Introduction Recombinant adeno-associated virus vectors (rAAV) are a major gene therapy platform for treatment of monogenic disorders, including hemophilia A. Valoctocogene roxaparvavec (AAV5-HLP-hFVIII-SQ) is an AAV5 vector delivering B-domain-deleted (BDD) human FVIII transgene controlled by a hybrid liver-selective promoter. In a phase 3 trial, a single treatment of AAV5-HLP-hFVIII-SQ (6e13 vg/kg) provided therapeutic expression of FVIII and bleeding control in adult severe hemophilia A patients. However, the mechanistic basis of transaminitis, variability and durability observed in clinical trial participants are not clear. Understanding the molecular changes in the liver are critical to identify appropriate immune modulatory strategies for safety, efficacy, and long-term durability of AAV gene therapy. The aim is to investigate liver gene expression profiles before and after AAV5-HLP-canine-BDD-FVIII (cFVIII) administration in a severe hemophilia A dog model.

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liverdirected AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

Introduction Adeno-associated virus (AAV)-based gene therapy can initiate host-immune responses to the AAV vector components or the transgenic protein product which could reduce AAV-mediated expression. Data from preclinical studies suggest that corticosteroid treatment may limit these immune responses and improve gene therapy outcomes.