique opportunities for treating a variety of diseases and for studies of fundamental mechanisms in cell biology. Our understanding of the basis of liver repopulation has come from studies of transplanted cells in animal models. A variety of studies established that transplanted hepatocytes as well as stem/progenitor cells survive, engraft, and function in the liver. Transplanted cells survive life-long, although cells do not proliferate in the normal liver. On the other hand, the liver is repopulated extensively when diseases or other injuries afflict native hepatocytes but spare transplanted cells. The identification of ways to repopulate the liver with transplanted cells has greatly reinvigorated the field of liver cell therapy. The confluence of insights in stem/progenitor cells, transplantation immunology, cryobiology, and liver repopulation in specific models of human diseases indicates that the field of liver cell therapy will begin to reap the promised fruit in the near future.     

Improved hepatic gene transfer by using an adeno-associated virus serotype 5 vector

Adeno-associated viral (AAV) vectors have been shown to direct stable gene transfer and expression in hepatocytes, which makes them attractive tools for treatment of inherited disorders such as hemophilia B. While substantial levels of coagulation factor IX (F.IX) have been achieved using AAV serotype 2 vectors, use of a serotype 5 vector further improves transduction efficiency and levels of F.IX transgene expression by 3- to 10-fold. In addition, the AAV-5 vector transduces a higher proportion of hepatocytes ( approximately 15%). The subpopulations of hepatocytes transduced with either vector widely overlap, with the AAV-5 vector transducing additional hepatocytes and showing a wider area of transgene expression throughout the liver parenchyma.     

Oligonucleotide-mediated gene targeting in human hepatocytes: implications of mismatch repair

Gene therapy using viral vectors for liver diseases, particularly congenital disorders, is besought with difficulties, particularly immunologic reactions to viral antigens. As a result, nonviral methods for gene transfer in hepatocytes have also been explored. Gene repair by small synthetic single-stranded oligodeoxynucleotides (ODNs) produces targeted alterations in the genome of mammalian cells and represents a great potential for nonviral gene therapy. To test the feasibility of ODN-mediated gene repair within chromosomal DNA in human hepatocytes, two new cell lines with stably integrated mutant reporter genes, namely neomycin and enhanced green fluorescent protein were established. Targeting theses cells with ODNs specifically designed for repair resulted in site-directed and permanent gene conversion of the single-point mutation of the reporter genes. Moreover, the frequency of gene alteration was highly dependent on the mitotic activity of the cells, indicating that the proliferative status is an important factor for successful targeting in human hepatocytes. cDNA array expression profiling of DNA repair genes under different cell culture conditions combined with RNA interference assay showed that mismatch repair (MMR) in actively growing hepatocytes imposes a strong barrier to efficient gene repair mediated by ODNs. Suppression of MSH2 activity in hepatocytes transduced with short hairpin RNAs (shRNAs) targeted to MSH2 mRNA resulted in 25- to 30-fold increase in gene repair rate, suggesting a negative effect of MMR on ODN-mediated gene repair. Taken together, these data suggest that under appropriate conditions nonviral chromosomal targeting may represent a feasible approach to gene therapy in liver disease.     

Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction

Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about approximately 5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only approximately 5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5'-bromo-2'-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic beta-galactosidase. Colabeling for beta-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.     

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.     

Potential of bioengineering processes for therapeutic repopulation of the liver with cells

Multiple unique aspects of liver biology make this organ an excellent paradigm for novel cell and gene therapy applications. In recent years, insights were obtained into how transplanted cells engraft and proliferate in the liver, including in the context of pre-existing disease. Also, a variety of animal models were studied to establish the basis of cell and gene therapy applications in specific disorders. Through ongoing research activity, additional mechanisms in liver repopulation have been uncovered, where manipulation of specific cell compartments and cellular processes,e.g., those aimed at extracellular matrix component receptors or soluble signals in transplanted and native cells can be exploited for enhancing cell engraftment and proliferation. Such studies demonstrate the possibility of applying biotechnology and/or bioengineering principles to organ replacement aimed at cell and gene therapy. Joining of these disciplines with research in stem cell biology, particularly in efforts concerning targeting of transplanted stem cells to given organs with achievement of lineage-specific cell differentiation and function, will be particularly important for future cell and gene therapy applications. This review offers an overview of relevant mechanisms in liver repopulation.     

Therapeutic liver repopulation for the treatment of metabolic liver diseases

Orthotopic liver transplantation is the treatment of choice for many inherited and acquired liver diseases. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. In contrast, hepatocyte suspensions can be isolated from a single donor liver can be transplanted into several hosts, and this procedure may help overcome the shortage in donor livers. In classic hepatocyte transplantation, however, only 1% of the liver mass or less can be replaced by donor cells. Recently though, we have used a mouse model of hereditary tyrosinemia to show that > 90% of host hepatocytes can be replaced by a small number of transplanted donor cells in a process we term "therapeutic liver repopulation". This phenomenon is analogous to repopulation of the hematopoietic system after bone marrow transplantation. Liver repopulation occurs when transplanted cells have a growth advantage in the setting of damage to recipient liver cells. Here we will review the current knowledge of this process and discuss the hopeful implications for treatment of liver diseases.     

