Study Abstract

Hemophilia B (HEMB), a single gene disorder affecting Factor IX, is an ideal target for gene therapies. In order to establish a relevant model for evaluating such therapeutics, primary hepatocytes from a patient with HEMB were evaluated for clotting defects and gene therapy correction in vivo in liver-humanized mice. For this, primary hepatocytes from a male HEMB donor were transplanted into male FRG® mice on the NOD background (FRGN). Primary hepatocytes from a non-hemophilic human male donor were transplanted into male FRGN mice as a control. Mice with human albumin levels of >4000 ug/mL, indicative of livers repopulated with >70% human hepatocytes, were compared. Humanized mice repopulated with control donor cells (n=4) had average circulating hFIX levels of 895 ±96.6 ng/mL, whereas mice repopulated with the HEMB donor (n=5) had 9-fold lower average circulating hFIX levels of 98.9±26.9 ng/mL. Measurement of partial thromboplastin time (PTT) in plasma samples from liver-humanized mice created with the control donor cells (n=9) revealed a mean PTT of 28.49 seconds. In HEMB liver-humanized mice (n=9), the average PTT was increased by 24% to 35.34 seconds. Thus, physiologically relevant defects in clotting could be modelled in liver-humanized mice reconstituted with a HEMB donor. To demonstrate that this model could be used to assess efficacy of gene therapies, HEMB mice were injected i.v with 2.5x10¹¹ copies per mouse of  liver-tropic AAV expressing hFIX (AAVDJ-CAG-hFIX). Two weeks after injection, increased expression of hFIX (343±66.6 ng/mL) and a significant decrease in PTT (30.25s, p<0.005) was observed compared to untreated HEMB liver-humanized mice. Even without significant optimization of AAV-mediated correction, there was no significant difference in clotting time between HEMB liver-humanized mice that received hFIX gene therapy and mice that were liver-humanized with hepatocytes from a control donor. Our findings demonstrate that primary human hepatocytes from donors with genetic disease such as hemophilia B can successfully repopulate FRG mice, recapitulate physiologic disease pathologies, and be used to evaluate relevant in vivo therapeutic approaches.