Introduction

Liver cirrhosis (LC) is a worldwide problem. In North America and Europe, the prevalence is approximately 1000 per million population, and mortality is approximately 100 per million, which is also the case in Japan.^1,2,3 Among many etiological factors such as hepatitis virus infection, alcohol and drug abuse, chronic infection by the hepatitis C virus (HCV) is the major cause of LC in Japan and in some southern European countries such as Italy and Spain. HCV-carrier frequency is estimated at 2% worldwide, and chronic hepatitis C progresses to LC and then hepatocellular carcinoma at a high incidence within two or three decades.

While several therapeutic approaches have been investigated for HCV infection, such as iron reduction, antioxidants and antiviral agents (Amantadine, Ribavirin),⁴ the most effective therapy is currently considered to be IFN treatment.^2,5,6 Among interferons (IFNs), IFN- and IFN- are widely used to treat HCV infection in clinics, whereas IFN- does not have a significant antiviral effect. In addition to the antiviral effect, several lines of evidence suggested that IFN- is also effective in curtailing LC in experimental animal models and clinical studies.^{6,7,8,9,10,11,12,13} IFN- is therefore expected to be particularly powerful in preventing the development of LC in patients with HCV infection.

In the conventional IFN regimen, the recombinant IFN- protein is subcutaneously injected for several months. However, the overall sustained virologic response rate is still limited to 20–30%.^2,6 Although dose escalation is expected to be effective in a resistant case,⁶ it is often impossible because of the severe adverse effects such as flu-like symptoms, leukopenia and mental depression. Since the half-life of the IFN- protein is 3 h in the blood circulation and only 0.01% of subcutaneously injected IFN- can reach the target organ (the liver),¹⁴ the administration of IFN- through a subcutaneous route requires higher IFN- levels in the serum than in the liver. Therefore, vector-mediated local IFN- production in the liver may be able to overcome the limitations of conventional IFN- protein therapy.

In this study, we examined whether a gene therapy strategy of targeting IFN- gene expression to the liver can be a better alternative to conventional IFN- protein administration to the subcutaneous space with respect to the distribution of the IFN- protein, period of expression and development of any adverse effect. An adenovirus vector was employed, since intravenous injection of the vector could express transgenes efficiently in the liver,¹⁵ and the biological effect of the adenovirus-mediated IFN- gene therapy was also evaluated in rats with dimethylnitrosamine (DMN)-induced LC.^16,17,18 This model showed the confinement of the IFN- expression to the liver and the therapeutic efficacy of suppressing liver injury and fibrosis. Moreover, we demonstrated the usefulness of the Cre-mediated regulation system for shutting off the IFN- transgene expression efficiently in vitro and in vivo. The study suggests for the first time the feasibility of IFN- gene therapy for HCV-associated liver diseases.

Results

Intravenous adenovirus vector injection into rats with DMN-induced cirrhotic liver

In rats with DMN treatment for 3 weeks, all liver specimens from 14 rats, which were killed for examination of LC development (n=5) and vector distribution (n=9), showed the collapse of parenchymal cells and the formation of regenerative nodules separated by extensive fibrous septa, which are similar to the characteristic pathological changes observed in human LC (Figure 1a). To examine the adenovirus-mediated gene transduction efficiency into the liver, AxCA-lacZ or ADVCA-AP was intravenously injected into rats with DMN-induced LC. The expression of lacZ and AP genes was mainly observed in the liver, especially in septal cells but not in the hepatocytes (Figure 1c and inset).¹⁵ Except for a faint expression in the spleen and kidney, X-gal staining was not detected in major organs such as the lung, heart, small intestine and testis. To confirm the preferential transgene expression in the liver, we examined the lacZ gene expression in various organs of cirrhotic rats transduced with AxCA-lacZ by the RT-PCR method. The expression of the lacZ gene was mainly observed in the liver, and faint expression was detected in the lung, kidney and small intestine (Figure 1e). In normal rats injected with saline, X-gal staining was detected in septal cells as well as in hepatocytes of the liver (Figure 1d) and faint staining was observed in the spleen. The lacZ gene expression data were consistent with the immunostaining of IFN- in the rats with DMN-induced LC or normal rats after the injection of AxCA-rIFN (data not shown).

Figure 1.

