©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy
Carolina González, David Tabernero, Maria Francesca Cortese, Rosario Casillas, Cristina Godoy, Sara Sopena, Ariadna Rando, Marçal Yll, Rosa Lopez-Martinez, Francisco Rodríguez-Frías, Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
David Tabernero, Josep Gregori, Mar Riveiro-Barciela, Sara Sopena, Josep Quer, Rafael Esteban, Maria Buti, Francisco Rodríguez-Frías, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
Maria Francesca Cortese, Josep Gregori, Rosario Casillas, Cristina Godoy, Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
Josep Gregori, Roche Diagnostics SL, Sant Cugat del Vallès 08174, Spain
Mar Riveiro-Barciela, Rafael Esteban and Maria Buti, Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
Author contributions: Rodríguez-Frías F designed the research; Cortese MF coordinated the research; González C and Tabernero D equally contributed to design the experiments; González C, Casillas R, Godoy C, Sopena S and Rando A performed the experiments; González C, Tabernero D and Gregori J analyzed data acquired during the experiments and interpreted the results; González C, Tabernero D, Cortese MF and Riveiro-Barciela M drafted the manuscript; Yll M, Lopez-Martinez R, Buti M, Quer J, Esteban R and Rodríguez-Frías F critically reviewed the manuscript.
Supported by the Instituto de Salud Carlos III, No. PI15/00856; and the European Regional Development Fund (ERDF), No. PI15/00856.
Institutional review board statement: The study was reviewed and approved by the Clinical Research Ethics Committee (CEIC) of Hospital Universitari Vall d’Hebron.
Conflict-of-interest statement: Josep Gregori is an employee of Roche Diagnostics, SL.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Maria Francesca Cortese, PhD, Research Scientist, Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 119-129, Barcelona 08035, Spain. firstname.lastname@example.org
Received: February 16, 2018
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.