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.

Grant Number: 5R01HL166462-03
NIH Institute/Center: NIH
Principal Investigator: Graca Almeida-Porada