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

Abstract

RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAibased therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adenoassociated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log 10 decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.

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

“…The strength of RNAi suppression then gradually decreased with time, presumably because of the loss of AAV vectors in the liver. 13 A second injection with dsAAV9/HBV-S1 at the age of 62 weeks caused a similar level of HBV suppression as the first treatment and prolonged HBV suppression in these transgenic animals ( Figure 4c). Mice injected with saline or dsAAV vector of the same serotype, but expressing the irrelevant GL2 shRNA, showed no change in serum HBV DNA titer over the 17-month observation period (Figures 4b and c).…”

“…14 We used HBV-S1 shRNA in this experiment, because HBV-S1, but not other HBV-specific shRNAs, delivered by these hepatotropic AAV vectors effectively suppresses HBV replication and gene expression in the ICR/HBV transgenic mouse model without apparent toxicities. 13,14 Our previous data showed that the dsAAV7, 8 and 9 were not crossreactive, and a longer anti-HBV effect could be achieved by the sequential use of dsAAV8/HBV-S1 and dsAAV9/HBV-S1. 14 Groups of viral titer-matched 6-week-old HBV transgenic mice were sequentially injected intravenously with the distinct AAV serotypes carrying HBV-S1 or the control luciferase-specific GL2 shRNA at the time points shown in Figure 4a.…”

“…13 To recapitulate the validity of the model, several basic features of this transgenic mouse line were studied. For example, Northern blotting analysis revealed significant levels of HBV mRNAs in the liver and kidney (Figure 1a).…”

“…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. 13 We demonstrated that administration of a double-stranded adeno-associated virus serotype 8 vector (dsAAV8), encoding a HBVtargeting shRNA, profoundly reduced the serum HBV titer and the liver HBV DNA, mRNA and proteins for at least 120 days without apparent toxicities. 13 However, the RNAi effect gradually decreased with time, presumably because of the liver injury and subsequent liver regeneration in the aged ICR/HBV mice.…”

“…13 We demonstrated that administration of a double-stranded adeno-associated virus serotype 8 vector (dsAAV8), encoding a HBVtargeting shRNA, profoundly reduced the serum HBV titer and the liver HBV DNA, mRNA and proteins for at least 120 days without apparent toxicities. 13 However, the RNAi effect gradually decreased with time, presumably because of the liver injury and subsequent liver regeneration in the aged ICR/HBV mice. 14 A longer anti-HBV effect can be achieved by the sequential use of AAV vectors of different serotypes expressing the same anti-HBV shRNA.…”