<i>In-Silico</i> Development of Effective Short Hairpin RNA Molecules Targeting the Transactivator Region of Human Hepatitis B Virus

Abstract

Background: Hepatitis B virus (HBV) infection remains a major global health challenge, implicated in approximately 80% of hepatocellular carcinoma (HCC) cases. The hepatitis B virus X protein (HBx) plays a critical role in both viral replication and oncogenesis. Novel therapeutic strategies are urgently needed to suppress HBV persistence and its oncogenic effects. RNA interference (RNAi) — a natural gene-silencing mechanism mediated by double-stranded RNAs — holds significant therapeutic potential. Objectives: Therefore, this study aimed to design effective short hairpin RNA (shRNA) molecules targeting the hepatitis B virus X (HBX) gene. Methods: Complete HBV genome sequences representing all major genotypes were retrieved from the NCBI GenBank database. Conserved regions of the HBX gene were identified via multiple sequence alignment using Clustal Omega. Candidate shRNAs were designed using three computational tools: The WI short interfering RNA (siRNA) Selection Program, BLOCK-iT RNAi Designer, and siRNA Wizard. These shRNAs were evaluated for GC content, thermodynamic stability, and secondary structure using CLC Genomics Workbench. Specificity was confirmed through BLAST analysis against the human genome to exclude potential off-target interactions. Results: Sixteen candidate shRNAs were generated from the design platforms and screened for optimal structural and functional characteristics. Three shRNAs demonstrated the most favorable properties, including optimal GC content, high sequence conservation across HBV genotypes, strong target accessibility, and absence of significant off-target homology. These candidates are predicted to efficiently silence HBX gene expression. Conclusions: Computational design of shRNAs targeting conserved HBX sequences identified three high-scoring candidates with potential therapeutic application. These molecules provide a basis for future experimental validation and may contribute to the development of shRNA-based therapies for HBV infection and HBV-associated HCC.