Persistent and stable gene expression by a cytoplasmic RNA replicon based on a noncytopathic variant Sendai virus
Persistent and stable gene expression by a cytoplasmic RNA replicon based on a noncytopathic variant Sendai virus
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DOI：
10.1074/jbc.m702028200
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发表时间：
2007-09-14
影响因子：
4.8
通讯作者：
Nakanishi, Mahito
中科院分区：
文献类型：
--
作者：
Nishimura, Ken;Segawa, Hiroaki;Nakanishi, Mahito
通过将外源基因整合到宿主染色体中，在哺乳动物细胞中实现了外源基因的持续稳定表达。然而，这种方法在实际应用中存在一些缺点。例如，蛋白质药物的大规模生产经常需要费力地扩增插入的基因以优化基因表达。外源DNA随机插入染色体有时也会导致正常组织细胞的恶性转化，从而引起医疗应用中的安全问题。在这里，我们报道了一种新型细胞质 RNA 复制子，能够稳定表达已安装的基因，而无需染色体插入。该系统基于非细胞病变变体仙台病毒株 Cl.151 的 RNA 基因组。我们发现这种变异病毒通过逃避视黄酸诱导基因I-干扰素调节因子3介导的抗病毒机制，与宿主细胞建立稳定的共生关系。使用仙台病毒 Cl 的克隆基因组 cDNA。 151，我们开发了一种安装有外源标记基因的重组RNA，它作为高拷贝复制子（约4 x 104拷贝/细胞）稳定地维持在细胞质中，而不干扰正常的细胞功能。标记基因的强表达在各种类型的培养细胞中持续超过6个月，在大鼠结肠粘膜中持续至少两个月，没有任何明显的副作用。这种稳定的 RNA 复制子是各种生物应用的潜在有价值的遗传平台。
Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl. 151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 x 104 copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications.