Inhibition of Hepatitis B virus replication using RNAi effectors targeting the viral X-gene
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Date
2018
Authors
Carmona, Sergio Catril
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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.
Description
A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand,
Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy.
Johannesburg, June 2018.