Modified apoptotic molecule (BID) reduces hepatitis C virus infection in mice with chimeric human livers.

Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.

TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice.

Tumor necrosis factor (TNF) family ligand TNF-alpha and Fas ligand (FasL) can trigger apoptosis in solid tumors, but their clinical usage has been limited by hepatotoxicity. TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the TNF family, and its clinical application currently is under a similar debate. Here, we report a recombinant soluble form of human TRAIL (114 to 281 amino acids) that induces apoptosis in tumor cells but not human hepatocytes. We first isolated human hepatocytes from patients and showed that the human hepatocytes expressed Fas but no TRAIL death receptor DR4 and little DR5 on the cell surface. Antibody cross-linked FasL, but not TRAIL, triggered apoptosis of the human hepatocytes through cleavage of caspases. We then examined TRAIL hepatotoxicity in severe combined immunodeficient/Alb-uPA chimeric mice harboring human hepatocytes. Intravenous injection of FasL, but not TRAIL, caused apoptotic death of human hepatocytes within the chimeric liver, thus killing the mice. Finally, we showed that repeated intraperitoneal injections of TRAIL inhibited intraperitoneal and subcutaneous tumor growth without inducing apoptosis in human hepatocytes in these chimeric mice. The results indicate that the recombinant soluble human TRAIL has a profound apoptotic effect on tumor cells but is nontoxic to human hepatocytes in vitro and in vivo.

Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application.

Compounds with in vitro anti-hepatitis C virus (HCV) activity are often advanced directly into clinical trials with limited or no in vivo efficacy data. This limits prediction of clinical efficacy of compounds in the HCV drug pipeline, and may expose human subjects to unnecessary treatment effects. The scid-Alb-uPA mouse supports proliferation of transplanted human hepatocytes and subsequent HCV infection. Cohorts of genotype 1a HCV-infected mice were treated with interferon alpha-2b(IFN-alpha), BILN-2061 (anti-NS3 protease), or HCV371 (anti-NS5B polymerase). Mice treated with 1350 IU/g/day IFN-alpha intramuscularly for 10 to 28 days demonstrated reduced viral titers compared with controls in all five experiments (P < .05, t test); viral titers rebounded after treatment withdrawal. A more pronounced antiviral effect with IFN-alpha was seen in genotype 3a-infected mice. Pilot studies with BILN2061 confirmed exposure to 10X replicon EC50 at trough and reduced viral titer over 2 log at 4 days. In a second 7-day study, mean HCV RNA titers dropped 1.1 log in BILN2061-treated animals, 0.6 log in IFN-treated mice, and rose 0.2 log in controls (P = .013, ANOVA). Pre-existing mutants with partial resistance to BILN2061 were identified by sequencing both the human inoculum and sera from treated mice. The polymerase inhibitor HCV371 yielded a decline in HCV titers of 0.3 log relative to vehicle-treated controls (P = NS). Performance of all three antiviral regimens in the chimeric mouse model paralleled responses in humans. In conclusion, this system may help selection of lead compounds for advancement into human trials with an increased likelihood of clinical success while broadening the tools available for study of the biology of HCV infection.