Intravenous injection of AxCA-lacZ into rats. (a, b) Hematoxylin–eosin staining ( 40) of liver from rats treated with DMN (a) or with saline (b) for 3 weeks. (c, d) X-gal reaction ( 100) of liver from rats treated with DMN (c) or with saline (d) for 3 weeks followed by AxCA-lacZ transduction. Rats were infused once via tail vain with AxCA-lacZ (1 10⁷ PFU). (c, inset) AP staining of the DMN-induced LC after the intravenous injection of ADVCA-AP (1 10⁷ PFU) ( 400). P, parenchyma; S, fibrous septa. Brown-stained cells were observed in fibrous septa. X-gal and AP staining was performed as described.^47,49 (e) RT-PCR analysis of organ distribution of lacZ gene expression 2 days after the intravenous injection of AxCA-lacZ. The untreated normal liver did not show any PCR band.

Full figure and legend (131K)

IFN- levels in the liver and serum following AxCA-rIFN injection

Based on in situ evidence suggesting the preferential expression of INF- in the liver, the IFN- levels and time course of its clearance were measured in the liver and serum following the intravenous injection of AxCA-rIFN. In total, 120 IU of the rat IFN- per gram of tissue was produced in the liver of DMN-injected rats 3 days after the injection of AxCA-rIFN, whereas no IFN- was detected in the serum (Figure 2). On the other hand, a significant level of rat IFN- transiently appeared in the liver and in the serum as well by the subcutaneous injection of 1 10⁵ IU of the recombinant rat IFN- (Figure 2), which corresponds to the dose range of conventional IFN- treatment in clinics. To assess the toxicity of the adenovirus-mediated IFN- gene therapy, hepatic transaminases were measured in DMN-injected rats 2 days after the injection of AxCA-rIFN. No significant difference was observed among the rats injected either with PBS, AxCA-lacZ or AxCA-rIFN (AST; 257.0135.2, 267.7171.7, 264.270.9, ALT; 226.3122.1, 233.3135.9, 211.764.3, respectively), suggesting that the IFN- gene expression did not cause significant hepatic toxicity.

Figure 2.

Time course of the rat IFN- expression in the liver and serum. After treatment with DMN for 3 weeks, rats were injected once with recombinant rat IFN- (open circle; 0.1 MIU, s.c., n=3), AxCA-rIFN (solid square; 1 10⁷ PFU, i.v., n=3), or AxCA-lacZ (open triangle; 1 10⁷ PFU, i.v., n=3).

Full figure and legend (25K)

Improvement of LC in DMN-treated rats by intravenous injection of AxCA-rIFN

We examined whether hepatic INF- expression could improve liver fibrosis in a DMN-injected rat, because the antifibrotic effect of IFN- protein has been shown in experimental animal models and clinical studies.^{6,7,8,9,10,11,12,13} The AxCA-rIFN-treated rats survived significantly longer than the rats injected with either AxCA-lacZ or PBS (P=0.0001), and five of nine IFN--transduced rats survived more than 100 days (Figure 3). Histological examination clearly revealed less fibrous connective tissue components in Glisson's sheath and pseudolobule formations in the livers of control and AxCA-rIFN-transduced rats than in the liver of the AxCA-lacZ-transduced rats, in which the extensive formation of fibrotic septa and thickened reticulin fibers joining central areas were observed (Figures 1a, 4a and b). The collagens were detected by Sirius red staining^19,20 in the bridging fibrous septa in the liver of AxCA-lacZ-transduced rats, whereas the liver of AxCA-rIFN-transduced rats was almost devoid of collagens (Figure 4c and d). The expression of -smooth muscle actin was reduced in the activated stellate cells of the DMN-treated rats transduced with AxCA-rIFN (Figure 4e and f). To quantitatively assess the liver fibrosis, we measured the hydroxyproline content of the liver²¹ and serum levels of transaminases (AST and ALT). The hydroxyproline and transaminases in AxCA-rIFN-treated rats 70 days after the injection were significantly lower than those in AxCA-lacZ- or PBS-injected rats 10 days after the injection (Table 1). Since it is known that the DMN-induced histopathological changes and hepatic transaminase elevation do not regress spontaneously in the rat LC model,^16,18,22 our observation suggested that IFN- gene expression can rescue the rats from DMN-induced hepatic damage and improve the histopathology of LC.

Figure 3.

Survival of rats transduced with AxCA-rIFN. DMN-treated rats were intravenously injected once with PBS (n=10), AxCA-rIFN (1 10⁷ PFU, n=9), or AxCA-lacZ (1 10⁷ PFU, n=10) after treatment of DMN for 3 weeks. Life-table analysis is presented as a Kaplan–Meier plot.

