[Frontiers in Bioscience 3, a11-15, February 1, 1998]|
TARGETED INHIBITION OF HEPATITIS B VIRUS GENE EXPRESSION: A GENE THERAPY APPROACH
Frank Y.-T. Tung and Steven W. Bowen
Department of Infectious Disease and Microbiology, University of Pittsburgh, Pittsburgh PA 15261
Received 1/21/98 Accepted 1/28/98
HBV is an enveloped, hepatotropic DNA virus that causes acute and chronic liver cell injury, inflammation and hepatocellular carcinoma (HCC). Primary HBV infection is usually self-limited, with clearance of viral antigens and infection from liver and blood and the development of lasting immunity to reinfection (1). However, 5-10% of individuals do not recover from primary infection, but develop a persistent, usually lifelong, hepatic infection (2). As in primary infections, such individuals may be asymptomatic or experience varying grades of chronic liver injury (3). Even though it is a minority outcome, persistent infection occupies a central role in the pathogenesis of HBV. In severe chronic hepatitis, liver transplant is the only curative remedy for patients suffering from liver failure. Unfortunately, reinfection of donor liver is almost unavoidable.
More than 250 million people throughout the world are chronically infected by this virus, serve as its reservoir, and have a 200-fold greater risk of developing HCC than their non-infected counterparts (4). HBV replicates episomally within infected cells by a process involving reverse transcription of an RNA pregenome (5). During prolonged infection, viral DNA sequences integrate into the host cell genome where they and the flanking cellular sequences are commonly rearranged in association with the development of HCC (6,7). Moreover, most of the mortality from HBV infection results in chronic rather than acute disease (8,9). Severe chronic hepatitis B frequently leads to premature death from liver failure (10). Viral replication itself does not appear to be cytotoxic; variation in the severity of liver damage between individuals has been attributed instead to differences in host immune responses to virally infected cells (11-14). However, the implicated target antigens and host effect mechanisms remain elusive. The only established therapy for chronic hepatitis B is interferon-alpha, with an efficacy of only 30-40% in highly selected patients (15).
Analysis of the complete nucleotide sequence of HBV genome indicated four major open reading frames (ORFs) which are encoded by minus-strand DNA. The coding regions for HBsAg revealed unanticipated complexity. The coding region for HBsAg (ORF S) proved to be the 3' portion of a large coding region: upstream of ORF S is an in-phase reading frame (Pre S) with two conserved in-phase ATG codons that can direct the synthesis of additional HBsAg-related proteins, termed pre-S1 and pre-S2. Similarly, the coding region for HBcAg (ORF C) is also preceeded by a short upstream in-phase ORF (termed pre-C). The ORF X encodes a transactivator protein which can trans-activate transcription from the HBV core promoter (16) as well as other viral promoters (17,18). Overlapping these coding regions is a large open reading frame, ORF P, which is believed to encode the viral polymerase.
One rational strategy for inhibiting virus replication would be to use nucleic acids of defined HBV gene sequences that would "hybridize" to virus genetic material, either regulatory signals or coding information, inside the infected cells and thereby block viral expression or replication in a very specific manner (19-21). Synthetic antisense oligonucleotides to HBV surface antigen have been used to inhibit HBV replication in vitro and in animal models, however, the antiviral effect is short-term and transient (22-24). Antisense RNA expressed by a retroviral vector is not immunogenic and will not elicit immune responses. One very attractive feature of using antisense nucleic acids for inhibition of HBV is the possibility that virus replication can be completely stopped without killing the host cells. Such an approach is especially attractive for cells that have a regenerative ability, such as liver cells. The progeny derived from antiviral gene protected liver cells may replace the damaged cells caused by viral gene expression or host immune responses.
Retroviral vectors have been widely used as a highly efficient method for gene transfer into eukaryotic cells and for gene therapy strategies(25,26). Retroviral vectors offer a number of advantages compared to other gene transfer vectors, including stable integration of the transferred genes and a lack of retrovirus protein coding sequences in the transfer vector. Thus, we describe here the construction of a novel retroviral vector to express HBV antisense RNA and its application to block HBV antigen expression in culture hepatic cells.