Abstract
Hemophilia A (HA) is a genetic disorder resulting from deficient levels of the coagulation protein factor VIII. HA is inherited through an X-linked recessive pattern and is characterized clinically by excessive and spontaneous bleeding, the severity of which corresponds inversely to the amount of circulating factor VIII. Patients with recurrent hemorrhaging also experience severe complications such as joint damage, chronic pain, arthritis, and damage to nerves and tissues. Current medical dogma mandates that HA patients receive costly lifelong intravenous injections of recombinant factor VIII, wherein limiting factors such as a short half-life and activation of inhibitory antibodies affect treatment efficacy. New research is exploring gene and cell therapy-based approaches to restore expression of factor VIII that is otherwise deficient due to mutations of the F8 gene in HA patients. In fact, previous studies have created constructs that synthesize factor VIII in mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) to levels nearing those of normal physiology. However, long-term engraftment of MSCs has not been proven and efficient engraftment of HSCs requires undesirable preconditioning of the patient. Therefore, new and effective gene and cell therapy is needed to persistently provide factor VIII for HA patients. The overall goal of this study is to develop liver organoids that will increase engraftment success and simultaneously provide a supply of therapeutic levels (above 1% of normal) of factor VIII. The use of HA patient specific induced pluripotent stem cells (HA-iPSCs) is revolutionary for avoidance of an immune response against the cells differentiated from these iPSCs and transplanted back to the patient. We have generated liver organoids by coculturing iPSC-derived endothelial cells and iPSC-derived hepatocyte progenitor cells. We hypothesized that HA-iPSCs derived organoids can be used to ectopically express the F8 gene and efficiently engraft upon being transplanted, and therefore offer more effective treatment strategies. We proposed two aims for our study: Aim I was to express factor VIII in normal human iPSCs and HA-iPSC derived hepatocyte and endothelial cells using a viral vector carrying the factor VIII gene; Aim II was to generate liver organoids using both HA-iPSC derived hepatocyte and endothelial cells, and evaluate their long-term engraftment in immunodeficient mice. Successful differentiation of HA-iPSCs into endothelial cells and concurrent expression of factor VIII, as shown by our data, demonstrate the potential use of HA-iPSCs derived endothelial cells and hepatocyte progenitor cells as stem cell based therapy to treat hemophilia A. Results obtained in this project provide a foundation to develop a novel and translational therapeutic approach for HA to greatly reduce the burden imposed by extremely expensive ($200,000/year) and life-long infusion of factor VIII.