Full figure and legend (15K)

Figure 4.

Improvement of DMN-induced rat LC by the injection of AxCA-rIFN. (a, b) Liver sections stained with Azan-Mallory ( 40). (c, d) Differential staining of collagenous and noncollagenous proteins with Sirius red and Fast green ( 100). (e, f) Immunohistochemical staining of the liver using antibody against -smooth muscle actin ( 40). (a, c, e) A rat injected with AxCA-rIFN, and survived from the DMN-induced LC. Killed 70 days after the vector injection. (b, d, f) A rat treated with DMN and then injected with AxCA-lacZ. Killed 10 days after the vector injection.

Full figure and legend (712K)

Table 1 - Markers of hepatic fibrosis and damage.

Full table

Expression of MMPs and TGF- in the liver transduced with AxCA-rIFN

To examine the antifibrotic action of IFN- gene therapy, the expression of metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP) related to matrix degradation during liver fibrosis was analyzed by the RT-PCR method (Figure 5). Interstitial collagenase (MMP-13) has been considered an essential enzyme for collagenolysis in liver fibrosis, and it was reported that expression of MMP-13 is elevated at peak fibrosis and drops rapidly in the recovery periods.^23,24 The MMP-2, which is stimulated by TGF-, is necessary for proliferation and infiltration of hepatic stellate cells in the process of fibrosis formation.^25,26,27 The expression of TIMP-1 increases in the process of liver fibrosis to promote progression of liver fibrosis by preventing degradation of secreted collagens and decreases in the recovery phase.²⁸ In this study, the expression of MMP-13, MMP-2 and TIMP-1 was downregulated in the liver treated with AxCA-rIFN compared with the PBS- or AxCA-lacZ-injected cirrhotic liver, supporting the antifibrotic action of the AxCA-rIFN.

Figure 5.

Expression of MMPs and TIMP in the liver. The expression of MMP-13, MMP-2 and TIMP-1 mRNA was analyzed in the DMN-treated rats injected with the vectors by the RT-PCR method.

Full figure and legend (97K)

Furthermore, since it is reported that overexpression of TGF- plays a pivotal role in the progression of fibrosis,⁹ we examined TGF- expression in the liver. The control value of TGF- in untreated normal rats was 5.51.0 ng/g (n=5). Among the rats with DMN-induced LC, AxCA-rIFN-transduced animals showed a lower TGF- expression level (40.27.4 ng/g) than did the PBS-injected rats (71.36.0 ng/g). TGF- immunoreactivity was detected in the liver of an AxCA-lacZ-transduced rat, showing evidence of LC, and by contrast, TGF- was not detected in the AxCA-rIFN-transduced rat (data not shown). The data suggested that IFN- gene transfer suppresses TGF- induction in the process of DMN-induced hepatic fibrogenesis.

Regulation of IFN- expression by Cre-loxP reaction

Although not apparent in our rat experiment, it is known from clinical experience that IFN protein treatment may cause acute adverse effects. Thus, to examine a shut-off system of IFN- expression based on the Cre recombinase–loxP reaction in the adenovirus vector, an adenovirus vector harboring the rIFN- gene between two loxP sites (AxCALNL–rIFN) was constructed (Figure 6a). The intravenous injection of AxCALNL–rIFN was able to produce approximately 120 IU/g of IFN- in the liver similar to the AxCA-rIFN injection (Figure 2 and Figure 6), suggesting both vectors have equal potential for treating cirrhotic liver. First, NBT-2 cells were transfected with AxCALNL-rIFN, and 2 days later a 100 times larger amount of an adenovirus vector expressing the Cre recombinase (AxCAN-Cre) was added to the culture medium to excise the IFN- gene from AxCALNL-rIFN DNA. As shown in Figure 6b, the 783 bp vector fragment containing the rat IFN- gene disappeared, and the loxP-flanked excised fragment (225 bp) was detected by PCR analysis in NBT-2 cells only after the treatment of AxCAN-Cre. Rat IFN- production was also significantly suppressed in the NBT-2 cells in vitro by the treatment of AxCAN-Cre (Figure 6c).

Figure 6.

