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Inhibition of hepatitis B virus expression and replication by RNA interference
  Hepatology 2003;37:764-770. Amir Shlomai et al, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Report from NATAP HIV Retrovirus Conference (Feb, 2003): RNA interference (RNAi): a recently discovered part of the machinery of the human cell, and a novel possibility for future therapy; Written by David Margolis, MD, University of Texas, Southwestern Medical Center. http://www.natap.org/2003/Retro/day53.htm
Hepatitis B virus (HBV) is a 3.2-kb DNA virus, replicating almost exclusively in the liver.1 Although effective recombinant vaccines are available, HBV infection is still a major global health problem: Each year, acute and chronic HBV infection causes about 1 million deaths. Among the 350 million people with chronic infection, the risk of dying from HBV-related diseases, such as end-stage cirrhosis and hepatocellular carcinoma (HCC) is between 15% to 25%.2 Since the early 1990s, chronically infected patients have been treated with recombinant interferons that are effective only in limited cases. Recently, nucleoside analogs, which directly affect viral replication by inhibition of its reverse transcriptase activity, were shown to be highly effective in the clearance of HBV-DNA from serum. However, the recurrence of viremia after cessation of therapy and the development of escape mutants with prolonged treatment remain major obstacles in achieving complete cure. Furthermore, nucleoside analogs, such as 3TC- lamivudine, impede viral replication but do not directly promote its eradication.
RNA interference (RNAi) is the process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous messenger RNA (mRNA).4 This process is mediated by 21 to 23 nucleotides, called small interfering RNAs (siRNA), cleaved from dsRNA. Although first discovered in Caenorhabditis elegans,5 it was soon after shown that RNAi can be induced in various mammalian cells by introducing synthetic 21nt siRNAs6 to obtain strong and specific suppression (knockdown) of gene expression. Recently, a new vector system called pSUPER (suppression of endogenous RNA), which directs the synthesis of siRNAs and persistently suppresses gene expression in mammalian cells, has been developed.
To evaluate the anti-HBV therapeutic potential of RNAi, we designed 2 pSUPER vectors, each targeted against a distinct 19nt sequence in the HBV genome. We have analyzed the levels of viral proteins and transcripts, as well as the viral replicative forms, in the presence of the constructed pSUPER vectors. We show that RNAi is an efficient approach in reducing the level of HBV transcripts and proteins and in suppression of HBV replication.
RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the target gene. Because it has been shown that RNAi can be accomplished in cultured mammalian cells by introducing small interfering RNAs (siRNAs), much effort has been invested in exploiting this phenomenon for experimental and therapeutic means. In this study, we present a series of experiments showing a significant reduction in hepatitis B virus (HBV) transcripts and proteins in cell culture, as well as in the viral replicative forms, induced by siRNA-producing vectors. The antiviral effect is sequence-specific and does not depend on active viral replication. In conclusion, our data suggest that RNAi may provide a powerful therapeutic tool, acting both on replication-competent and on replication-incompetent HBV.
Discussion by Authors
HBV infection is still a major health problem, even though effective vaccines have been available for the last 20 years.2 Interferon and lamivudine therapy for chronic hepatitis B carriers are reported to result in long-term remissions in a significant percentage of patients24,25; however, these treatments have some drawbacks, including possible serious side effects in the case of interferon or recurrence of viremia after cessation of therapy and development of escape mutants after a long period of lamivudine treatment.
Therefore, using RNAi as an anti-HBV tool seems to have some important advantages: First, specifically targeting the viral transcripts and proteins severely impairs its replication and promotes its eradication, without activating nonspecific cellular responses, hence minimizing undesirable side effects. In addition, the numerous potential targets for RNAi along the viral genome make it possible to target conserved regions, limiting the viral ability to create escape mutants. The potential to introduce a few siRNAs targeted against different sequences simultaneously further limits this ability and makes it possible to treat chronically infected people with diverse circulating HBV genomes. Moreover, the ability of siRNAs to reduce the levels of viral transcripts and proteins even in the absence of active viral replication makes it a good candidate as an adjuvant therapy to lamivudine, which acts only on the replication-competent HBV.
Even though RNAi therapy shares a major disadvantage with lamivudine therapy, namely, the inability to affect the covalently closed circular DNA pool that resides in the liver cell nuclei of chronically infected people, it seems that its ability to severely affect multiple steps in the viral life cycle is a significant progress. Obviously, the siRNA-encouraging results should be tested in animal models. To this end, developing an efficient delivery method is needed. Methods to be considered include injection of synthetic siRNAs or siRNA expressing vectors into the blood stream27 or directly into the liver. The expected stability of siRNA expression vectors over the synthetic siRNAs may ensure a long enough expression for efficient eradication of the virus. In conclusion, our present results open a new avenue for treating HBV infection, which remains a common and serious disease.