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
Processing time: 90 Days and 12.5 Hours
Hepatitis B virus (HBV) infection can be controlled with current treatments, but cure is not achieved due to persistence of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. This minichromosome forms a viral reservoir that is a source of residual viral replication and expression of viral proteins; thus, it has a key role in liver disease progression. To surmount this circumstance, new anti-HBV therapeutic approaches are under development, with gene therapy being a promising option. Among these approaches, small interference RNA (siRNA) can be used to silence specific genes at the post-transcriptional level through a sequence-specific interaction with target mRNAs, resulting in inhibition of viral protein expression. Among all the HBV proteins, Hepatitis B X protein (HBx), coded by the HBV X gene (HBX), is a determining factor in the infection. It regulates cccDNA expression and interacts with several cellular pathways, facilitating liver disease progression. Of particular note, because of its location near the co-terminal 3’ end, all HBV transcripts include the HBX sequence. Hence, it could be a valuable target for a hypothetical curative treatment based on gene therapy. In this sense, identification of hyper-conserved regions within HBX is needed to define a new gene therapy system that would be effective whatever the patient’s clinical stage or HBV genotype.
Although antiviral therapy can suppress viral replication, the risk of liver disease progression and development of hepatocellular carcinoma (HCC) remains due to cccDNA-related expression of viral antigens. Interference with expression of the viral proteins could be helpful to limit progression of the disease, and siRNAs would be valid tools in this sense. To design an effective siRNA, an appropriate target must be found. The HBX sequence is included in all the viral transcripts due to its co-terminal localization in the viral genome. siRNAs targeting hyper-conserved regions of this gene would interfere with expression of all the viral proteins. Furthermore, as these regions are conserved in the spectrum of clinical disease phases and viral genotypes, it would be a valid therapeutic approach for a wide range of situations. This could profoundly limit the risk of HCC, particularly in patients with low viremia due to antiretroviral efficacy.
Considering the essential role of HBx in viral infection and its potential utility as target for gene therapy, the aim of this study was to identify hyper-conserved regions within the HBV genome encompassing the HBX 5’ coding region and the upstream non-coding region (included in all HBV transcripts) in samples from HBV-infected patients in various clinical stages and with different viral genotypes. The regions identified might be feasible targets for a gene therapy able to inhibit viral protein expression in a wide spectrum of clinical and virological circumstances, thus limiting liver disease progression and the risk of HCC.
The study included 27 treatment-naïve chronic hepatitis B monoinfected patients in different clinical stages and with several HBV genotypes (from A-F and H). A serum sample from each patient with viremia > 3.5 log IU/mL was analyzed. The HBX 5’ end region [nucleotide (nt) 1255-1611] was PCR-amplified and later analyzed using next-generation sequencing (NGS). The sequences (reads) obtained after sequencing underwent an in-house bioinformatics filtering procedure, and haplotypes with a relative frequency ≥ 0.25% were maintained in the analysis. Haplotypes were genotyped by discriminant analysis with the same regions extracted from the 102 full-length patterns. Conservation of the quasispecies sequences was determined by calculating the information content (IC), based on Shannon’s uncertainty, of each position in a multiple alignment of all different sequences found in the patients. Sliding window analysis was then carried out to locate the fragment of at least 25 nt or 10 aa (which corresponds to the length of a possible target for siRNA therapy) with the highest IC within the multiple alignments, moving forward in steps of 1 (nt or aa). This method enables detection of conserved regions within the 5’ HBX gene by directly analyzing the viral quasispecies obtained with NGS.
After applying the quality filter, 1333069 haplotype sequences were obtained. Genotyping analysis highlighted a complex mixture of HBV genotypes. By studying the nt conservation, we identified two hyper-conserved nucleotide regions in HBX. The first one, between nt 1255 and 1286, corresponded to a non-coding region, whereas the second one, consisting of 3 conserved fragments (spanning an overall portion between 1519 and 1603), coincided with a coding region. Of note, the fragment between nt 1563 and 1602 was also conserved at the amino acid level, identifying a region between residues 63 and 76, which included a portion of a Kunitz-like domain. These results highlight new potential targets for gene therapy, mainly based on siRNA. Of note, in vitro and in vivo functional studies of the specific siRNAs should be performed to test their potential usefulness for therapy.
Gene therapy represents a highly promising therapeutic tool to achieve a cure against HBV infection. Several sequence-specific treatment systems are currently in development, and identification of conserved sequences would provide useful therapeutic targets. Detection of a target present in all the clinical disease stages and HBV genotypes could lead to development of a therapy that would be effective in a wide range of situations. Considering the key role of HBx in viral infection and disease progression, we focused the study on analyzing conservation of the HBX gene. Of note, considering the high variability previously observed in the 3’end of HBX, we speculated that the 5’end could be a better subject for study. Moreover, thanks to the co-terminality of this viral gene, a siRNA targeting this gene could interfere with all the viral transcripts. Here, we investigated conservation of a portion of the HBV genome encompassing the HBX 5’ coding region and upstream non-coding region, both of which are included in all HBV transcripts. By NGS analysis, we identified two hyper-conserved regions in our region of interest in serum samples from HBV patients with different clinical and virological characteristics. This new therapeutic tool could have relevant applicability in clinical practice. Together with inhibition of the expression of one of the main viral proteins involved in HBV replication and disease progression, it could block the expression of the other viral antigens, thus profoundly interfering with disease evolution and the appearance of HCC. Furthermore, the NGS method developed here could be used to find other hyper-conserved regions within the HBV genome that could be potential targets for gene therapy based on siRNA.
This study describes a method that can be used to find other conserved sequences in the HBV genome, making it a starting point in the search for other possible targets for gene therapy. Here, the hyper-conserved regions were found by directly analyzing the viral quasispecies sequences obtained using NGS. These regions can then be used to produce siRNA molecules for in vitro and in vivo testing of antiviral activity.