PROJECT SUMMARY ABSTRACT
Gene therapy for the X-linked bleeding disorder hemophilia holds much promise to accomplish a lasting cure.
Four clinical trials utilizing adeno-associated viral (AAV) gene transfer to the livers of males with severe
hemophilia are currently being investigated in multiple Phase III clinical trials. Hemophilia A (deficiency in factor
VIII, FVIII), the more common form of the disease (~80% of patients), has traditionally been more difficult to treat
by gene therapy because FVIII is a large molecule and not efficiently expressed and secreted. Nonetheless,
initial results demonstrated complete correction of the disease. However, FVIII levels declined substantially over
time, raising worrying questions about durability, and patients also experienced prolonged mild hepatotoxicity
despite steroid drug treatment during the first year of gene therapy. Multiple recent observations raise serious
questions about the safety of hepatic gene therapy for hemophilia A. These urgently need to be addressed so
that this promising approach can be safely applied to patients and to achieve sustained correction. For instance,
the reasons for hepatotoxicity and for the decline in FVIII expression are unclear, highlighting critical gaps in our
knowledge of the interactions between the vector and hepatocytes and between the FVIII expression and
hepatocytes, as well as the role of the immune system in long-term outcome. There is also renewed concern
about insertional mutagenesis. We will address these basic and mechanistic questions related to the biology of
AAV and FVIII. The central hypothesis of this proposal is that multiple interconnected features of AAV and FVIII
biology limit durability of therapeutic expression and pose serious safety concerns. Further, we postulate that
unraveling these mechanisms will allow for design of vectors and protocols that minimize these problems, thus
resulting in lasting therapy and enhanced safety. The program combines expertise in FVIII biology, cellular stress
responses, immunology, and AAV vector biology and is structured into 3 scientific Projects, an administrative
Core and 2 scientific Cores. Project 1 (Kaufman) seeks to overcome FVIII protein misfolding and cell toxicity.
Project 2 (Xiao) will uncover the mechanisms that lead to formation of subgenomic AAV vector particles that
form during vector production through nuclease and recombination activities. Project 3 (Herzog) will define the
mechanisms of innate and adaptive immune responses to AAV-FVIII gene transfer. The objectives of the three
projects will be supported by an administrative core (Core A), a core that provides human hepatocytes for in vitro
and in vivo studies (Core B), and a core that performs development and molecular analysis of AAV vectors (Core
C). Overall, this project applies the expertise of the individual investigators towards addressing major
unanswered questions in FVIII biology, gene therapy for hemophilia, liver-directed gene transfer, and molecular
and immunobiology of AAV vectors.