Interferon Gene Transfer by a Hepatitis B Virus Vector Efficiently Suppresses Wild-Type Virus Infection

Proc. Natl. Acad. Sci. USA Vol. 96, pp. 10818–10823, September 1999 Medical Sciences Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection ULRIKE PROTZER*, MICHAEL NASSAL*†,PEI-WEN CHIANG*‡,MICHAEL KIRSCHFINK§, AND HEINZ SCHALLER*¶ *Zentrum fu¨r Molekulare Biologie Heidelberg and §Department of Immunology, University of Heidelberg, Im Neuenheimer Feld, D-69120 Heidelberg, Germany; and †University Hospital, Department of Internal Medicine II͞Molecular Biology, University of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany Communicated by Peter H. Duesberg, University of California, Berkeley, CA, July 13, 1999 (received for review February 25, 1999) ABSTRACT Hepatitis B viruses specifically target the concepts include gene therapy and the use of defective or liver, where they efficiently infect quiescent hepatocytes. Here attenuated viruses to block wild-type viral infection (3). Im- we show that human and avian hepatitis B viruses can be munomodulatory cytokines such as IL-12, IFN-␥ or tumor converted into vectors for liver-directed gene transfer. These necrosis factor-␣ potently suppress hepatitis B virus (HBV) vectors allow hepatocyte-specific expression of a green fluo- replication in an HBV transgenic mouse model (10, 11), rescent protein in vitro and in vivo. Moreover, when used to whereas IL-12 and the Th1 cytokines IFN-␥ and IL-2 seem to transduce a type I interferon gene, expression of interferon play an important role for viral clearance in chronically efficiently suppresses wild-type virus replication in the duck infected patients (12). However, systemic application of cyto- model of hepatitis B virus infection. These data suggest local kines is limited by severe side effects (7, 9). Local production cytokine production after hepatitis-B-virus-mediated gene of cytokines after liver-directed gene transfer should provide transfer as a promising concept for the treatment of acquired a more efficient and better-tolerated alternative, and hepatitis liver diseases, including chronic hepatitis B. B virus-based vectors might be particularly suitable for this approach. Persistent viral infections can be viewed as acquired genetic Here we show that in vitro infection of primary human diseases and therefore as a major challenge for the application hepatocytes with recombinant HBV carrying a gene coding for of gene therapy. Generally, treatment will require transient a green fluorescent protein (GFP) (13) leads to clearly de- rather than permanent effector gene expression, such as tectable GFP expression. Because the availability and in- stimulation of the immune system against the infectious agent fectibility of these cells is limited (14, 15), and no feasible in (1). Appropriate gene-delivery systems should specifically vivo infection system exists for the human virus, we took target the infected tissue or cell type, should allow for control advantage of the duck hepatitis B virus (DHBV) model, which of duration and strength of effector gene expression, and, readily allows for infection studies with primary hepatocytes ideally, should be administered by injection into the blood- and whole animals (16). We demonstrate in vivo infection of stream (1, 2). To treat infectious diseases of the liver, none of hepatocytes after injection of recombinant DHBV-GFP into a the currently available vector systems meets all of these peripheral vein, and we prove that cells preinfected with the criteria (3). wild-type virus can be superinfected with the recombinant Hepatitis B viruses, or hepadnaviruses, are small enveloped virus. As a first step toward therapeutically useful hepadna- DNA viruses with distinct features that make them attractive virus vectors, we constructed a recombinant DHBV carrying candidates as vectors for gene therapy of acquired liver dis- the duck homologue of IFN-␣, which efficiently suppressed eases. They selectively target the liver after inoculation into the wild-type virus replication. bloodstream, and they efficiently infect quiescent hepatocytes. Viral gene expression is directed by hepatocyte-specific pro- MATERIALS AND METHODS moter-enhancer elements (4, 5), and, in contrast to retrovi- ruses, genome replication via reverse transcription does not Plasmid Constructs. HBV͞DHBV constructs contain, un- require integration of the hepadnaviral DNA into the host der control of the cytomegalovirus immediate early promoter- genome (6); rather it establishes a stable episomal transcrip- enhancer, a terminally redundant genome of HBV, subtype tion template. The feasibility, however, of a hepadnaviral ayw 1 (pCH-9͞3091, HBV nucleotides 3091 to 3182-1 to vector system has not yet been demonstrated. 