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

造血异常致病基因的阐明及阵发性睡眠性血红蛋白尿基因治疗的进展

基本信息

批准号：
14370299
负责人：
TANI Kenzaburo
金额：
$ 8.96万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

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.

阵发性夜间血红蛋白尿（PNH）已被证明是由磷脂酰肌醇聚糖A（猪-A）基因突变引起的。由于PNH是一种多能性造血干细胞疾病，除溶血外，还具有血栓形成、易感染、发育不全、致白血病和预后差等特点，因此被认为是细胞治疗和基因治疗的目标。在本研究项目中，我们从基因治疗的角度分析PNH。在过去两年的研究中，我们得出了以下结果。(1)利用VSV-G伪慢病毒载体（V-LV）将猪- a基因高效转导人造血干细胞，成功实现了GFP基因高效转导人造血干细胞。(2)利用V-LV构建了猪- a表达载体（PIG-LV），将猪- a基因转入GPI锚定缺陷的K562白血病细胞。我们证实了cd55和CD59的恢复表达。(3)在获得IRB对我们的研究方案的许可和2例PNH患者的知情同意后，我们从患者身上采集骨髓细胞，移植到免疫缺陷NOG（NOD/SCID/γ - cnull）小鼠骨髓中，制造PND模型小鼠。(4)利用PIG-LV体外将猪- a基因转染PNH患者造血干细胞。(5)近年来关于PNH的报道提示除猪- a外可能有其他基因参与PNH的发病机制，我们正在构建表达随机核酶的V-LV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在人造血细胞和白血病细胞中都能高效表达。maxizyme通过选择性破坏急性淋巴细胞白血病的致病基因bcr/abl基因来表达抗白血病的作用。