Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype.
Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype.
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DOI：
10.1182/blood.v97.10.3004
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发表时间：
2001-05
期刊：
影响因子：
20.3
通讯作者：
J. Nishimura;Ken L. Phillips;R. Ware;S. Hall;Lee Wilson;T. Gentry;T. Howard;Y. Murakami;M. Shibano;T. Machii;E. Gilboa;Y. Kanakura;J. Takeda;Taroh Kinoshita;W. Rosse;Clay A. Smith
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文献类型：
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作者：
J. Nishimura;Ken L. Phillips;R. Ware;S. Hall;Lee Wilson;T. Gentry;T. Howard;Y. Murakami;M. Shibano;T. Machii;E. Gilboa;Y. Kanakura;J. Takeda;Taroh Kinoshita;W. Rosse;Clay A. Smith
阵发性睡眠性血红蛋白尿症(PNH)是一种克隆性造血干细胞疾病，其特征是由于血细胞亚群中糖基磷脂酰肌醇锚定蛋白(GPI-APs)的缺乏而导致补体介导的溶血。X连锁磷脂酰肌醇糖链A类(PIG-A)基因的获得性突变似乎是PNH的特征和致病原因。为了开发PNH的基因治疗方法，构建了一个名为MPIN的逆转录病毒载体，该载体含有PIG-A互补DNA、内部核糖体进入位点和神经生长因子受体(NGFR)作为选择标记。转导MPIN基因后，PNH患者的外周血单核细胞(PB)和PNH患者外周血(PB)中的单个核细胞均能高效稳定地表达PIG-A和NGFR基因。PIG-A在这些细胞系中的表达稳定恢复了GPI-AP的表达。将MPIN导入1例PNH患者的骨髓单个核细胞，获得髓系/红系集落和红系细胞。这些转导的红系细胞恢复了GPI-APs的表面表达和对溶血的抵抗力。这些结果表明，MPIN能够有效而稳定地恢复多种PIG-A缺陷型造血细胞的GPI-AP的功能。此外，MPIN还可转导正常供者外周血CD34(+)细胞，表明MPIN可转导原始人类祖细胞。这些发现为确定MPIN能否在多潜能干细胞中恢复PIG-A功能奠定了基础，从而为PNH提供了一种潜在的新治疗选择。
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan-class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A-deficient B-cell line (JY5), a PIG-A-deficient K562 cell line, an Epstein-Barr virus-transformed B-cell line (TK-14(-)) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A-deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34(+) cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.