Enhancing immune regulation in gene therapy for hemophilia

In recent years, 3 gene therapy protocols for the treatment of the bleeding disorders hemophilia A (coagulation factor FVIII deficiency) and hemophilia B (FIX deficiency) have received regulatory approval, all using in vivo adeno-associated viral (AAV) gene transfer into hepatocytes. We were the first to show that this approach induces regulatory T cells (Treg) and promotes immune tolerance to the transgene product, a phenomenon that has since been replicated in many different animal models. First evidence that this concept applies to humans has now been obtained in a hemophilia A patient whose antibody against FVIII was eliminated by AAV gene transfer. However, we found that the liver is also capable of priming CD8+ T cell responses against the transgene product (through forming dendric cell (DC) - Kupffer cell (KC) - T cell networks). We uncovered critical innate signals and mechanisms that lead to T cell activation, defined a role for levels of gene expression, and developed adjunct immune modulation protocols that further improve immune regulation, reduce responses to viral capsid, and enhance safety of therapy. Here, we will continue combining our team’s expertise in gene transfer, immunology, and liver biology to define intrahepatic immune mechanisms and promote tolerance induction. Additionally, we will incorporate new models of high translational relevance and study the effects caused by hepatocyte steatosis. Ultimately, we aim for these studies to help eliminate the considerable inter-patient variability in efficacy, toxicity/immune response, and durability of gene therapy. Approved drugs and clinical trials utilize diverse capsids and very different vector doses. Realizing that efficacy in mice cannot be extrapolated to humans, we developed models with primary human hepatocytes (PHHs) to better predict clinical performance of candidate capsids. A major unknown is the impact of steatotic liver disease, which has become highly prevalent in the population, including among hemophilia patients. Our preliminary data show increased transduction and transgene expression in steatotic PHH. We hypothesize that steatosis significantly impacts transduction and innate immune activation with capsid-dependent outcomes. We seek to 1. define the transduction patterns and virus-host cell interactions of AAV vectors in normal vs steatotic (fatty) human liver using “humanized” mice, a human liver 5-cell organoid model, and machine perfused human livers. 2. Define the mechanisms that link innate immune responses and antigen trafficking to transgene product-specific immune responses in AAV transduced liver. 3. Develop optimal immune regulation in gene therapy for hemophilia A by transient administration of an immunocytokine. This proposal addresses specific aspects of hemophilia treatment as well as questions of broader significance for liver-directed gene therapy. Moreover, addressing the complexity of the liver’s immune system (incl. innate immune mechanisms, balance between adaptive immunity and tolerance, impact of steatosis) is of considerable general significance.