Abstract
Hemophilia A (HA) is an X-linked recessive disorder characterized by a severe lack of coagulation factor VIII (FVIII). This lack of FVIII leads to bleeding crises following even minor injuries (Ferreira et al., 2015). Prolonged bleeding crises can result in joint and muscle damage, and ultimately death if left untreated (Everett, Cleuren, Khoriaty, & Ginsburg, 2014). This disorder affects 1 in 5,000 males in the United States (US). At least 40% of these US cases are severe, defined as having less than 1% of FVIII in the blood plasma (Franchini, 2013; Park et al., 2015). Current treatment for HA consists of regularly scheduled injections of recombinant FVIII (Rangarajan et al., 2017). These FVIII injections help prevent bleeding events, but must be administered for the duration of the patients’ lives. Although these treatments help mitigate symptoms for HA, FVIII injections are not a cure. A long-term treatment, beyond the current treatments, needs to be developed to better care for those suffering from HA.
Researchers have previously considered treating HA by transplanting liver sinusoidal endothelial cells (LSECs), which are the cell type that naturally produces FVIII (Fomin et al., 2013). These cells were transplanted from healthy donor mice into HA model mice. Results showed a FVIII level in the plasma of transplanted mice of approximately 10%, a level that denotes mild cases of HA in humans (Follenzi et al., 2008; Fomin et al., 2013). Fomin et.al have also successfully transplanted human LSECs into HA model mice, a significant milestone in the translation of this treatment into a cure for human patients (Fomin et al., 2013). Although LSECs have shown promising results in their ability to provide a therapy for HA, these cells are limited by the shortage of liver donors, difficulty to harvest, and invasive transplantation procedures (Fomin, Togarrati, & Muench, 2014). Other researchers attempted to utilize skin fibroblast cells transfected with the bddF8 gene, a gene that produces FVIII with the B-domain removed since the B- domain decreases FVIII expression (Roth, Tawa, O'Brien, Treco, & Selden, 2001). The results from this study were promising during the first six months following treatment, but FVIII levels decreased back to less than 1% in the HA model mice thereafter (Roth et al., 2001). This research provided promising results in treatments for HA, but long-term efficiency of these transplantations still need to be optimized before their wide-spread use.
Currently, there is no cure for HA and the treatment isn’t entirely ideal, making it important for researchers to seek alternative treatment for this rare disorder. The primary goal of the research in Dr. Zhou’s lab is to develop an autologous stem cell-based therapy to functionally correct HA. This can be achieved through gene editing of HA patients’ induced pluripotent stem cells (HA-iPSC) with CRISPR-Cas9 to produce functional FVIII and differentiating them into endothelial cells for transplantation. To determine the accuracy of the designed gene editing, we utilized qRT-PCR, PCR, and gel electrophoresis. We found that multiple HA-F8-iPSC clones integrated the bddF8 gene and were capable of expressing FVIII as detected by both an ELISA and qRT-PCR. To analyze the therapeutic effect of HA-F8-ECs transplanted into HA mice, we utilized an FVIII antigen ELISA to detect FVIII levels in the plasma of the mice. Further research is needed to properly determine the therapeutic effect of transplantation. The endothelial cells derived from the autologous hiPSCs edited to express F8 with CRISPR/Cas9 have the potential to treat HA by producing FVIII constantly upon transplantation without the need for intravenous injections.