Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo.
Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo.
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逆转录病毒介导的人腺苷脱氨酶基因序列转移到培养细胞和体内小鼠造血细胞中。
DOI：
10.1073/pnas.83.8.2566
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发表时间：
1986
影响因子：
11.1
通讯作者：
R. Mulligan
中科院分区：
文献类型：
--
作者：
D. Williams;S. Orkin;R. Mulligan
腺苷脱氨酶（腺苷氨基水解酶，EC 3.5.4.4; ADA）的缺乏导致严重的联合免疫缺陷，这种疾病可能通过基因转移到造血细胞中来纠正。我们已经构建了逆转录病毒含有人ADA cDNA和显性选择标记，突变的二氢叶酸还原酶基因（DHFR*）编码甲氨蝶呤耐药。单独插入人ADA cDNA（DHFR*-ADA）或插入猴病毒40（SV 40）启动子（DHFR*-SVADA）。尽管用任一构建体感染的NIH 3 T3细胞产生人ADA活性，但用DHFR*-SVADA获得了显著更高的水平。用DHFR*-SVADA感染培养物中的鼠淋巴样细胞导致人酶的表达水平远高于小鼠内源性水平。ADA活性也增加感染后的人ADA缺陷型B细胞系。用DHFR*-SVADA病毒感染的同基因骨髓重建的致死性辐照小鼠在总脾DNA或脾造血干细胞（CFU-S）衍生集落中含有未重排的整合前病毒DNA。然而，未检测到人ADA。RNA分析表明，相对较低的和可变的表达从逆转录病毒长末端重复序列，并没有检测到的表达从内部SV 40启动子。这些数据表明，在体内培养的细胞和CFU-S之间存在内在的生物学差异。
Deficiency of the enzyme adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4; ADA) leads to severe combined immunodeficiency, a disorder that potentially could be corrected by gene transfer into hematopoietic cells. We have constructed retroviruses containing human ADA cDNA and a dominant selectable marker, a mutated dihydrofolate reductase gene (DHFR*) encoding methotrexate resistance. Human ADA cDNA was inserted alone (DHFR*-ADA) or with a simian virus 40 (SV40) promoter (DHFR*-SVADA). Although NIH 3T3 cells infected with either construct produced human ADA activity, substantially greater levels were attained with DHFR*-SVADA. Infection of murine lymphoid cells in culture with DHFR*-SVADA led to expression of human enzyme at a level well above the mouse endogenous level. ADA activity was also increased after infection of a human ADA-deficient B-cell line. Lethally irradiated mice that were reconstituted with syngeneic marrow infected with the DHFR*-SVADA virus contained unrearranged, integrated proviral DNA in total spleen DNA or in spleen hematopoietic stem cell (CFU-S)-derived colonies. Nevertheless, no human ADA was detectable. RNA analysis showed relatively low and variable expression from the retroviral long terminal repeat, and no detectable expression from the internal SV40 promoter. These data suggest that intrinsic biologic differences exist between cultured cells and CFU-S in vivo.