• Introduction: The sodio-taurocholic acid cotransport polypeptide (NTCP) is a 9-transmembrane domain protein. NTCP is mainly expressed in the basement membrane side of hepatocytes (blood sinus side), which promotes the absorption of bile acid in portal vein blood into the liver, and is associated with cholestasis, liver fibrosis, and nonalcoholic fatty liver disease. In addition, it is also a functional receptor for human hepatitis B virus (HBV) and hepatitis D virus (HDV). The two marketed drugs targeted are synthetic polypeptides of HBV Pre-S1 structure, which can competitively bind to NTCP to block the entry of hepatitis virus and bile acids into liver cells. There are also oligonucleotide drugs that target NTCP, and ADAR-based RNA editing introduces NTCP nonsense mutations to relieve cholestasis.
• Targeting strategy: The exons 1-6 of mouse Ntcp gene that encode the whole molecule (ATG to STOP codon), including 3’UTR were replaced by human counterparts in B-hNTCP mice. The promoter and 5’UTR of the mouse gene are also replaced. The human NTCP expression is driven by human NTCP promoter, while mouse Ntcp gene transcription and translation will be disrupted.
• mRNA and protein expression analysis: 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. NTCP was detectable in both the liver and kidney of wild-type mice and homozygous B-hNTCP mice, as the antibody used cross-reacts with both human and mouse NCTP.
• In vivo efficacy: Human NTCP targeted nucleic acid drug was efficacious in B-hNTCP mice after drug administration.
• Application: This model can be used to evaluate the efficacy and safety of drugs targeting NTCP

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

Strain specific analysis of NTCP mRNA expression in wild-type C57BL/6JNifdc and 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.

Sex specific analysis of NTCP mRNA expression in heterozygous male and female B-hNTCP mice by RT-qPCR. Liver 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 PCR with mouse or human NTCP primers. Both mouse Ntcp and human NTCP mRNA expression are higher in female mice compared to that in male mice. Values are expressed as mean ± SEM.

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 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.