Genetically edited hepatic cells expressing the NTCP-S267F variant are resistant to hepatitis B virus infection

Takuro Uchida; Seung Bum Park; Tadashi Inuzuka; Min Zhang; Joselyn N Allen; Kazuaki Chayama; T Jake Liang

doi:10.1016/j.omtm.2021.11.002

Genetically edited hepatic cells expressing the NTCP-S267F variant are resistant to hepatitis B virus infection

Mol Ther Methods Clin Dev. 2021 Nov 10:23:597-605. doi: 10.1016/j.omtm.2021.11.002. eCollection 2021 Dec 10.

Authors

Takuro Uchida^{1

2}, Seung Bum Park¹, Tadashi Inuzuka¹, Min Zhang¹, Joselyn N Allen¹, Kazuaki Chayama^{3

4}, T Jake Liang¹

Affiliations

¹ Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
² Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
³ Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
⁴ RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.

Abstract

The sodium-dependent taurocholate co-transporting polypeptide (NTCP)-S267F variant is known to be associated with a reduced risk of hepatitis B virus (HBV) infection and disease progression. The NTCP-S267F variant displays diminished function in mediating HBV entry, but its function in HBV infection has not been fully established in more biologically relevant models. We introduced the NTCP-S267F variant and tested infectivity by HBV in genetically edited hepatic cells. HepG2-NTCP clones with both homozygous and heterozygous variants were identified after CRISPR base editing. NTCP-S267F homozygous clones did not support HBV infection. The heterozygote clones behaved similarly to wild-type clones. We generated genetically edited human stem cells with the NTCP-S267F variant, which differentiated equally well as wild-type into hepatocyte-like cells (HLCs) expressing high levels of hepatocyte differentiation markers. We confirmed that HLCs with homozygous variant did not support HBV infection, and heterozygous variant clones were infected with HBV equally as well as the wild-type cells. In conclusion, we successfully introduced the S267F variant by CRISPR base editing into the NTCP/SLC10A gene of hepatocytes, and showed that the variant is a loss-of-function mutation. This technology of studying genetic variants and their pathogenesis in a natural context is potentially valuable for therapeutic intervention against HBV.

Keywords: CRISPR base editing; HBV; NTCP; S267F polymorphism; stem cell-derived hepatocyte.