Application of in-vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action

Abstract

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 <i>in vitro</i> primary hepatocyte cultures to predict <i>in vivo</i> liver-directed AAV expression in different species. We assessed whether <i>in vitro</i> AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model <i>in vivo</i> 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 <i>in vitro</i> correlated with <i>in vivo</i> 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 <i>in vivo</i>. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using <i>in vitro</i> primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.