Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma & affects 1/30,000 males. Patients suffer from recurrent bleeds in soft tissues leading to physical disability in addition to life threatening bleeds. Current treatment (based on FIX infusion) is transient & plagued by increased risk for blood-borne infections (HCV, HIV), high costs & limited availability. This has fueled a search for gene/cell therapy based alternatives. Gene therapy with viruses is beset with problems of safety & increased immunogenicity. Being the natural site of FIX synthesis, the liver is expected to provide immune-tolerance & easy circulatory access. Liver transplantation is a successful, long-term therapeutic option but is limited by scarcity of donor livers & chronic immunosuppression; making iPSC-based cell therapy an attractive prospect. As part of this project, we plan to generate iPSCs from hemophilic patients that will then be genetically corrected by inserting DNA coding for FIX. After validation for correction, we will then differentiate these iPSCs into liver cells that can be transplanted into our mouse model of hemophilia that is capable of accepting human hepatocytes and allowing their proliferation. These mice exhibit disease symptoms similar to human patients & we propose that by injecting our corrected liver cells they will exhibit normal clotting as measured by various biochemical & physiological assays. If successful, this will provide a long-term cure for hemophilia & other liver diseases.
So far, we have generated multiple stem cell lines from two hemophilia B patients through cells in their peripheral blood. We have further sequenced the coding regions of the FIX gene in these patients to identify the mutations responsible for the disease. We have corrected the responsible single nucleotide change in two stem cell lines for one patient and are working towards correcting the other patient. For the other patient, we are currently planning to use a universal correction strategy that will bypass the need to sequence and identify the mutations each time. This will allow the integration of a functional and corrected F9 gene sequence under its native regulatory sequence. We have also generated a new mouse model for the disease that is deficient in 4 genes, making it permissive to the transplantation and expansion of human hepatocytes in its liver. We demonstrate the hemophilic nature of this new mouse strain through prolonged clotting time, absence of circulating protein and other biochemical assays. This hemophilic mousewill allow us to screen for symptoms of disease alleviation upon administration of patient derived hepatocytes. We are currently optimizing our differentiation protocol and conducting preliminary studies where these in vitro differentiated cells will be transplanted in our mouse model.