Towards Cell Therapy for FPD/AML with Patient-Specific IPS Cells

Abstract 4677

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell type. This makes them a valuable source of cells for treating genetic diseases. If the disease causing mutation can be corrected in patient-specific iPSCs and the cells differentiated to therapeutically relevant cell types and transplanted back, the symptoms of the disease may be alleviated or possibly cured. Hematopoietic diseases are an attractive target due to the ability of hematopoietic stem cells (HSCs) to reconstitute the entire hematopoietic system. However, directing the differentiation of iPSCs towards a HSC fate has proven difficult. Our lab aims to (1) Derive iPSCs from patients with a hematopoietic disease and correct the mutant gene through gene targeting. This will provide iPSCs that could be used in treating the patients. (2) Develop methods that direct the differentiation of iPSCs towards a HSC fate. This will be accomplished through novel iPSC reporter lines that we will use in small molecule and RNAi screens to identify factors that direct iPS cell differentiation towards a HSC fate.

Familial Platelet Disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disorder caused by a mutation in the gene RUNX1 resulting in thrombocytopenia, platelet defects, and a predisposition to AML. Our first goal is to derive iPSCs from FPD/AML patients and correct the genetic defect using zinc finger nuclease mediated gene targeting. This will provide a potential source of cells for therapy. Towards this goal, we have obtained fibroblasts from four members of a FPD/AML family and derived iPSCs from two of the individuals. Currently we are correcting the RUNX1 mutation in these iPSC lines using zinc finger nuclease mediated gene targeting. We have developed a pair of zinc finger nucleases that bind and cleave RUNX1, resulting in gene targeting frequencies of 80–90% in 293 cells and iPSCs respectively.

Our second goal is to develop RUNX1 iPSC reporter lines for use in screening small molecule and RNAi libraries to find methods of directing iPSC differentiation to the HSC fate. RUNX1 is a key player in the development of adult hematopoiesis and is expressed at the earliest stages of hematopoiesis. We have targeted fluorescent proteins and a luciferase cassette into the RUNX1 locus of wild type iPSC lines using the RUNX1 zinc finger nucleases at high efficiency. We will use these RUNX1 reporter iPSC lines in screens to identify inducers of RUNX1 expression soon. We believe such inducers have the potential to direct iPSCs towards a HSC fate.