ESTABLISHING AN IN VITRO MODEL FOR CRISPR-CAS9 SCREENING OF INTERFERON EFFECTORS IN HBV INFECTION

Hepatitis B virus (HBV) is a hepatotropic virus that can cause both acute and chronic liver infection. According to the World Health Organization, 254 million people were living with chronic hepatitis B virus (CHB) infection in 2022, with approximately 1.2 million new cases each year. While vaccination prevents new infections, CHB still leads to cirrhosis and hepatocellular carcinoma, contributing to over one million deaths annually and underscoring the need for improved therapies (World Health Organization 2025). Interferon-alpha (IFN- α), including its pegylated forms, is approved for CHB treatment, but its efficacy is limited to a small subset of patients, and its mechanism of action remains poorly understood. Indeed, interferon therapy can activate different immune cell types, but only a fraction of CHB patients achieve durable clinical benefit, underscoring the need to better understand the molecular basis of its limited efficacy (Ye and Chen 2021). We hypothesize that specific interferon-stimulated genes (ISGs) act as key effectors of the antiviral response and sought to identify them via a CRISPR-Cas9 knockout screen targeting ~2,000 ISGs. Identifying the ISGs that mediate antiviral activity may provide insights into how interferon responses can be potentiated, both to improve patient outcomes and to increase our understanding of their mechanism of action. In parallel, we aim to determine, in the context of HBV infection, which interferon subtype elicits the most robust in vitro response, thereby providing the most suitable condition for the screening. A successful genetic screen requires an in vitro model that supports efficient HBV infection and retains intact IFN responsiveness. Since no such model was readily available, we systematically evaluated candidate cellular models and identified HepG2-NTCP cells cultured with 2.5% DMSO as optimal. This condition promotes hepatocyte-like differentiation and supports both robust HBV infection and strong response to IFN-α. In addition to infection and IFN sensitivity, effective screening requires a reliable method to detect and enrich HBV-infected cells. After evaluating a reporter virus approach, we developed a robust single-cell detection method based on intracellular staining of HBV core antigen (HBcAg). In conclusion, our study establishes a reliable experimental system for genetic screening of IFN effectors in HBV infection and provides a biologically relevant system for dissecting the antiviral mechanisms of interferon against HBV.

Grantor dc:publisher

Università degli Studi di Milano

Year dc:date

2025

Author dc:creator

• MARCHESE, SILVIA

Contributors dc:contributor

• tutor: R. De Francesco ; co-tutor: L. Manganaro ; coordinator: G. Norata
• S. Marchese
• DE FRANCESCO, RAFFAELE
• NORATA, GIUSEPPE DANILO

• Settore BIOS-15/A - Microbiologia

Statement dc:rights

• info:eu-repo/semantics/openAccess
• license:Creative commons
• license uri:http://creativecommons.org/licenses/by-sa/4.0/

Language dc:language

eng

Handle dc:identifier

https://hdl.handle.net/2434/1204341

OAI identifier oai:identifier

oai:air.unimi.it:2434/1204341