Cooley's anemia (CA) will be cured in an animal model of beta thalassemia major by therapeutic cloning, genome modification, and replacement cell therapy Therapeutic cloning will be used to derive primary beta/0 thalassemic embryonic stem (ES) cell lines that will be genetically modified to correct their defect. Corrected thalassemic ES cells will be used for replacement cell therapy after differentiation into hematopoietic progenitors and transplantation back into CA animals. Initial experiments will utilize a knockout mouse model of primary beta-thalassemia that reproduces most of the pathology of the disorder (PNAS 92 9259-9263). The model was created by targeted deletion of 16 kilobases of DNA that removes both adult mouse beta-globin genes. Later experiments will utilize a novel mouse model of CA generated by targeted gene replacement of the adult murine alpha-globin genes with human alpha-globin and the adult mouse beta-globin genes with a human gamma- to beta-globin gene switching cassette that contains a primary beta thalassemic allele. Newborn mice homozygous for the knockin allele will survive solely on human fetal hemoglobin at birth. The mice will succumb to CA during the first weeks of life once the fetal to adult hemoglobin switch is complete. Cooley's anemia ES cell lines will be established from developing blastocysts isolated from either heterozygous mating pairs or from nuclear transfer of fibroblast nuclei isolated from newborn CA mice into enucleated mouse eggs. Identical Cooley's anemia mice will be cloned from these ES cells by injection of CA ES cells into tetraploid blastocysts. Cloning after modification of the CA ES cells will be utilized to test genetic therapies designed to correct the thalassemia. Therapeutic benefit will be assessed by a direct comparison of the anemia in mice cloned from the modified ES cells to clones produced from the unmodified CA ES cells. Hematopoietic stem cells (HSCs) isolated from the bone marrow of cloned mice generated from the corrected CA ES cells will be used to cure sublethally conditioned isogeneic mice cloned from the diseased ES cells Finally, in vitro differentiation of the corrected CA ES cells will be used to generate hematopoietic progenitors in cell culture Conditioned CA mouse clones will be cured by the injection of these isogeneic corrected hematopoietic progenitors. Successful completion of these studies will delineate the basic steps required for curing many heritable hematopoietic disorders in humans, namely, therapeutic cloning to establish autologous ES cell lines, correction of diseased allele(s) in the ES cells, and hematopoietic progenitor replacement cell therapy.
