Using human liver tissue equivalents to optimize AAV-mediated GT and better define age-related clinical risks

PROJECT SUMMARY Gene therapy (GT) clinical trials using AAV vectors are poised to fulfill the promise of a safe, affordable, lifelong correction of bleeding disorders following a single treatment. Still, clinical trials using AAV vectors to treat hemophilia A (HA) in adults have underscored the hurdles, such as the presence of pre-existing AAV antibodies, and unexpected risk of hepatoxicity in these patients. Importantly, this toxicity was not seen in preclinical animal studies, highlighting the dangers of extrapolating data from animal models to humans. Since the next step for GT to treat severe HA will be implementation of this approach in children, it is crucial to predict, as accurately as possible, unforeseen risks in this population. Currently, is unknown whether the unexpected immune/ inflammatory responses seen are due to the use of AAV as a delivery vehicle, or they are caused by the forced expression of FVIII within hepatocytes, which are not the native site of FVIII production. However, since similar toxicity has not been seen in AAV clinical trials for hemophilia B (hepatocytes are the natural site of FIX production), it is rational to posit that ectopic FVIII expression likely plays a role. In addition, preclinical data have also shown that, at the high doses used, AAV, long assumed to be largely episomal, may exhibit significant levels of host genome integration that could potentially drive clonal expansion and hepatocellular carcinoma (HCC), the risk of which increases as a result of hepatocyte proliferation. These are critical questions to safely extend the use of these potentially curative treatments to the pediatric population, in whom the higher proliferation and more primitive state of the liver may increase these risks. The overall goal of the present proposal is to utilize a human liver tissue equivalent (hLTE) platform to answer these questions and to determine the impact recipient age has on these variables. We will use hLTE to test the overall hypothesis that FVIII expression can be improved, the pre-existing immunity to AAV overcome, and the toxicity seen in clinical trials avoided, by optimizing the codon usage and/or sequence of the fVIII transgene to minimize the unfolded protein response and ER stress and/or by targeting transduction to hepatic endothelium, the native site of FVIII synthesis. Specifically, we will use a physiologically relevant hLTE platform to: 1) define age-dependent impact of AAV transduction vs. hepatocyte-targeted FVIII expression on human liver biology and function, the potential to trigger innate immunity, and whether optimizing the codon usage and sequence content of the fVIII transgene can prevent this undesired immune/inflammatory response; 2) test whether targeting AAV transduction to hepatic endothelium will improve FVIII expression, prevent hepatic inflammation/immunity, preserve liver function, and protect AAV from existing anti-capsid immunity; and 3) investigate if genomic integration frequency will be higher at younger ages, due to increased cell cycling, and whether targeting hepatic endothelial cells will decrease the potential for genotoxicity. It is hoped that these studies will identify the means to maximize the efficacy and safety of human liver-targeted AAV GT for HA and thereby pave the way for its use in pediatric patients.