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

阵发性睡眠性血红蛋白尿(PNH)是由磷脂酰肌醇多糖A类(PIG-A)基因突变引起的。由于PNH是一种多能造血干细胞的紊乱，不仅以溶血为特征，而且还具有血栓形成、易感染性、发育不良、白血病原性和预后不良等特点，因此被认为是细胞治疗和基因治疗的靶点。在本研究项目中，我们从基因治疗的角度分析了PNH。在过去两年的研究中，我们取得了以下成果。(1)利用VSV-G伪型慢病毒载体(V-LV)将PIG-A基因高效导入人造血细胞，并成功地将GFP基因导入人造血干细胞。(2)利用V-LV构建了PIG-A表达载体(PIG-LV)，并将PIG-A基因导入GPI锚点缺陷型K562白血病细胞。我们证实了CD55和CD59的恢复表达。(3)在征得本研究所研究方案的许可和两名PNH患者的知情同意后，取患者的骨髓细胞移植到免疫缺陷NOG(NOD/SCID/γ)小鼠的骨髓中，建立PND模型小鼠。(4)利用PIG-LV体外将PIG-A基因导入PNH患者的造血干细胞。(5)最近关于PNH的报道表明，除PIG-A外，其他基因可能参与了PNH的发病过程，我们一直在构建表达V-LV的随机核酶文库，以确定致病基因。(6)利用新开发的Maxizyme核酶，我们证明了Maxizyme在人的造血细胞和白血病细胞中都能高效表达。通过选择性破坏急性淋巴细胞白血病的致病基因bcr/abl基因，最大限度地发挥抗白血病作用。

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.