Inhibition of Hepatitis B virus replication using RNAi effectors targeting the viral X-gene

Abstract

Globally, an estimated 257 million people were living with chronic hepatitis B virus (HBV) infection in 2015. The epidemic is concentrated predominantly in sub-Saharan Africa and Southeast Asia. Long-term complications of chronic HBV infection are cirrhosis and hepatocellular carcinoma. Currently licensed therapies have limited efficacy in eradication of HBV. Therefore, the need for new therapeutic modalities remains. RNA interference (RNAi) is a highly conserved sequence-specific gene silencing pathway. RNAi is activated by short interfering RNAs (siRNAs) which have therapeutic potential and can be exploited against HBV. RNAi effectors may be synthetically produced or derived from expressed RNA sequences. This approach requires efficient and safe delivery of these effectors. The aim of this thesis was to determine whether HBV replication could be inhibited by exploiting the RNAi pathway. We targeted HBV X open reading frame (ORF), a highly conserved sequence that overlaps all viral transcripts and codes for HBx, a viral protein critical for replication and persistence. We developed and evaluated novel lipid based vectors to deliver siRNAs. Effective, short hairpin RNAs (shRNA), long hairpin RNAs (lhRNA) and synthetic siRNAs were selected in vitro. Viral- and non-viral vectors (NVV) were developed for delivery. These included recombinant adenoviral vectors, lipid-based pegylated-siRNA nanoparticles and cationic lipid vectors. These therapeutic complexes were assessed in a model of transient infection, using hydrodynamic tail vein injection, and one with features of chronic infection, using HBV transgenic mice.

We demonstrated effective HBV inhibition by RNAi effectors. Molecular analysis revealed intracellular processing of the shRNAs was more effective than lhRNAs. Highly effective shRNAs demonstrated processing of the intended strand. HBV inhibition did not elicit innate immune responses. Assembly of cationic lipid-based NVV with anti-HBV synthetic siRNA was successful and resulted in HBV inhibition in vitro and in vivo models. This body of work provides substantial evidence that different RNAi modalities can suppress HBV replication culture and animal models of acute and chronic HBV infection. HBx ORF was shown to be a highly suitable target. Despite these optimistic findings, several challenges still remain for RNAi-based therapies to reach the clinic. Safe and effective delivery strategy is an important future objective.