Methods/Results: Mouse and human plasma C5 protein levels were measured in our non-humanized and liver-humanized mouse models, respectively, using species-specific ELISA.  Both the FRG and FRGN strains express functional mouse C5 whereas our recently developed congenic FRGN strain that is homozygous for the NOD Hc0 mutation does not (hereafter referred to as FRGN C5KO). Accordingly, plasma from FRGN C5KO mice lacks classical complement activity in a sensitized sheep red blood cell hemolytic assay. In liver-humanized FRG, FRGN and FRGN C5KO strains, human plasma C5 protein is present at levels ~10-fold above that of mouse plasma C5 protein in non-humanized mouse complement-sufficient strains. Consistent with this large difference in C5 protein levels, hemolytic activity in the plasma from liver-humanized mice, including liver-humanized FRGN C5KO mice, is ~28 fold higher than in plasma from non-humanized, complement-sufficient mice. These findings suggest that even in complement-sufficient mouse hosts, the overwhelming majority of complement activity in liver-humanized mice is of human origin. To begin to assess whether efficient in vivo delivery of GalNAc-conjugated molecules to human hepatocytes in xenografted mice is possible, the expression of the asialoglycoprotein receptor (ASGPR1) in primary human donor cells prior to transplant and following stable liver engraftment in FRG strains was evaluated. Flow cytometry revealed that cell surface ASGPR1 expression is present in ~80% of the primary cryopreserved hepatocytes from each of our donors tested. Importantly, ³95% of engrafted donor human hepatocytes express high levels of cell surface ASGPR1 for at least 150 days after transplant.  To knockdown human C5 expression, liver-humanized mice were subcutaneously dosed with 5-10mg/kg of a C5-targeted siRNA containing a triantennary GalNAc conjugate and enhanced stabilization chemistry.  A single dose of 5mg/kg or 10mg/kg of C5 siRNA was sufficient to knockdown human plasma C5 protein levels ~70% by day 10 post-injection. In 5 of 7 of singly dosed animals, knockdown of human C5 protein in plasma was maintained for at least 25 days. We are currently evaluating whether multiple doses of the siRNA in liver-humanized mice is toxic to human hepatocytes by measuring human-specific ALT1 expression using a custom ELISA that we developed.  

Conclusion: The high levels of expression of ASGPR1 in human hepatocytes in chimeric livers combined with the efficient and durable knock down of human C5 achieved with a single dose of galNAc siRNA demonstrate that FRG® liver-humanized mice are an ideal preclinical model for evaluating the targeting efficiency and toxicity of GalNAc-conjugated therapeutics.