Elucidation of pathogenic genes for abnormal hematopoiesis and the development of gene therapy in paroxysmal nocturnal hemoglobinuria

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Paroxysmal nocturnal hemoglobinuria(PNH) has been demonstrated to be induced by genetic mutation of phosphatidylinositol glycan-class A(PIG-A). As PNH is the disorder of pluripotent hematopoietic stem cells and characterized not only by hemolysis, but also thrombosis, easy-infectibilitu, hypoplasia, leukemogenicity and poor prognosis, the disease is considered to be the target of cell therapy and gene therapy. In this research project, we analyzed PNH from the standpoint of gene therapy. During the last 2 research years, we produced the following results. (1)To transduce PIG-A gene into human hematopoietic cells efficiently, we made a use of VSV-G pseudotype lentivirus vector(V-LV) and successfully demonstrated the efficient transduction of GFP genes into human hematopoietic stem cells. (2)We constructed PIG-A expression vector(PIG-LV) using V-LV and transduced the PIG-A gene into GPI anchor deficient K562 leukemia cells. We demonstrated the recovered expression of CD 55 and CD59. (3)After obtaining the permission of our research protocol by our IRB and the informed consents from two PNH patients, we harvested bone marrow cells from the patients and transplanted to the marrow of immunodeficient NOG(NOD/SCID/γcnull) mice to make PND model mice. (4)We transduced PIG-A gene into the hematopoietic stem cells of PNH patients in vitro using PIG-LV. (5)Recent report on PNH suggested the additional genes besides PIG-A might be involved in the pathogenesis of PNH, we have been constructing random ribozyme expressing V-LV library to determine the pathogenetic gene. (6)Using newly-developed ribozyme of maxizyme, we demonstrated that the maxizyme was expressed efficiently in human hematopoietic cells as well as leukemia cells. Also the maxizyme could express antileukemia effects by the selective destruction of bcr/abl gene, pathogenic gene of acute lymphocytic leukemia.