Myeloablation is not required to select and maintain expression of the drug-resistance gene, mutant MGMT, in primary and secondary recipients
Myeloablation is not required to select and maintain expression of the drug-resistance gene, mutant MGMT, in primary and secondary recipients
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DOI：
10.1016/s1525-0016(03)00141-2
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发表时间：
2003-07-01
影响因子：
12.4
通讯作者：
Gerson, SL
中科院分区：
文献类型：
--
作者：
Bowman, JE;Reese, JS;Gerson, SL
造血祖细胞的基因转导是基因治疗临床前发展的一个重要里程碑。在输注转导的干细胞之前进行骨髓消融处理会导致显著的宿主发病率。相比之下，耐药基因转移利用了体内对转导干细胞的明智选择，随着时间的推移，只达到了所需的转导和表达水平。O-6-苄基鸟嘌呤(BG)抗性突变体O-6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因是一种有效的转基因细胞选择基因。使用两种不同的突变mgmt，G156A和P140K，它们在BG抗性上的差异为1：20，我们询问长期再生和二次小鼠再生细胞是否可以在非去髓性受体中被转导、移植和选择，以及突变的MGMT是否会继续在二次受体再生细胞中表达。我们发现，在严格的药物选择竞争下，表达更多BG耐药变异体P140K-MGMT的细胞比表达G156A-MGMT的祖细胞更富集。此外，MFG逆转录病毒载体将突变的MGMT基因转移到长期再填充细胞中，这些细胞在未去髓的初级受体中选择性浓缩后，再繁殖次级小鼠，并继续表达转基因。因此，MFG突变的MGMT载体转导可再生的造血干细胞，既可用于化疗耐药，也可用于丰富第二治疗基因。
Gene transduction of hematopoietic progenitors capable of reconstituting both primary and secondary recipients is an important milestone in preclinical development of gene therapy. Myeloablation conditioning prior to infusion of transduced stem cells causes significant host morbidity. In contrast, drug-resistance gene transfer utilizes judicious in vivo selection of transduced stem cells over time, reaching only the level of transduction and expression required. The O-6-benzylguanine (BG)-resistant mutant O-6-methylguanine-DNA methyltransferase (MGMT) gene is a potent selection gene for transduced cells. Using two different mutant MGMTs, G156A and P140K, that vary in BG resistance by a factor of 1:20, we asked whether long-term repopulating and secondary mouse-repopulating cells could be transduced, transplanted, and selected for in the nonmyeloablated recipient and whether the mutant MGMT would continue to be expressed in secondary recipient repopulating cells. We found that under stringent drug-selection competition, cells expressing the more BG-resistant variant, P140K-MGMT, were enriched over G156A-MGMT-expressing progenitors. In addition, the MFG retroviral vector transmitted the mutant MGMT gene to long-term repopulating cells that, after selective enrichment in the nonmyeloablated primary recipient, repopulated secondary mice and continued to express the transgene. Thus, MFG mutant MGMT vectors transduce repopulating hematopoietic stem cells that may be used both for chemotherapeutic drug resistance and to enrich for second therapeutic genes.