Investigating the Human-mouse Interspecies Barrier to AAV-driven Gene Therapy Using a Mouse with a Humanized Liver

Gene therapy with recombinant adeno-associated viral (AAV) vectors is becoming a frequent therapeutic modality for the treatment of monogenic diseases. One of the main challenges in gene therapy using the AAV capsid is that transduction rates obtained in preclinical studies using rodent and non-human primate models are not always predictive of transduction rates in humans. Clinical trials using rAAV8 to deliver factor IX (FIX) gene demonstrated much lower plasma FIX levels in human patients than that shown in rodents and non-human primates (NHPs). In contrast, AAV5-mediated expression of B-domain deleted Factor VIII translated relatively well from preclinical species to humans. In this study, we compare the expression of beta-chorionic gonadotrophin (bCG) reporter gene intravenously delivered by AAV5 or AAV8 at 6e13vg/kg in the Phoenix Bio (PXB) mouse; a mouse with a chimeric liver, almost fully repopulated with human hepatocytes. Sex and age matched PXB and Severe Combined Immunodeficiency (SCID) mice were euthanized 5 weeks after vector administration. The liver bCG DNA, RNA and protein were analyzed in histological liver specimens to compare the behavior of both capsids. In addition, the interspecies-tropism was investigated by immunostaining and quantifying the number of bCG-positive mouse and human-like hepatocytes in PXB chimeric livers. On average, PXB livers were 90% humanized based on human-albumin levels as well as percent of CK18/8 (clone DC10)-immunopositive hepatocytes. Regardless of the AAV serotype, PXB livers transduced poorly when compared to SCID mice. Based on the number of hepatocytes with nuclear bCG DNA content detected by in situ hybridization (ISH), AAV5 and AAV8 transduced 1.5 times more humanized hepatocytes than murine ones. However, based on quantitative immunohistochemistry (IHC), AAV5 similarly transduced 1% of humanized and murine hepatocytes while AAV8 distinctly transduced 15 times more residual murine hepatocytes than humanized hepatocytes. Despite having a preferential tropism for mouse hepatocytes, AAV8 serotype was more efficient in transducing humanized hepatocytes (3%). For both capsids, the loss of hepatic transduction efficiency was evident at RNA and protein levels. Our DNA and RNA results suggest that a higher dose could help increase transcription and translation rates. Irrespectively, low transduction efficiency to AAV gene therapy in PXB could be also attributed to a suboptimal response to AAV-directed gene therapy from the female human donor used for the PXB liver engraftment