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)^–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.     

Long-term expression of the human glucocerebrosidase gene in vivo after transplantation of bone-marrow-derived cells transformed with a lentivirus vector

BACKGROUND: Gaucher disease is a lysosomal storage disorder resulting from a deficiency of glucocerebrosidase (GC). Recently, lentivirus vectors have been developed for efficient gene transfer into hematopoietic stem cells (HSCs). A recombinant lentivirus vector was used to evaluate the transduction of the human GC gene into murine bone-marrow-derived HSCs and its expression in their progeny. METHODS: Murine HSCs were transduced with lentivirus vector (lenti-EF-GC; MOI = 10-100). We transplanted female wild-type C57BL/6J mice with genetically modified male HSCs via the tail vein. RESULTS: We show that intravenous transplantation of transduced HSCs has therapeutic potential. Enzyme activity was increased two- to three-fold in various tissues, especially in the hematopoietic system. Numerous transplanted HSCs survived for 6 months and were shown by PCR to contain the provirus genes; the Y chromosome was identified by FISH analysis in the cells of female mouse recipients. CONCLUSIONS: The recombinant lentiviral vector transduces HSCs that are capable of long-term gene expression in vivo. This approach is potentially useful for the treatment of patents with Gaucher disease and other lysosomal storage disorders.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

High sensitivity of neonatal rat hepatocytes to retroviral-mediated gene transfer and their transplantation into the spleen of adult rat.

To optimize conditions for retroviral-mediated transfer of a recombinant gene to hepatocytes, the pMNSM-Tk-lacZ vector, which we had constructed to express the bacterial beta-galactosidase gene, was transduced to rat hepatocytes under various conditions, and the expression of beta-galactosidase activity was examined by cytochemical staining. Compared to the hepatocytes of adult rats, those of newborns were about 50-100 times more sensitive to transduction with the beta-galactosidase gene in vitro. The sensitivity was high in the newborn hepatocytes when the virus was infected on days 1 and 2 after initiation of culture. However, the sensitivity to infection did not correlate with the DNA synthetic activity. The gene transfer was feasible not only to hepatocytes in monolayer culture but also to those in spheroid culture. The spheroidal aggregates containing hepatocytes transduced with the beta-galactosidase gene could be transplanted into the spleen of syngenic adult rat, although the expression was very low.     

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector

BACKGROUND: Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. METHODS: Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. RESULTS: In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. CONCLUSIONS: In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: PBL can mediate the efficient transduction of hepatocytes in vivo after a single intravenous injection of recombinant retroviruses. This approach is feasible in humans.     

Development and application of gene therapy technologies

The success of hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency (X-SCID) was a major breakthrough in the field of gene therapy. However, two patients treated with this gene therapy developed leukemia at a later time, and retroviral vector-mediated gene transfer was considered to trigger leukemogenesis; i.e. insertional mutagenesis caused activation of LMO 2 gene, which was one step toward leukemia development. To cope with this serious problem, basic studies are required to improve the safety of retroviral vectors and to develop the method for site-specific integration of transgenes. In addition, we have to develop technologies such as selective amplifier genes (SAGs), the system for selective expansion of transduced cells, in order to obtain therapeutic efficacy of hematopoietic stem cell gene therapy in many other disorders. Moreover, clinical applications of AAV vector are promising from the standpoint of safety issue, because this vector is derived from non-pathogenic virus. AAV vector is appropriate for gene transfer into neurons, muscles, and hepatocytes. For example, gene therapy for Parkinson's disease is investigated using AAV vectors. Genetic manipulation is also one of the indispensable technologies in the field of regeneration medicine, and further promotion of basic research is important.     

Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with beta-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 x 10(12) vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression.     

Cell transplantation causes loss of gap junctions and activates GGT expression permanently in host liver

Cell transplantation into hepatic sinusoids, which is necessary for liver repopulation, could cause hepatic ischemia. To examine the effects of cell transplantation on host hepatocytes, we transplanted Fisher 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. Within 24 h of cell transplantation, areas of ischemic necrosis, along with transient disruption of gap junctions, appeared in the liver. Moreover, host hepatocytes expressed gamma-glutamyl transpeptidase (GGT) extensively, which was observed even 2 years after cell transplantation. GGT expression was not associated with alpha-fetoprotein activation, which is present in progenitor cells. Increased GGT expression was apparent after transplantation of nonparenchymal cells and latex beads but not after injection of saline, fragmented hepatocytes, hepatocyte growth factor, or turpentine. Some host hepatocytes exhibited apoptosis, as well as DNA synthesis, between 24 and 48 h after cell transplantation. Changes in gap junctions, GGT expression, DNA synthesis, and apoptosis after cell transplantation were prevented by vasodilators. The findings indicated the onset of ischemic liver injury after cell transplantation. These hepatic perturbations must be considered when transplanted cells are utilized as reporters for biological studies.     

CD8(+) T-cell responses to adeno-associated virus capsid in humans

Hepatic adeno-associated virus (AAV)-serotype 2 mediated gene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid-specific memory CD8(+) T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid-specific CD8(+) T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.