Persistence of the HBV covalently closed circular DNA (cccDNA) genome in the nuclei of infected hepatocytes plays a critical role in the establishment of chronic HBV infection in over 296 million people worldwide. Currently approved antivirals against HBV do not lead to cure, as they fail to silence or clear the cccDNA minigenome.

In our previous research, we identified a highly guanine-rich sequence located in the HBV PreC/C promoter which forms a DNA secondary structure known as a Guanine-quadruplex (G4). This G4 structure consists of Hoogsteen hydrogen-bonded guanosines that stack upon one another in a specific manner, acting as a transcriptional regulator. We aim to target this G4 located in the PreC/C promoter region using a humanized single-domain antibody (sdAb) to disrupt HBV RNA transcription and affect cccDNA stability.

Using phage display technologies, we identified 11 sdAbs that can target the G4 sequence present PreC/C promoter region of HBV. Through biophysical characterization, we determined that the strongest binder (S10) has a 90nM affinity for the 22nt HBV PreC/C G-quadruplex forming oligo. Additionally, S10 shows strong target specificity for the PreC/C G4 when compared to G4s derived from the human genome with 70-90% sequence similarity. Structural insights into the sdAb-HBV G4 complex were obtained through size-exclusion chromatography small-angle x-ray scattering (SEC-SAXS), providing valuable information about the antibody-antigen binding.

To assess the ability of S10 to target HBV, ChIP-qPCR experiments were performed on HBV-infected HepG2-NTCP-A3 cells that stably express S10. ChIP-qPCR data shows that S10 can effectively immunoprecipitate HBV, showing enrichment at the HBV PreC/C G4 locus and cccDNA locus. Additionally, ChIP-seq is currently in progress to determine target specificity of S10.

The capability of S10 to distinguish between different sequences and secondary DNA structures offers insights into its potential exploitation in future therapeutic strategies for HBV.