The goal of my thesis project is to develop a new therapy that will achieve life-long treatment of hemophilia and potentially other genetic diseases. Hemophilia A and B are X-linked diseases caused by dysfunction or absence of blood coagulation factors VIII (FVIII) and IX (FIX), respectively. More than 20,000 men in the US have hemophilia, and hundreds of thousands more worldwide. Hemophilia is currently treated by protein replacement therapy, but this is extremely costly (>$100,000 per patient per year) and up to 25% of severe hemophiliacs develop anti-FVIII or -IX antibodies that neutralize the protein replacement. The development of a safe and effective gene therapy for hemophilia is a long-standing goal for the disease. Despite some clinic success, gene therapy has not become a widespread treatment. The failures and problems of gene and cell therapy for genetic disease have been largely due to instability of the treatment, immunogenicity of the vector, and the development of vector-mediated oncogenesis. To overcome these serious limitations, I propose to exploit T cells as a cellular vehicle for hemophilia gene therapy. I hypothesize that lentiviral vectors can be used to introduce a FIX gene into human T cells, and that transfer of the T cells back into a hemophiliac will result in safe and sustained expression of FIX and correction of the disease. My hypothesis is based on the stunning clinical success of chimeric antigen receptor (CAR) T cell therapy in treating human leukemia. CAR T cells are generated by removing T cells from a patient, and transducing them with a lentiviral vector encoding a CAR that recognizes leukemic cells. The engineered T cells are transferred back to patients where they wipe out tumor cells. Dozens of patients have been treated with CAR T cells, and no vector-related adverse events have been reported. Moreover, LV-engineered T cells have been found to last for years in patients. To test my hypothesis, I will: (1) Generate lentiviral vector-mediatd FIX- secreting human T cells (in vitro), (2) Carry out studies to optimize the ability of the FIX-secreting T cells to be transferred into animals and mediate long-lasting production of circulating FIX, and (3) Evaluate the use of the CRISPR/Cas9 system to insert a FIX transgene into a safe harbor locus of the genome of human T cells. This will be the first time that T cells are utilized as a cellular vehicle for hemophilia gene therapy. Importantly, because lentiviral vector-modified T cells have been used in humans, there is already substantial safety data. Thus, if the outcome of this project establishes the effectiveness of the approach in animal models of hemophilia, it will be feasible to move this strategy to the treatment of human hemophilia. In addition to its relevance for human health, this F31 application will also support my training as a scientist. Through these studies I will gain expertise in the burgeoning area of human T cell therapy, as well as in genetic engineering, CRISPR/Cas9 technology, and hemophilia. I will also gain general expertise in hematology and immunology. This training will be invaluable towards my goal of becoming an independent investigator.
The studies in this project will generate and test a novel gene and cell therapy for hemophilia B. The strategy I will evaluate has been designed to overcome the limitations and problems of existing approaches. If successful, these studies could lead to a safe and long-lasting new treatment for the hemophilias as well as other genetic diseases.
