Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs

Abstract

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Although a preventive vaccine is available, the therapeutic options for chronically infected patients are limited. It has been shown that RNA interference can prevent HBV gene expression and replication in vivo when HBV expression vectors are delivered simultaneously with small interfering RNA (siRNA) or siRNA expression constructs. However, the therapeutic potential of siRNAs to interrupt ongoing HBV replication in vivo has not been established. Here, we show that expression of HBVspecific siRNAs in the liver of HBV transgenic mice by recombinant adenoviruses can suppress preexisting HBV gene expression and replication to almost undetectable levels for at least 26 days. These results demonstrate that efficiently delivered siRNAs should be able to silence HBV in chronically infected patients.adenovirus vector ͉ RNA interference C hronic hepatitis B virus (HBV) infection causes Ͼ1 million deaths each year due to cirrhosis of the liver and hepatocellular carcinoma (www.cdc.gov͞hepatitis and www.who.int͞ mediacentre͞factsheets͞fs204͞en). Hence, there is a need for alternative ways to treat this persistent infection. RNA interference (RNAi) has rapidly emerged as a technology for regulating mammalian gene expression because it provides a means of sequence-directed degradation of specific RNAs (1-8). In the case of HBV, published in vivo hydrodynamic transfection studies have shown that simultaneous delivery of HBV expression plasmids and HBV-specific small interfering RNAs (siRNAs) (or siRNA-expressing constructs) to the mouse liver can prevent the induction of HBV gene expression and replication (9-11). To expand on those studies, we have examined the therapeutic potential of RNAi for the treatment of chronic HBV infection where ongoing viral gene expression and replication are established in the liver before siRNA delivery.Several variables that have not been previously addressed could affect the utility of RNAi for the treatment of an ongoing HBV infection. First, in the context of an established infection, viral RNAs may be protected within complexes or nucleocapsid structures as has been reported for hepatitis D virus (1), Rous sarcoma virus (12), and respiratory syncytial virus (RSV) (13). This is particularly relevant to HBV, which replicates in capsids after encapsidation of the viral pregenomic RNA. A second variable is how efficiently preexisting viral RNA can be reduced and to what extent RNA suppression can block viral DNA replication. Finally, established viral gene expression may allow for the induction of viral RNAi-defense mechanisms as reported for plant viruses (14) and flockhouse nodavirus (15).It has been shown that RNAi is capable of reducing mammalian gene expression in vivo. Genes such as fas (16), caspase-8 (17), agouti-related peptide (18), tyrosine hydroxylase (19), and ras (20, 21) have been targeted for degradation in mice, and corresponding changes in phenotype were observed. Yet, the actual degree o...

Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA

“…In addition to the AAV vector used in this study, two recent studies used recombinant adenoviral vectors carrying shRNAs and examined their in vivo silencing effect in similar HBV transgenic mice model. Uprichard et al 20 noted significant suppression of serum HBsAg and HBeAg as well as hepatic HBV transcripts and replicative DNA for at least 20-26 days after injection of 2-5 Â 10 9 PFUs of recombinant adenoviruses expressing HBV-specific shRNAs. The HBV suppression effect was much less significant in another study using adenoviral vectors as shRNAexpressing vehicle, resulting in only 10-fold reduction of HBsAg and two to fivefold reduction of HBeAg and circulating HBV virion in transgenic mice.…”

“…22,51 This concern was actually addressed in one study, which used HBV transgenic mice deficient in IFN-g and IFN-a/b receptor expression to rule out the possibility that HBV suppression was due to the adenoviral vector-induced cytokine effect. 20 Nevertheless, we believe that dsAAV2/8 vector used in this study is a much more appropriate vehicle than adenoviral vector in conducting shRNA studies in HBV transgenic mice. Our result showed that injection of up to 1 Â 10 12 vector genomes of dsAAV2/8 vector did not induce expression of IFN-a/b, IFN-g and TNF-a ( Figure 4), indicating that these previously known HBV inhibitory cytokines were not involved in the RNAi-mediated viral inhibition in our study.…”

“…Two recent reports demonstrated that shRNAs delivered by recombinant adenoviral vector could suppress HBV replication and gene expression in transgenic mice, an animal model more clinically relevant to chronic HBV in terms of containing heavily ongoing HBV replication in all hepatocytes. 20,21 However, as a strong stimulator for both innate and adaptive immune responses, recombinant adenoviral vector by itself could interrupt HBV replication and gene expression in liver of transgenic mice through induction of several inflammatory cytokines, 22 thereby complicating the analysis of RNAimediated repression effects carried by this vector. In clinical application, adenoviral vector is also not an ideal vehicle for chronic HBV gene therapy, because this vector sustains only transient gene expression and might cause severe toxicities at high doses.…”

RNA interference-mediated prevention and therapy for hepatocellular carcinoma

“…In an HBV transgenic mouse model mimicking chronic infection, (Uprichard et al, 2005) have shown that pol III driven siRNA delivered to the liver by recombinant adenovirus suppressed preexisting HBV gene expression and replication for at least 26 days. These results demonstrate the potential of viral vectors for delivery to the liver and long-term RNAi-mediated suppression of target messages when expression systems are used to produce intracellular siRNAs.…”

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus

“…7 RNA interference (RNAi) therapy using chemically synthesized small interfering RNA and plasmid-or adenovirus-encoded small hairpin RNA (shRNA) have shown promising anti-HBV effects in cell cultures 8,9 and animal models. [10][11][12] However, because of the transient nature of the suppression and the inefficient liver transduction rate, these approaches are unlikely to generate durable anti-HBV effects in more stringent HBV infection conditions, such as in chronically infected patients, as virtually all hepatocytes are infected. We have previously reported generation of a transgenic murine model carrying a complete HBV genome on the ICR mouse background (denoted hereafter ICR/HBV), and demonstrated that, throughout their lifetime, these mice produced consistently high levels of serum HBV DNA, with titers comparable with those found in chronic HBV patients.…”