Regulation of IFN- expression by Cre–loxP reaction. (a) Structure of AxCALNL-rIFN. PCR primers are shown by arrows together with the Cre recombination products. (b) PCR detection of the IFN- transgene fragment cleaved by superinfection with AxCAN-Cre (moi 3000) 2 days after the transduction with AxCALNL-rIFN (moi 30). The PCR primers amplified a fragment containing Cre (1275 bp), a fragment containing rIFN- flanked by two loxP sequences on both ends (783 bp) and the corresponding fragment with the loxP-rIFN-loxP portion excised off by Cre (225 bp). (c, d) Rat IFN- expression measured by ELISA in NBT-2 cells in vitro (c) and in the rat liver in vivo (d) showing an effective suppression of the IFN- transgene expression. Open circle, AxCALNL-rIFN (n=3); solid triangle, AxCALNL-rIFN+AxCAN-Cre (n=3); open square, AxCA-lacZ (n=3).

Full figure and legend (122K)

Next, normal rats were sequentially transduced via the tail vein, first with AxCALNL-rIFN and then 2 days later with AxCAN-Cre. Rat IFN- expression was again significantly decreased in the liver of the rats injected with AxCAN-Cre as compared to the rats without AxCAN-Cre injection (Figure 6d).

Discussion

Among preclinical models of liver fibrosis, those induced by the chronic carbon tetrachloride (CCl₄) intoxication or ligation of bile duct are often used. However, the reversibility of hepatic fibrosis has been reported in these models.²⁴ Therefore, we employed a DMN-induced model, in which fibrosis and hepatic transaminase elevation do not spontaneously regress in rat LC.^16,18,22 This is the first preclinical study demonstrating the advantage and feasibility of IFN- gene therapy for LC, and the efficacy and safety of the approach need to be reproduced in other LC models in different animals.

Several strategies have been recently proposed for the gene therapies of LC: a blockade of TGF- signaling using the dominant negative form of the TGF- receptor prevented liver fibrogenesis and dysfunction;²⁹ transduction with hepatocyte growth factor (HGF) gene inhibited fibrogenesis and hepatocyte apoptosis in cirrhotic liver;³⁰ and an adenovirus-mediated expression of telomerase RNA and urokinase-type plasminogen activator also exerted inhibitory effects against fibrogenesis.^31,32 However, none of those strategies has been examined for an antiviral effect on HCV. We found that the AxCA-rIFN vector inhibited HCV replication in vitro (unpublished data), and the anti-HCV effect may add an advantage to the IFN- gene therapy for HCV-induced LC. It is also noted that HGF transgenic mice developed a broad array of tumors, suggesting caution should be used when considering HGF as a future therapeutic agent.³³

The subcutaneous route of IFN protein injection in conventional IFN therapy is often associated with systemic toxicity, because it requires an approximate 10–20-fold higher level of IFN concentration in the systemic circulation than in the liver.¹⁴ In this study, we found that an adenovirus-mediated IFN- gene delivery can lead to a significant IFN- production in the liver but not in the serum. The locally produced IFN- will rapidly bind to the receptor in the liver tissue, and only a few IFN- may escape to the systemic circulation. We detected no significant systemic toxicity in the rats after injection of AxCA-rIFN. Moreover, Figure 2 suggested that a single injection of the IFN- vector led to a more sustained IFN- level and a larger area under the concentration curve than did the subcutaneous injection of the IFN- protein. The cost of the standard IFN regimen in Japan is about 2 million yen (approximately 15 000 US dollars) per patient for 6 months under the Current National Health Insurance Program, which also limits access to therapy for patients who need more IFN because of a significant amount of virus in the serum. The IFN- gene therapy may offer a better cost–benefit than the conventional IFN protein therapy.

Since it is known from clinical experience that IFN protein treatment may cause acute adverse effects, a safety device should be considered so that vector-induced IFN- production can be terminated when a significant adverse effect appears. Several regulatory systems have been proposed for gene transfer: prokaryotic repressor–operator-based approaches (lac, tet) were applied to transgenic mice to control certain gene expression, and recently, a chemically induced dimerization-based approach (FKBP) was shown to be useful for in vivo gene regulation because of its high induction ratio. Among these methods, the Cre–loxP system has an advantage in its simplicity: only a Cre-expressing adenovirus vector is necessary to shut off the gene. To the best of our knowledge, this report is the first to demonstrate that a Cre-mediated regulation system is useful for shutting off gene expression in a gene therapy model in vivo.

