Gene transfer of adenosine deaminase into primitive human hematopoietic progenitor cells.
Gene transfer of adenosine deaminase into primitive human hematopoietic progenitor cells.
复制标题
将腺苷脱氨酶基因转移到原始人类造血祖细胞中。
DOI：
10.1089/hum.1991.2.3-203
复制
发表时间：
1991
影响因子：
4.2
通讯作者：
Caskey,CT
中科院分区：
文献类型：
--
作者：
Cournoyer,D;Scarpa,M;Mitani,K;Moore,KA;Markowitz,D;Bank,A;Belmont,JW;Caskey,CT
遗传性腺苷脱氨酶(ADA)缺陷导致严重的联合免疫缺陷，被普遍认为是发展人类躯体基因治疗的最佳模式。纠正ADA缺乏症和其他淋巴-造血疾病的理想靶点是造血干细胞。我们使用重组人白介素3和重组人白介素6的组合来刺激原始人类造血祖细胞在与辐射细胞共培养期间的增殖，该细胞产生了包装在两性颗粒中的高滴度的ADA转导逆转录病毒载体。在一系列的9个实验中，平均83%的克隆祖细胞(CFU-E和CFU-GM)被发现获得了经聚合酶链式反应分析确定的转移序列。此外，在两个实验中，从转导后9周的长期骨髓培养中获得的克隆性祖细胞中，24-44%的克隆祖细胞含有载体序列。后者来源于所谓的长期培养启动细胞(LTC-IC)，LTC-IC是可能与造血干细胞相关的原始细胞。此外，转导的ADA酶在正常和ADA缺乏的红系集落中都有表达，在长期骨髓培养至少2周的非贴壁细胞中也有表达，水平接近正常红系细胞的内源性ADA水平。这些结果表明，通过逆转录病毒基因转移可以将ADA编码序列有效地导入承诺和原始的人造血祖细胞，这将导致转导的酶在承诺造血祖细胞的后代中得到充分表达。
The inherited deficiency in adenosine deaminase (ADA), which results in severe combined immunodeficiency, is generally regarded as an optimal model for the development of human somatic gene therapy. The ideal target for the correction of ADA deficiency and other lympho-hematopoietic disorders would be the hematopoietic stem cell. We have used a combination of recombinant human interleukins-3 and -6 to stimulate the proliferation of primitive human hematopoietic progenitor cells during a period of co-cultivation with irradiated cells producing high titers of an ADA-transducing retroviral vector packaged in amphotropic particles. In a series of nine experiments, an average of 83% of the clonogenic progenitors (CFU-E and CFU-GM) were found to have acquired the transferred sequence as determined by polymerase chain reaction analysis. In addition, in two experiments, 24–44% of the clonogenic progenitors derived from long-term myeloid cultures 9 weeks post-transduction were found to contain vector sequence. The latter cells are derived from so-called “long-term culture-initiating cells” (LTC-IC), which are primitive cells probably related to hematopoietic stem cells. Moreover, the transduced ADA enzyme was found to be expressed in both normal and ADA-deficient erythroid colonies, and in the nonadherent cells of long-term bone marrow culture for at least 2 weeks at levels that approximate the endogenous ADA levels of normal erythroid cells. These results indicate that the ADA coding sequence can efficiently be introduced by retroviral gene transfer into both committed and primitive human hematopoietic progenitor cells, and that this will result in adequate expression of the transduced enzyme in the progeny of committed hematopoietic progenitors.