Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis

Abstract

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.

FIG. 1

Structure of HD-IFN and Ad-IFN and expression of mIFN-α2 in vitro. (A and B) mIFN-α2 expression cassette used in this study. The mIFN-α2 gene was cloned downstream of the TTR promoter (prom.) and enhancer (enh.) followed by the polyadenylation signal of bovine growth hormone (GH pA). Int., intron. (A) To generate the HD-IFN vector, the expression cassette was cloned in the SmiI site of the STK120 backbone vector, which contains the following sequence: the left-terminus Ad5 internal terminal repeats (ITR) and packaging signal (ψ); a fragment of the human hypoxanthine guanine phosphoribosyltransferase (HPRT); a human fragment of the C346 cosmid; and the right-terminus Ad5 ITR sequence. (B) To derive the Ad-IFN vector, the expression cassette was recombined in the E1 region of pHVAd1 vector as described in Materials and Methods. (C) The human cell lines Huh-7 and HeLa were transduced with either HD-IFN (open bars) or Ad-IFN (solid bars) at a multiplicity of infection of 10. Secretion of mIFN-α2 into the cell culture medium was measured by the VSV cythopathic inhibition assay as described in Materials and Methods.

FIG. 2

HD-IFN-induced biological response. C57/B6 mice were injected i.v. with HD-IFN at different doses (2 × 10⁸, 9 × 10⁸, and 1.8 × 10⁹ PFU-E), Ad-IFN (2 × 10⁹ PFU), or Ad-βgal (2 × 10⁹ PFU) or mode injected and euthanized at 1 week p.i. (A) Northern blot analysis was performed with 20 μg of total liver RNA from two representative mice per group. Membrane was hybridized with a radiolabeled mIFN-α2 probe. (B) Intrahepatic mIFN-α2 content, expressed as units of mIFN-α2 per gram of liver. ud, undetectable. (C) mIFN-α2 levels present in the serum, expressed as units per milliliter, were measured at the time of autopsy in the VSV inhibition assay as described in Materials and Methods. (D) Northern blot analysis for expression of 2′5′OAS. (E) Total liver RNA was also analyzed for TNF-α expression in an RNase protection assay. (F) The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization of RNA loading in each lane. (G) The intensities of the 2′5′OAS, TNF-α, and GAPDH bands were quantified with a PhosphorImager and are expressed as the ratio of 2′5′OAS to TNF-α corrected for GAPDH.

FIG. 3

Hepatic protection in acute hepatitis induced by MHV-3. Groups of five C57/B6 mice were injected i.v. with Ad-IFN (3 × 10⁹ PFU), Ad-βgal (3 × 10⁹ PFU), or HD-IFN (3 × 10⁹ PFU-E) or mock injected. Untreated mice were used as controls. At day 7 p.i., mice were infected with an i.p. injection of MHV-3 (100 PFU), and transaminase (GPT) levels present in the serum were measured 3 days later. (A) mIFN-α2 present in the serum at 7 days p.i., measured in the VSV inhibition assay. Bars represent individual animals. The horizontal line indicates the limit of detection of the assay. (B) Transaminase (GPT) levels (expressed as units per liter) present in the serum after MHV-3 infection in the same groups of mice as in panel A.

FIG. 4

Hepatic protection in acute hepatitis induced by MHV-3 in the absence of circulating mIFN-α2. C57/B6 mice were mock injected or injected with HD-IFN at the dose of 3 × 10⁸ PFU-E as described in the legend to Fig. 3. Samples were collected at 3 days after MHV-3 infection and analyzed. (A) Transaminase (GPT) levels (expressed as units per liter) present in serum were determined at the time of autopsy (3 days after MHV-3 infection). (B) Northern blot analysis was performed with 20 μg of total RNA from a liver fraction from each mouse. Membrane was hybridized with a radiolabeled 2′5′OAS probe. (C) Northern blot analysis with a 250-bp probe from the 3′ end of the MHV-3 genome, obtained from plasmid DE25 (21). (D) MHV-3 particles present in a liver, expressed per milligram of liver weight. Total liver RNA was also analyzed in an RNase protection assay for expression of inflammatory cytokines such as TNF-α (E) and IFN-γ (F) and for a marker for T-lymphocyte activation, CD3ɛ (G). (H) The housekeeping gene GAPDH was used for normalization. cont., controls.

FIG. 5

Liver histology. (A and B) Liver samples from two animals preinjected with HD-IFN. The hepatic parenchyma is normal; no morphologic patterns of hepatic injury are detectable. (C and D) Liver samples from two animals preinjected with Ad-βgal. (E and F) Liver samples from two animals mock injected. In C, D, E, and F, several areas of zonal necrosis in the hepatic parenchyma are depicted. A main central core of liquefactive necrosis characterizes these areas where nuclei are undetectable, and apoptotic cells with mainly pyknotic nuclei surround them. Hematoxylin and eosin staining. Bars, 35 μm.

FIG. 6

ConA-induced hepatitis. HD-IFN injection protects C57/B6 mice from ConA challenge. C57/B6 mice were injected i.v. with HD-IFN at different dosages (6 × 10⁸ or 3 × 10⁸ PFU-E) or with Ad-IFN at 2 × 10⁹ PFU and challenged with ConA (0.3 g/mouse) 30 days p.i. At 24 h after ConA challenge, transaminases (GPT) (expressed as units per liter) present in the serum were measured. The basal level of serum GPT was measured in untreated mice which were used as controls (cont.). Data are the mean ± standard deviation of serum GPT measured in four animals.