Several delivery systems for targeting the liver were reported. Eto and Takahashi³⁴ reported that hepatitis B virus production was inhibited by asialoglycoprotein receptor-directed IFN, and Protzer et al³⁵ demonstrated that IFN gene transfer by a hepatitis B virus vector efficiently suppressed wild-type virus replication in a duck model of hepatitis B virus infection. However, the hepatitis B virus vector has a low gene transfer efficiency in the liver as compared with that of an adenovirus. When adenovirus vectors were systemically injected into rodents with normal liver, 80–90% of the vector is found in the liver and most vectors target hepatocytes.^36,37 Several reports showed that adenovirus-mediated transgene expression was preferentially shown in septal cells rather than in hepatocytes in cirrhotic rats,^15,38,39 and Nakamura et al¹⁵ have hypothesized that the reduction of intralobular hemodynamics by the shunt formation between portal and central veins resulted in the shift of gene expression from hepatocytes to septal cells in cirrhotic rats. In this study also, the expression of lacZ and AP genes was mainly detected in the fibrous septa of cirrhotic liver after the intravenous injection of vectors. Therefore, an adenovirus might be particularly suitable for gene therapy for LC, because it has a high efficiency of gene transduction into the connective tissues of fibrous septa by the systemic injection.

A major concern regarding the adenovirus vectors is their immunogenicity, because a repeated injection of the vector may be necessary to eradicate HCV infection. Adenoviral proteins can induce a toxic/anaphylactic reaction, which seems to depend on the amount of adenovirus vectors.^40,41 In our strategy, a large amount of adenovirus vector may not be required, because cytokines such as IFN are effective in a small dose. In fact, in this study the injection of 1 10⁷ PFU (compatible with 2 10⁹ PFU in human) AxCA-rIFN resulted in significant IFN- production in the rat liver, a level comparable to that produced by subcutaneous injection of 100 000 IU of recombinant IFN- protein. The intrahepatic arterial infusion, instead of a systemic administration, could further reduce the amount of adenovirus vectors. Less immunogenic vectors such as the 'gutless' adenoviral vector^42,43,44 and polycation-based synthetic nonviral vectors⁴⁵ are being developed in several laboratories at a rapid pace. The present study showed the feasibility and potential advantages of IFN- gene therapy for HCV-associated liver diseases.

Materials and methods

Replication-defective recombinant adenoviruses

Replication-defective recombinant adenoviral vectors carrying either rat IFN- cDNA (AxCA-rIFN), -galactosidase (AxCA-lacZ) or alkaline phosphatase (ADVCA-AP) gene were prepared as described.^46,47 The recombinant adenoviruses are Ad5 defective with a deletion in the E3 region and have the CAG promoter, which is a hybrid of the cytomegalovirus immediate-early enhancer sequence and the chicken -actin/rabbit -globin promoter.⁴⁸ AxCALNL-rIFN, which carries the rat IFN- cDNA between two loxP sequences downstream of the CAG promoter, was constructed to regulate the IFN- expression by Cre–lox reaction (Figure 6a).

Animal model

As a model of LC, we used DMN-injected LC rats, an established animal model of persistent liver fibrosis with pathophysiological findings closely resembling those of human LC.^16,17,18,22 A measure of 1% DMN dissolved in saline was injected into 48 Sprague–Dawley male rats (4-week old, 120–130 g in body weight) intraperitoneally at 1 ml per kg weight for 3 consecutive days per week for 7 weeks. After the DMN injection for 3 weeks, five rats were killed for examination of LC development. Another three rats received subcutaneous injection of the recombinant IFN- protein. The remaining 40 rats were injected via the tail vein with a single infusion of 200 l of either PBS (n=13), 1 10⁷ PFU of AxCA-rIFN (n=12), AxCA-lacZ (n=13) or ADVCA-AP (n=2). Three rats from each group (ADVCA-AP: n=2) were killed at day 2 for the analysis of vector distribution. Several organs including the liver, lung, heart, spleen, kidney, small intestine and testis were fixed in 10% formalin for histological examination and histochemical staining of AP or frozen immediately in liquid nitrogen for the measurement of hydroxyproline and TGF-1, and for the histochemical staining of X-gal. The remaining rats in each PBS- (n=10), AxCA-rIFN- (n=9) and AxCA-lacZ-injected group (n=10) were observed for survival.

