Synthetic messenger RNA as a tool for gene therapy
Synthetic messenger RNA as a tool for gene therapy
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DOI：
10.1089/hum.2006.17.1027
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发表时间：
2006-10-01
影响因子：
4.2
通讯作者：
Weissman, Sherman M.
中科院分区：
文献类型：
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作者：
Rabinovich, Peter M.;Komarovskaya, Marina E.;Weissman, Sherman M.
在生物医学应用中，用DNA转染人细胞具有插入诱变的风险。用mRNA转染避免了这个问题;然而，基于在特殊载体中的初步DNA模板克隆的mRNA的体外生产是一个费力且耗时的过程。我们报告了一种有效的无载体的方法，从聚合酶链反应生成的DNA模板的mRNA生产。对于所有测试的细胞类型，mRNA比DNA更容易转染，并且其表达在细胞群中高度均匀。即使是对DNA转染相对抵抗的细胞类型也可以很好地表达转染的mRNA。通过改变输入RNA的量，可以在很宽的范围内控制mRNA的表达水平。细胞可以有效地同时装载几种不同的转录本。为了测试这种方法的潜在临床应用，我们用编码针对CD 19的嵌合免疫受体的mRNA转染人T淋巴细胞，CD 19是白血病和淋巴瘤中广泛表达的表面抗原。转染的mRNA赋予T细胞针对来自同一供体的CD 19（+）靶点的强大细胞毒性。这些结果表明，该方法可用于产生针对恶性细胞的自体T淋巴细胞。
Transfection of human cells with DNA in biomedical applications carries the risk of insertional mutagenesis. Transfection with mRNA avoids this problem; however, in vitro production of mRNA, based on preliminary DNA template cloning in special vectors, is a laborious and time-consuming procedure. We report an efficient vector-free method of mRNA production from polymerase chain reaction-generated DNA templates. For all cell types tested mRNA was transfected more readily than DNA, and its expression was highly uniform in cell populations. Even cell types relatively resistant to transfection with DNA could express transfected mRNA well. The level of mRNA expression could be controlled over a wide range by changing the amount of input RNA. Cells could be efficiently and simultaneously loaded with several different transcripts. To test a potential clinical application of this method, we transfected human T lymphocytes with mRNA encoding a chimeric immune receptor directed against CD19, a surface antigen widely expressed in leukemia and lymphoma. The transfected mRNA conferred powerful cytotoxicity to T cells against CD19(+) targets from the same donor. These results demonstrate that this method can be applied to generate autologous T lymphocytes directed toward malignant cells.