PROJECT SUMMARY/ABSTRACT Hepatic in vivo gene transfer with adeno-associated viral (AAV) vectors for treatment of the X-linked bleeding disorder hemophilia has advanced to multiple Phase I/II and 4 Phase III clinical trials, all seeking to stably restore hemostasis through long-term expression in hepatocytes. As opposed to other treatment modalities, gene therapy has the potential to cure the disease, thus eliminating the need for frequent injections of coagulation factors or other medicines. However, it has been a major challenge to accomplish sustained correction of hemophilia A (deficiency in coagulation factor VIIII, FVIII, representing ~80% of hemophilic patients). FVIII is difficult to express at high levels. Hence, high vector doses are required. Nonetheless, FVIII activity in the normal to super-physiological range was achieved in clinical trial. While this result was widely celebrated in 2017, it has since become clear that FVIII expression at these high levels is not stable. A gradual decline in all patients was observed in years 2-4, down to the lower end of the therapeutic range, raising serious concerns about durability. Other studies begin to show similar outcome, whereas lower FVIII levels, resulting from more modest vector doses, appear to be sustained. Hepatotoxicity and treatment with steroids were typical features during the first year of high-dose gene transfer. There is now a growing realization that basic science studies are paramount to achieve safety and efficacy. Our preliminary data strongly support the hypothesis that the closely interlinked cellular stress and innate immune responses to FVIII and the vector (which are substantially impacted by vector design) limit stability of therapeutic expression and also drive adaptive immunity. It is well established that expression of FVIII at high levels results in the accumulation of unfolded protein in the ER, cellular stress and toxicity, aggregation, und induction of the unfolded protein response (UPR). Our data show that hepatic AAV gene transfer, while having the potential to induce immune tolerance to transgene products, also activates innate immune pathways and cytokine responses that can lead to cellular immune responses. AAV vectors introduce pathogen-associated molecular patterns (PAMPs). Cellular stress and innate immunity are interlinked, and damage-associated molecular patters (DAMPs) drive immunity. In fact, we see gradual loss of FVIII expression in a hemophilia A mouse models despite a lack of antibody formation. This project specifically proposes to: i) Define the mechanisms by which “subgenomic particles” cause innate and adaptive immune responses to AAV- FVIII in primary human hepatocytes/innate immune cells and in murine models; ii) define immune response mechanisms against FVIII in hepatic AAV gene transfer as a function of vector and transgene design; and iii) define the mechanisms that lead to gradual loss of FVIII expression and develop protocols for sus...