Organ distribution of lacZ gene expression

After DMN injection for 3 weeks, the Sprague-Dawley male rats were intravenously injected with 1 10⁹ PFU of AxCA-lacZ. Total RNA was extracted 2 days later from various organs such as the lung, heart, liver, spleen, kidney and small intestine using Isogen^® reagent (Nippon Gene, Tokyo, Japan) and 2 g of RNA was used for cDNA synthesis. Amplification of the lacZ cDNA (250 bp) was carried out using 1 l of synthesized cDNA in a 50 l PCR mixture containing 20 pmol of the forward primer (5'-GATAGATCCCGTCGTTTTAC-3'), reverse primer (5'-TGAGGGGACGACGACAGTAT-3'), 1.5 mM MgCl₂, 0.2 mM dNTPs and 1 U of recombinant Taq DNA polymerase. In total, 30 cycles of the PCR were carried out at 95°C for 1 min, 60°C for 1 min and 72°C for 2 min. The PCR products were electrophoresed on a 2% agarose gel, transferred onto a nylon membrane (Hybond N, Amarsham Biosciences Corp., Piscataway, NJ, USA) and hybridized with a ³²P-labeled lacZ gene in 50% foramide, 5 Denhardt's solution, 0.1% SDS, 5 SSPE, and 100 g/ml of salmon testis DNA at 42°C for 16 h. The membranes were then washed in 0.1 SSC and 0.1% SDS. As a positive control for each RNA preparation and RT-PCR, the glyceraldehydes 3-phosphatase dehydrogenase (G3PDH) sequence was also amplified simultaneously in separate tubes using the forward (5'-TGCACCACCAACTGCTTAG-3') and the reverse primers (5'-GGATGCAGGGATGATGTTC-3'). For amplification of G3PDH, PCR was performed for 25 cycles.

Measurement of rat IFN-, 2'-5'-oligoadenylate synthetase (2'-5'AS) and TGF-1

Rat IFN- was measured by enzyme-linked immunosorbant assay (ELISA) using anti-rat IFN- polyclonal antibody (Access Biomedical Diagnostic Research Laboratories Inc., San Diego, CA, USA). TGF-1 was measured by enzyme assay at a clinical reference laboratory (SRL, Tokyo, Japan). 2'-5' AS activity was determined by a radioimmunoassay kit (Eiken, Tokyo, Japan).

Immunohistochemical examination

Immunohistochemical analysis was performed using an antibody against rat IFN- (Access Biomedical Diagnostic Research Laboratories, Inc., -smooth muscle actin (American Research Product, Tokyo, Japan), or TGF-1 (Promega, Tokyo, Japan) and developed using ABC kit (Nichirei, Tokyo, Japan).

Expression of MMPs and TIMP

Liver tissues were collected from DMN-treated rats 10 days after the injection of PBS or AxCA-lacZ, and 70 days after the injection of AxCA-rIFN. The expression of MMP-13, MMP-2 and TIMP-1 was examined by the RT-PCR method as described above using the following primer sets: MMP-13, forward (5'-TGACTATGCG-TGGCTGGAA-3') and reverse primers (5'-AAGCTGAAATCTTGCCTTGGA-3') (355 bp); MMP-2, forward (5'-ACCATCGCCCATCATCAAGT-3') and reverse primers (5'-CGAGCAAAAGCATCATCCAC-3') (348 bp); TIMP-1, forward (5'-CCGCAGACGGCGTTCT-GCAA-3') and reverse primers (5'-TCGAGACCCAAGGGATTGCC-3') (525 bp). In all, 35 cycles (MMP-2: 30 cycles) of PCR were carried out at 94°C for 30 s, 58°C (MMP-13: 52°C) for 30 s and 72°C for 30 s, and the PCR products were electrophoresed on a 2% agarose gel.

Regulation of IFN- expression by Cre-loxP reaction

NBT-2 cells (rat bladder cancer cell line, 1 10⁵ cells) were transfected with AxCALNL-rIFN at an moi of 30, and 2 days later the cells were transfected with AxCAN-Cre at an moi of 3000. The Sprague-Dawley rats were transduced with 200 l of AxCALNL-rIFN (1 10⁷ PFU) through the tail vein, and 2 days later AxCAN-Cre (1 10⁹ PFU) was transduced via the same route. Two days later, the rats were killed to examine rat IFN- expression by ELISA. The status of the vector DNA was examined by PCR 2 and 6 days after the injection. The sequences of the forward and reverse primers were 5'-GTGGTATTTGTGAGCCAGGG-3', and 5'-TACAGCTC-CTGGGCAACGTG-3', respectively (Figure 6a). In total 35 cycles of the PCR were carried out at 94°C for 30 s, 57°C for 30 s and 72°C for 60 s, and the PCR products were electrophoresed on a 1% agarose gel.

