799. Expressed Long Hairpin RNA Sequences Inhibit HBV Replication In Vivo without Inducing an Interferon Response
Authors: Abdullah Ely, Sergio Carmona, Carol Crowther, Steven Mufamadi, Samantha Barichievy, Marc Weinberg, Patrick Arbuthnot
Journal: Molecular Therapy (2006)

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the global population and HBV carriers are at an increased risk for cirrhosis and hepatocellular carcinoma. Licensed therapies against HBV are only partially effective and development of improved treatment modalities remains an important medical objective. Exploitation of the RNA interference (RNAi) pathway shows promise as a novel antiviral treatment strategy for the management of HBV. Traditionally short hairpin RNAs (shRNAs) of 21–23 bp in the stem region have been used to induce RNAi- mediated gene silencing and avoid inducing the sequence non-specific interferon response. The use of longer (> 30 bp) hairpin RNAs affords the advantage of generating multiple siRNA from a single Dicer template, which target different sites. This potentially limits the problem of viral escape mutants that evade the silencing effects of a single siRNA. We generated U6 promoter-driven cassettes that encode long hairpin RNAs (lhRNAs) comprising a 60 bp stem. The duplex RNA was designed to target the HBx sequence of the A1 subgenotype of HBV. G:U pairings, which occurred at approximately every 6 bp, were incorporated in the stem sequence to circumvent induction of the interferon response by RNA-dependent protein kinase and RNase L pathways. One of the two lhRNA targeted to HBx successfully inhibited markers of HBV replication in cultured cells and in the murine hyperdynamic model of HBV replication. HBV surface antigen, HBV mRNA and viral particles were diminished by approximately 80% by the lhRNA. Knockdown was comparable to that achieved by a shRNA targeted against a region covered by the lhRNA and was observed over a period of 1 week. Control lhRNA targeted to the HIV-1 Tar region did not knock down HBV gene expression significantly. As an index of activation of the interferon response genes, OAS1, MxA and IFN-β mRNA concentrations were measured in transfected cells and in hepatocytes of mice treated using the hydrodynamic injection procedure. None of the lhRNA cassettes induced expression of these genes. These data indicate that U6 promoter-driven lhRNA are capable of effectively inhibiting HBV replication without inducing the unwanted interferon response. These lhRNA sequences have potential therapeutic application and may be useful to counter viral escape. Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the global population and HBV carriers are at an increased risk for cirrhosis and hepatocellular carcinoma. Licensed therapies against HBV are only partially effective and development of improved treatment modalities remains an important medical objective. Exploitation of the RNA interference (RNAi) pathway shows promise as a novel antiviral treatment strategy for the management of HBV. Traditionally short hairpin RNAs (shRNAs) of 21–23 bp in the stem region have been used to induce RNAi- mediated gene silencing and avoid inducing the sequence non-specific interferon response. The use of longer (> 30 bp) hairpin RNAs affords the advantage of generating multiple siRNA from a single Dicer template, which target different sites. This potentially limits the problem of viral escape mutants that evade the silencing effects of a single siRNA. We generated U6 promoter-driven cassettes that encode long hairpin RNAs (lhRNAs) comprising a 60 bp stem. The duplex RNA was designed to target the HBx sequence of the A1 subgenotype of HBV. G:U pairings, which occurred at approximately every 6 bp, were incorporated in the stem sequence to circumvent induction of the interferon response by RNA-dependent protein kinase and RNase L pathways. One of the two lhRNA targeted to HBx successfully inhibited markers of HBV replication in cultured cells and in the murine hyperdynamic model of HBV replication. HBV surface antigen, HBV mRNA and viral particles were diminished by approximately 80% by the lhRNA. Knockdown was comparable to that achieved by a shRNA targeted against a region covered by the lhRNA and was observed over a period of 1 week. Control lhRNA targeted to the HIV-1 Tar region did not knock down HBV gene expression significantly. As an index of activation of the interferon response genes, OAS1, MxA and IFN-β mRNA concentrations were measured in transfected cells and in hepatocytes of mice treated using the hydrodynamic injection procedure. None of the lhRNA cassettes induced expression of these genes. These data indicate that U6 promoter-driven lhRNA are capable of effectively inhibiting HBV replication without inducing the unwanted interferon response. These lhRNA sequences have potential therapeutic application and may be useful to counter viral escape.