Abstract

Background

The establishment of chronic HBV infection in over 297 million people is due in part through the virus’ highly stable covalently closed circular DNA (cccDNA) persisting in the nuclei of infected hepatocytes. Thus, curing HBV will require direct targeting of the minigenome and detailed structural knowledge of the cccDNA to determine vulnerabilities.

Purpose

Previous studies have demonstrated that human protein Sp1 interacts with the HBV genome at the preCore/Core promoter - a critical interaction for viral replication. We have recently discovered that the Sp1-binding region of HBV pre-Core forms a highly ordered G-quadruplex (G4) secondary structure which presents a novel therapeutic anti-HBV target.

Method

Using phage display technologies, we have identified 11 G-quadruplex binding single domain antibodies that can target the G4 present within the cccDNA. Using recombinant protein expression we characterized the strongest binder (S10) and its interaction with a 22nt HBV pre-Core G-quadruplex forming oligo. Using MicroScale Thermophoresis (MST), the binding affinity (KD) between S10 and the target G4 was determined to be ~218 nM, which is 100x stronger for folded G4 versus unfolded oligos of the same sequence. Using oligos of (~60-90%) sequence similarity, it was observed that S10 has a KD for the target G4 that was at least 10x higher than similar sequences. To determine the effect of S10 on HBV replication, we have transduced the HepG2-NTCP-A3 cell line to express these sdAbs and are in the process of evaluating antiviral effects and target specificity for cccDNA.

Result(s)

Using biophysical in-vitro approaches, S10 has shown a great potential in being able to discriminate against different G4’s, while having a high degree of affinity as well as high complex stability.

Conclusion(s)

The ability of these G4 binding single domain antibodies to discriminate between different sequences or secondary DNA structure provides an insight into how they can be exploited in future HBV therapeutic strategies.

Disclosure of Interest

None Declared

Details

Title
A61 TARGETING A GUANINE QUADRUPLEX IN THE HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA GENOME USING SINGLE DOMAIN ANTIBODIES
Author
D'souza, S 1 ; G Balderas Figueroa 2 ; Badmalia, M D 2 ; Patel, T R 3 ; Coffin, C 4 

 Microbiology, Immunology, and Infectious Diseases, University of Calgary Cumming School of Medicine, Calgary 
 Chemistry and Biochemistry, University of Lethbridge, Lethbridge 
 Microbiology, Immunology, and Infectious Diseases, University of Calgary Cumming School of Medicine, Calgary; Chemistry and Biochemistry, University of Lethbridge, Lethbridge; Discovery Lab Faculty of Medicine and Dentistry, University of Alberta, Edmonton 
 Microbiology, Immunology, and Infectious Diseases, University of Calgary Cumming School of Medicine, Calgary; Department of Medicine, University of Calgary Cumming School of Medicine, Calgary, Canada 
First page
34
Publication year
2023
Publication date
Mar 2023
Publisher
Oxford University Press
ISSN
25152084
e-ISSN
25152092
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
3170158772
Copyright
ڣ The Author(s) 2023. Published by Oxford University Press on behalf of the Canadian Association of Gastroenterology. This work is published under https://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.