NTCP: Mutations in NTCP disrupt bile acid transport and confer susceptibility to HBV/HDV hepatic infection.

• Gene Information: Human SLC10A1 gene, encoding NTCP (sodium-taurocholate cotransporting polypeptide) is located on chromosome 14.
• Protein Expression: NTCP protein is specifically and predominantly expressed on the basolateral membrane of hepatocytes in the liver.
• Signaling Pathway: Functional impairment of NTCP impairs hepatic uptake of circulating bile acids, resulting in elevated serum bile acid levels and disordered bile acid homeostasis. Meanwhile, NTCP on the hepatocyte membrane mediates binding and internalization of HBV/HDV viral particles, enabling targeted viral infection of human liver cells.
• Therapeutic Application: Entry inhibitors (e.g., bulevirtide) that block NTCP function, together with NTCP expression suppressors, constitute a promising therapeutic strategy for the treatment of chronic hepatitis B (HBV), chronic hepatitis D (HDV), as well as the clinical management of cholestatic liver diseases.

NTCP

• The exons 1-6 of mouse Ntcp gene that encode the whole molecule (ATG to STOP codon), including the promoter, 5’UTR and 3’UTR were replaced with the human counterparts in B-hNTCP mice.
• Human NTCP expression is driven by human NTCP promoter, while mouse Ntcp gene transcription and translation will be disrupted.

• Human NTCP mRNA was exclusively detectable in homozygous B-hNTCP mice but not in wild-type mice. Human sequences were confirmed via Sanger sequencing.

Strain specific analysis of NTCP mRNA expression in wild-type C57BL/6JNifdc and homozygous B-hNTCP mice by RT-PCR. Liver RNA were isolated from wild-type C57BL/6JNifdc (+/+) and homozygous B-hNTCP mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human NTCP primers. Mouse Ntcp mRNA was only detectable in wild-type mice. Human NTCP mRNA was exclusively detectable in homozygous B-hNTCP mice but not in wild-type mice. Human sequences were confirmed via Sanger sequencing.

• The mRNA expression levels of both endogenous mouse Ntcp and humanized human NTCP were higher in female mice compared to that in male mice.

Sex specific analysis of NTCP mRNA expression in heterozygous male and female B-hNTCP mice by RT-qPCR. Liver total RNA was isolated from 12-week-old heterozygous male and female B-hNTCP mice (H/+, n=5 for males, and n=3 for females), then cDNA libraries were synthesized by reverse transcription, followed by qPCR with mouse or human NTCP primers. The mRNA expression levels of both endogenous mouse Ntcp and humanized human NTCP were higher in female mice compared to that in male mice. Values are expressed as mean ± SEM.

• NTCP was detected in liver, but weakly detected in kidney of both homozygous mice and wild-type mice, as the antibody used cross-reacted with both human and mouse NTCP.

Western blot analysis of NTCP protein expression in homozygous B-hNTCP mice and B-NDG hNTCP mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+), B-NDG mice (+/+), homozygous B-hNTCP mice (H/H) and B-NDG hNTCP mice (H/H), and then analyzed by western blot with cross reactive anti-NTCP antibody (Abcam, ab131084). 40 μg total proteins were loaded for western blot analysis. NTCP was detected in liver, but weakly detected in kidney of both homozygous mice and wild-type mice.

• The human NTCP mRNA in the treatment group were significantly reduced compared to the control group, demonstrating that B-hNTCP mice provide a powerful preclinical model for in vivo evaluation of human NTCP-targeted nucleic acid drugs.

The inhibitory efficiency of the NTCP-targeted small nucleic acid drug in heterozygous B-hNTCP mice. B-hNTCP mice were randomly divided into 2 groups (n=5, 12 weeks old, male). The human NTCP-targeted nucleic acid drug (SR122 analog, synthesized according to patents) and saline were administered to the mice individually. The nucleic acid drug was administered in the form of saline aqueous solution. The mice were sacrificed on day 14. (A) The schematic diagram of experimental processing. (B) The expression of human NTCP mRNA in liver. The human NTCP mRNA in the treatment group were significantly reduced compared to the control group. Values are expressed as mean ± SEM.