Published online May 21, 2018. doi: 10.3748/wjg.v24.i19.2095
Peer-review started: February 17, 2018
First decision: March 9, 2018
Revised: March 26, 2018
Accepted: May 6, 2018
Article in press: May 6, 2018
Published online: May 21, 2018
To detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5’ region that could be candidates for gene therapy.
The study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5’ end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed.
NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5’ end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain.
Two hyper-conserved regions detected in the HBX 5’ end may be of value for targeted gene therapy, regardless of the patients’ clinical stage or HBV genotype.
Core tip: Hepatitis B virus (HBV) is not cured with classic treatments, and liver disease can progress by persistence and expression of covalently-closed circular DNA. Gene therapy with small interference RNA may be an effective approach to ensure inhibition of viral expression and disease progression, and hepatitis B virus X gene (HBX) transcripts could be optimal targets for this therapy. This study includes patients with different HBV genotypes and clinical stages to cover many clinical and virological situations. Using next-generation sequencing, we found two hyper-conserved HBX regions, candidates for small interference RNA therapy, which could enable pan-genotypic inhibition of HBV expression, regardless of the patients’ disease status.