3182͞1 to 84, numbering from the core initiation codon) (17), Chronic viral hepatitis affects approximately 800 million or a terminally redundant genome of DHBV, subtype 16 (18) people and is the principal cause in the world of chronic liver (pCD16, DHBV nucleotides 2520 to 3021͞1 to 2816) (19, 20), disease, liver cirrhosis, and hepatocellular carcinoma (7). respectively (Fig. 1). The respective helper constructs Currently, the only therapy for chronic hepatitis that has a pCH3142 for HBV (21) and pCD4 for DHBV (20) are nearly lasting beneficial effect is systemic treatment with interferon identical except that they lack part of the 5Ј-proximal RNA (IFN)-␣; a sustained response is achieved in only one-third of packaging signal ␧, which makes them encapsidation-deficient patients with chronic hepatitis B and in only one-fifth of (Fig. 1). patients with chronic hepatitis C (7). Nucleoside analogues Marker constructs pCH-S-GFP and pCD-S-GFP were ob- provide a therapeutic alternative leading to a rapid decrease in tained by replacing DNA fragments containing the small serum HBV DNA levels and to histologic improvement of liver envelope (S) gene (in pCH-S-GFP from XhoI, position 1409, disease (8). However, short-term treatment leads to a rapid to NsiI, position 2347; in pCD-S-GFP from KpnI, position relapse of disease and long-term treatment often results in the selection of resistant viral variants (7); these outcomes em- Abbreviations: DHBV, duck hepatitis B virus; GFP, green fluorescent phasize the need for novel therapeutic approaches (7, 9). New protein; HBV, hepatitis B virus; IFN, interferon; moi, multiplicity of infection; PDH, primary duck hepatocytes; p.i., postinfection; r, The publication costs of this article were defrayed in part by page charge recombinant; vp, DNA-containing enveloped viral particles. ‡Present address: Dept. of Pediatrics, University of Michigan, MSRBI, payment. This article must therefore be hereby marked ‘‘advertisement’’ in Room 3520, 1150 W. Medical Center Drive, Ann Arbor, MI 48109. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. E-mail: hshd@ PNAS is available online at www.pnas.org. zmbh.uni-heidelberg.de. 10818 Downloaded by guest on September 28, 2021 Medical Sciences: Protzer et al. Proc. Natl. Acad. Sci. USA 96 (1999) 10819 cells (23) and chicken hepatoma LMH cells (24), respectively, were cotransfected at 30–40% confluence by using the calci- um-phosphate method with 50 ␮g of the respective expression construct and 25 ␮g of the helper construct per each 15-cm dish. Cell culture medium containing recombinant virions was collected from days 3 to 6 posttransfection; it was then concentrated 10- to 50-fold by precipitation with 6.5% poly- ethylene glycol 20,000͞0.9% NaCl at 0°C and stored in PBS͞ 10% glycerol at Ϫ20°C until further use. Wild-type HBV was produced by transfecting 25 ␮g of plasmid pCH-9͞3091 ac- cordingly. Virus titers, measured as DNA-containing enveloped viral particles (vp), were determined by density-gradient centrifugation and dot-blot analysis relative to an HBV- or DHBV-DNA standard (19). Isolation of Primary Hepatocytes. Primary hepatocytes were isolated by a standard two-step collagenase perfusion and subsequent differential centrifugation (50 ϫ g). Surgical human liver biopsies were obtained (after informed consent of the donor) and, after sealing of smaller vessels, perfused via a large branch of the portal vein. Two- to three-week-old Peking ducks and 16- to 20-week-old CH57BL͞6 mice were perfused via the portal vein. Primary human and mouse hepatocytes (2.5 ϫ 105 cells per cm2 and 4 ϫ 105 cells per cm2, respectively) were seeded onto collagen type I (Sigma Aldrich) coated tissue culture plates in a maintenance medium (25) with 10% FCS and maintained with 5% FCS. Primary duck hepatocytes (PDHs) (2.5 ϫ 105 cells per cm2) were seeded and maintained without FCS on untreated cell culture dishes (25). DHBV- infected duck hepatocytes were obtained from ducks experi- mentally infected the first day after hatching with 100 ␮lof 9 FIG. 1. Plasmid constructs used for the production of recombinant duck serum containing 10 DHBV16 virions, resulting in hepadnaviruses. The parental plasmids pCH-9͞3091 (HBV) and infection of virtually all hepatocytes (26). Sinusoidal endothe- pCD16 (DHBV) are based on terminally redundant hepadnavirus lial and Kupffer cells in the hepatocyte cultures were identified genomes (thick black lines) functionally mimicking the circular DNA by their receptor-mediated uptake of fluorescence-labeled genomes formed by reverse transcription of the RNA pregenomes acetylated low-density lipoprotein [Di-I-AcLDL, Paesel and [sinuous lines with A(n) representing the poly(A) tails]. Numbers refer Lorei, Duisburg, Germany (27)] and by phagocytosis

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