Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the treatment of hemoglobinopathies such as Cooley's anemia (CA). In the parent grant a novel human or- and 13U-globin gene knockin animal model of CA is being produced. This CA mouse model will be used to derive 13U thalassemic ES cell lines that will be genetically modified to correct their defect. Corrected thalassemic ES cells will be used for replacement cell therapy after in vitro differentiation into hematopoietic progenitors. Thus, the laboratory mouse will be used to model therapeutic cloning to cure CA. In this Supplement application, human ES cell line WA01 from the NIH Human Embryonic Stem Cell Registry will be utilized to model the genetic correction of CA in human cells. Specifically, wild-type human ES cells will be genetically modified to generate new """"""""diseased"""""""" cell lines that harbor common thalassemic mutations of the human 13 U-globin gene. These thalassemic human ES cell lines will be corrected by transgene addition and homologous recombination. Finally, in vitro differentiation of the corrected human ES cells will be used to generate hematopoietic progenitors in cell culture. Successful completion of the studies described in this supplement will broaden existing knowledge of human ES cell clonal isolation and expansion, targeting efficiencies especially of non-transcribed genes, and robustness for repeated serial targeting. Furthermore, these experiments will produce practical tools such as corrective DNA constructs that could be utilized therapeutically in future ES cells derived from patients suffering from hemoglobinopathies. ? ? ?