Statistical analysis

Statistical differences between variables were analyzed by the unpaired t-test. Survival distributions were calculated by the Kaplan–Meier method and were analyzed using the log-rank test. P-value <0.01 was considered significant.

References

1. Alter MJ, Mast EE. The epidemiology of viral hepatitis in the United States. Gastroenterol Clin North Am 1994; 23: 437–455.
2. Hoofnagle JH, di Bisceglie AM. The treatment of chronic viral hepatitis. N Engl J Med 1997; 336: 347–356. | Article | PubMed | ISI | ChemPort |
3. Schalm SW, Fattovich G, Brouwer JT. Therapy of hepatitis C: patients with cirrhosis. Hepatology 1997; 26: 128S–132S.
4. Bonkovsky HL. Therapy of hepatitis C: other options. Hepatology 1997; 26: 143S–151S.
5. Di Bisceglie AM et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med 1989; 321: 1506–1510. | PubMed | ChemPort |
6. Poynard T et al. Meta-analysis of interferon randomized trials in the treatment of viral hepatitis C: effects of dose and duration. Hepatology 1996; 24: 778–789.
7. Fort J et al. Effects of long-term administration of interferon alpha in two models of liver fibrosis in rats. J Hepatol 1998; 29: 263–270.
8. Camps J et al. Randomised trial of lymphoblastoid alpha-interferon in chronic hepatitis C. Effects on inflammation, fibrogenesis and viremia. J Hepatol 1993; 17: 390–396.
9. Castilla A, Prieto J, Fausto N. Transforming growth factors beta 1 and alpha in chronic liver disease. Effects of interferon alfa therapy. N Engl J Med 1991; 324: 933–940. | PubMed | ISI | ChemPort |
10. Dufour JF, DeLellis R, Kaplan MM. Regression of hepatic fibrosis in hepatitis C with long-term interferon treatment. Dig Dis Sci 1998; 43: 2573–2576. | Article | PubMed | ISI | ChemPort |
11. Okanoue T et al. Interferon therapy lowers the rate of progression to hepatocellular carcinoma in chronic hepatitis C but not significantly in an advanced stage: a retrospective study in 1148 patients. Viral Hepatitis Therapy Study Group. J Hepatol 1999; 30: 653–659.
12. Schvarcz R et al. Histological outcome in interferon alpha-2b treated patients with chronic posttransfusion non-A, non-B hepatitis. Liver 1991; 11: 30–38.
13. Yagura M et al. Changes of liver fibrosis in chronic hepatitis C patients with no response to interferon-alpha therapy: including quantitative assessment by a morphometric method. J Gastroenterol 2000; 35: 105–111.
14. Uemura I. Pharmacokinetics of recombinant human interferon alpha in rat. Kiso to Rinsho 1985; 19: 205–212.
15. Nakamura T, Akiyoshi H, Saito I, Sato K. Adenovirus-mediated gene expression in the septal cells of cirrhotic rat livers. J Hepatol 1999; 30: 101–106. | Article | PubMed |
16. Jezequel AM et al. A morphological study of the early stages of hepatic fibrosis induced by low doses of dimethylnitrosamine in the rat. J Hepatol 1987; 5: 174–181.
17. Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med 1993; 328: 1828–1835. | Article | PubMed | ISI | ChemPort |
18. Jenkins SA et al. A dimethylnitrosamine-induced model of cirrhosis and portal hypertension in the rat. J Hepatol 1985; 1: 489–499. | PubMed | ISI | ChemPort |
19. Lopez-De Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem 1985; 33: 737–743. | PubMed | ChemPort |
20. Bruck R et al. Prevention of hepatic cirrhosis in rats by hydroxyl radical scavengers. J Hepatol 2001; 35: 457–464.
21. Woessner JF. The determination of hydroxyproline in tissue and protein samples containing small proportion of imino acid. Arch Biochem Biophys 1961; 93: 440–447. | Article | PubMed | ISI | ChemPort |
22. Fujimoto J, Kaneda Y. Reversing liver cirrhosis: impact of gene therapy for liver cirrhosis. Gene Ther 1999; 6: 305–306. | Article |
23. Lee HS et al. Expression of matrix metalloproteinases in spontaneous regression of liver fibrosis. Hepatogastroenterology 2001; 48: 1114–1117. | PubMed | ChemPort |
24. Watanabe T et al. Gene expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride. J Hepatol 2000; 33: 224–235. | PubMed |
25. Overall CM, Wrana JL, Sodek J. Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression. J Biol Chem 1991; 266: 14064–14071. | PubMed | ISI | ChemPort |
