Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma and 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 is transient, plagued by increased risk for blood-borne infections (HCV, HIV), high costs and limited availability.
In the last three years of this grant, we established the feasibility of hepatocyte transplantation as a therapeutic approach for hemophilia B. We transplanted cadaveric human hepatocytes into our quadruple knock-out hemophilic mice and found that these human hepatocytes could engraft and expand in the mouse liver for up to a year. The transplanted animals also showed dramatic improvement in their clotting phenotype attesting to the feasibility of this option. Our objective here was to extend cell therapy to patient iPSC-derived hepatocytes to mitigate the immune risks and the dire need for transplantable livers.
Pursuing this direction, we procured blood samples from 2 hemophilia B patients (HB001 and HB002) and derived iPSCs from them. We established their pluripotency and genomic stability and sequenced the FIX to identify the disease causing mutations in each case, then used the CRISPR/Cas9 technology to correct the mutation and to restore functionality of the FIX gene/protein. We developed 2 different approaches for this: one where we only changed the defective nucleotide and another where we inserted the full length FIX cDNA under control of the endogenous promoter bypassing the need to sequence every patient. We differentiated these autologous iPSC lines into “hepatocyte like cells/HLCs” using our in-house differentiation protocol. We demonstrated correction of the genetic defect through multiple approaches whereby expression of FIX is restored in these HLCs in culture. Finally we transplanted these in vitro differentiated HLCs into our mouse model and will be testing for their in vivo presence.