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

• P2A and the human CDS that encodes human NTCP signal peptide, extracellular domain, transmembrane, cytoplasmic domain and 3’ UTR is inserted in the exon 3 of mouse Ntcp gene. The human NTCP protein expression will be driven by endogenous mouse Ntcp promoter, while mouse Ntcp gene transcription and translation will be disrupted. 

• Human NTCP mRNA was exclusively detectable in homozygous B-NDG hNTCP mice but not in wild-type B-NDG mice.

Strain specific analysis of NTCP mRNA expression in wild-type B-NDG mice and B-NDG hNTCP mice by RT-PCR. Liver RNA were isolated from wild-type B-NDG mice (+/+) and homozygous B-NDG 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 B-NDG mice. Human NTCP mRNA was exclusively detectable in homozygous B-NDG hNTCP mice but not in wild-type B-NDG mice.

• The NTCP mRNA expression level in homozygous B-NDG hNTCP mice was comparable to wild-type B-NDG mice.

Species specific analysis of NTCP mRNA expression in wild-type B-NDG mice and homozygous B-NDG hNTCP mice by RT-qPCR. Liver were collected from wild-type B-NDG mice (+/+) (male, n=4, 12-week-old) and homozygous B-NDG hNTCP mice (H/H) (male, n=4, 12-week-old). The human NTCP mRNA expression level in homozygous B-NDG hNTCP mice was comparable to that in wild-type mice. (A) Relative NTCP mRNA expression detected using mouse Ntcp primers, which were also capable of amplify human NTCP sequences as this primer pairs targeting the unreplaced region in 5’UTR. (B) Relative human NTCP mRNA expression detected using human-specific NTCP primers in wild-type and homozygous B-NDG hNTCP 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.

• Serum total bile acids showed no significant differences between wild-type B-NDG mice and homozygous B-NDG hNTCP mice.

Total bile acid analysis in wild-type B-NDG mice and homozygous B-NDG hNTCP mice. Serum was collected from wild-type B-NDG mice (+/+, n=15, 13-week-old) and homozygous B-NDG hNTCP mice (H/H, n=20, 13-week-old). Serum total bile acid in homozygous B-NDG hNTCP mice showed no significant differences between wild-type mice and homozygous B-NDG hNTCP mice. Values are expressed as mean ± SEM. Significance was determined by unpaired t test.  *P < 0.05, **P < 0.01, ***P < 0.001.

• 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 homozygous B-NDG hNTCP mice. B-NDG hNTCP mice were randomly divided into 2 groups (n=5, 13-week-old, male). The human NTCP-targeted nucleic acid drug (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.