Gene replacement therapy of congenital bone marrow disorders is a long sought treatment goal and a great challenge to experimental hematology. In this project we focus on gene replacement for Fanconi Anemia and Diamond Blackfan Anemia. Both diseases require successful gene insertion and function in hematopoietic stem cells (HSC) and their progeny. Successful gene transfer into HSC requires selection of transfected and functioning HSC by the host. This will occur in Fanconi Anemia because HSC lacking the Fanconi gene die in the marrow. In Diamond Blackfan Anemia, the selection occurs at the level of the erythroid progenitor and precursor cells. We will use two classes of retrovirus vectors; moloney leukemia based vectors and HIV based lentivirus vectors. These will be tested in vitro and in two in vivo models; the NOD SCID mouse and in rhesus monkey. We have shown that it is imperative to utilize short incubations preferably in cold temperatures in order to protect the HSC from toxicity induced by the procedures themselves. These new incubation conditions will be tested in the experimental models. Appropriate vectors will be produced at very high concentrations in our GMP vector laboratory and the scale up to large human samples will be carried out in our GMP cell manipulation core laboratory.
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