RETROVIRAL TRANSFER OF P47-PHOX INTO MURINE CELLS AND HUMAN MYELOID CELL LINES

这个非临床的IIDEA项目的目的是研究 使用逆转录病毒载体转移基因， 常染色体隐性遗传性慢性肉芽肿病（p47-phox）进入人类 造血细胞能够生产逆转录病毒的细胞系的开发 感染人类血细胞的过程将为研究 该蛋白在前体和分化的造血细胞中的表达 细胞含有蛋白质编码序列的p47-phox的cDNA已被克隆。 克隆到称为pLXSN的逆转录病毒载体中。两种小鼠生产细胞 已经建立了株系，其中一株产生亲嗜性逆转录病毒， 一种是感染啮齿类动物细胞，另一种是产生嗜异性逆转录病毒 能够感染多种哺乳动物和鸟类细胞。的 含有正常（有义）和 反向（反义）方向用于感染两种白血病细胞系， HL-60和U937 RNA和蛋白质分析显示逆转录病毒 将p47-phox基因转移到这些细胞中。此外，爱泼斯坦巴尔病毒 来自正常个体和AR-CGD患者的转化B淋巴细胞 也被感染了。这些细胞系似乎也被 基于蛋白质和RNA分析的逆转录病毒。 由转移的基因产生的蛋白质仍然是有功能的， 中求进工作总来源于AR-CGD的EBV转化的B淋巴细胞系 患者细胞可能有助于研究缺陷的纠正， 正常人的这些细胞产生少量的超氧化物， 在功能和分子水平上进行了研究。表达 转p47-phox基因小鼠骨髓及造血重建 在灵长类动物和人类研究之前， 可以继续了初步实验表明， 将该基因转移到小鼠骨髓中。

The purpose of this non-clinical, IIDEA project is to study the feasibility of using retroviral vectors to transfer the gene which is abnormal in autosomal recessive Chronic Granulomatous Disease(p47-phox) into human hematopoietic cells. Development of retrovirus producer cell lines capable of infecting human blood cells will provide the opportunity to study expression of this protein in precursor and differentiated hematopoietic cells. A cDNA for p47-phox containing the protein coding-sequence has been cloned into a retroviral vector known as pLXSN. Two murine producer cell lines have been established, one producing an ecotropic retrovirus capable of infecting rodent cells and the other producing amphotropic retrovirus capable of infecting a wide range of mammalian and avian cells. The amphotropic virus containing p47-phox in both normal(sense) and reverse(antisense) orientations was used to infect two leukemia cell lines, HL-60 and U937. RNA and protein analysis demonstrated retrovirally transferred p47-phox genes in these cells. In addition, Epstein Barr Virus transformed B lymphocytes from a normal individual and an AR-CGD patient were also infected. These lines also appear to have been transduced by the retroviruses based on protein and RNA analysis.Assays to determine whether the protein made from the transferred gene is functional are still in progress. EBV transformed B lymphocyte cell lines derived from AR-CGD patient cells may be helpful in studies of correction of the defect since these cells in normals produce a small amount of superoxide and can be studied at the functional as well as molecular level. Expression of transferred p47-phox in murine bone marrow and hematopoietic reconstitution of an irradiated mouse will be necessary before primate and human studies can proceed. Preliminary experiments indicate that it is possible to transfer this gene into murine bone marrow.