We will develop a human sickle cell mouse model for designing and testing gene therapy treatments of sickle cell disease and for functional studies of sickling in vivo. The sickle cell mouse line will be produced using a human beta globin locus yeast artificial chromosome containing the beta-S gene (beta-S-YAC) and a human alpha globin gene YAC (alpha-YAC) in a mouse homozygous for murine alpha and beta globin gene knockout mutations. This line will synthesize exclusively, or mostly, human hemoglobin S (Hb S) in the adult and human fetal globin (Hb F) and Hb S in the fetus. Hb F synthesis will rescue fetuses from lethal red cell sickling that would result from exclusive production of Hb S. We will extend the usefulness of this model by developing a novel approach for targeted mutagenesis transgenes directly fertilized oocytes derived from transgenic mice. The goal of the method is to provide an easy means to substitute , in vivo, the human alpha or beta-S genes with alleles encoding mutant alpha or beta-S chains that may inhibit or induce sickling. We will utilize the bacteriophage P1 Cre/lox and the yeast FLP/FRT site-specific recombination systems to catalyze gene replacement. Utilization of the sickle cell line, in conjunction with this targeted mutagenesis method, to study in vivo inhibition of sickling will have a major impact on designing vectors for gene therapy of sickle cell disease, as well as enhancing the ability of other investigators to study the pathophysiology of sickling. The sickle cell mouse model will also be employed for testing the expression of trans-acting factors that potentially reverse the developmentally- regulated fetal gamma globin to adult beta globin switch as an alternative form of sickle cell gene therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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University of Washington
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Constantinou, Varnavas C; Bouinta, Asimina; Karponi, Garyfalia et al. (2017) Poor stem cell harvest may not always be related to poor mobilization: lessons gained from a mobilization study in patients with ?-thalassemia major. Transfusion 57:1031-1039
Gori, Jennifer L; Butler, Jason M; Kunar, Balvir et al. (2017) Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates. Stem Cells Transl Med 6:864-876
Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia (2016) Optimizing autologous cell grafts to improve stem cell gene therapy. Exp Hematol 44:528-39
Li, Li B; Ma, Chao; Awong, Geneve et al. (2016) Silent IL2RG Gene Editing in Human Pluripotent Stem Cells. Mol Ther 24:582-91
Karponi, Garyfalia; Psatha, Nikoletta; Lederer, Carsten Werner et al. (2015) Plerixafor+G-CSF-mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy. Blood 126:616-9
Vierstra, Jeff; Reik, Andreas; Chang, Kai-Hsin et al. (2015) Functional footprinting of regulatory DNA. Nat Methods 12:927-30
Qi, Heyuan; Liu, Mingdong; Emery, David W et al. (2015) Functional validation of a constitutive autonomous silencer element. PLoS One 10:e0124588
Liu, Mingdong; Maurano, Matthew T; Wang, Hao et al. (2015) Genomic discovery of potent chromatin insulators for human gene therapy. Nat Biotechnol 33:198-203
Polak, Paz; Karli?, Rosa; Koren, Amnon et al. (2015) Cell-of-origin chromatin organization shapes the mutational landscape of cancer. Nature 518:360-364
Watts, Korashon L; Beard, Brian C; Wood, Brent L et al. (2014) No evidence of clonal dominance after transplant of HOXB4-expanded cord blood cells in a nonhuman primate model. Exp Hematol 42:497-504

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