26. Poncelet AC, Schnaper HW. Regulation of human mesangial cell collagen expression by transforming growth factor-beta 1. Am J Physiol 1998; 275: F458–F466. | PubMed | ISI | ChemPort |
27. Ikeda K et al. In vitro migratory potential of rat quiescent hepatic stellate cells and its augmentation by cell activation. Hepatology 1999; 29: 1760–1767. | Article | PubMed | ISI | ChemPort |
28. Iredale JP et al. Tissue Inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology 1996; 24: 176–184. | PubMed | ISI | ChemPort |
29. Qi Z et al. Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat. Proc Natl Acad Sci USA 1999; 96: 2345–2349. | Article | PubMed | ChemPort |
30. Ueki T et al. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat Med 1999; 5: 226–230. | Article | PubMed | ISI | ChemPort |
31. Rudolph KL et al. Inhibition of experimental liver cirrhosis in mice by telomerase gene delivery. Science 2000; 287: 1253–1258. | Article | PubMed | ISI | ChemPort |
32. Salgado S et al. Liver cirrhosis is reverted by urokinase-type plasminogen activator gene therapy. Mol Ther 2001; 2: 545–551.
33. Takayama H et al. Diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth factor/scatter factor. Proc Natl Acad Sci USA 1997; 94: 701–706. | Article | PubMed | ChemPort |
34. Eto T, Takahashi H. Enhanced inhibition of hepatitis B virus production by asialoglycoprotein receptor-directed interferon. Nat Med 1999; 5: 577–581. | Article | PubMed |
35. Protzer U et al. Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection. Proc Natl Acad Sci USA 1999; 96: 10818–10823. | Article | PubMed | ChemPort |
36. de Roos WK et al. Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver. Gene Ther 1997; 4: 55–62. | Article | PubMed | ChemPort |
37. Ferry N, Heard JM. Liver-directed gene transfer vectors. Hum Gene Ther 1998; 9: 1975–1981. | PubMed | ChemPort |
38. Garcia-Banuelos J et al. Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors. Evidence of cirrhosis reversion. Gene Ther 2002; 9: 127–134. | Article | PubMed | ISI | ChemPort |
39. Nakatani T et al. Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers. Gut 2000; 47: 563–570. | Article | PubMed | ChemPort |
40. Akli S et al. Transfer of a foreign gene into the brain using adenovirus vectors. Nat Genet 1993; 3: 224–228. | Article | PubMed | ISI | ChemPort |
41. Hermens WT, Verhaagen J. Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats: preferential survival of transduced astroglial cells in nude rats. Hum Gene Ther 1997; 8: 1049–1063. | PubMed | ChemPort |
42. Anderson WF. Human gene therapy. Nature 1998; 392: 25–30. | Article | PubMed | ISI | ChemPort |
43. Haecker SE et al. In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes. Hum Gene Ther 1996; 7: 1907–1914. | PubMed | ISI | ChemPort |
44. Lieber A, He CY, Kirillova I, Kay MA. Recombinant adenoviruses with large deletions generated by Cre-mediated ex-cision exhibit different biological properties compared with first-generation vectors in vitro and in vivo. J Virol 1996; 70: 8944–8960. | PubMed | ISI | ChemPort |
45. Langer R. Drug delivery and targeting. Nature 1998; 392: 5–10. | Article | PubMed | ISI | ChemPort |
46. Miyake S et al. Efficient generation of recombinant adenoviruses using adenovirus DNA–terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA 1996; 93: 1320–1324. | Article | PubMed | ChemPort |
47. Aoki K et al. Restricted expression of an adenoviral vector encoding Fas ligand (CD95L) enhances safety for cancer gene therapy. Mol Ther 2000; 1: 555–565. | Article | PubMed | ChemPort |
48. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991; 108: 193–199. | Article | PubMed | ISI | ChemPort |
49. Aoki K et al. Polyethylenimine-mediated gene transfer into pancreatic tumor dissemination in the murine peritoneal cavity. Gene Ther 2001; 8: 508–514. | Article | PubMed | ISI | ChemPort |

Acknowledgements

This work was supported in part by a grant-in-aid for the 2nd Term Comprehensive 10-year Strategy for Japan, and by grants-in-aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan.