In vitro evolution of enhanced RNA replicons for immunotherapy
In vitro evolution of enhanced RNA replicons for immunotherapy
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DOI：
10.1038/s41598-019-43422-0
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发表时间：
2019-05-06
影响因子：
4.6
通讯作者：
Weiss, Ron
中科院分区：
文献类型：
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作者：
Li, Yingzhong;Teague, Brian;Weiss, Ron
自我复制（复制子）RNA是一个有前途的基因治疗的新平台，但应用仍然受到大多数细胞类型中表达持续时间短和体内转基因表达水平低的限制。为了解决这些缺点，我们开发了一种体外进化策略，并确定了委内瑞拉马脑炎（VEE）复制子的非结构蛋白（nsPs）中的六个突变，这些突变促进了细胞中的亚基因组表达。nsP 2和nsP 3中的两个突变增强转基因表达，而nsP 3中的三个突变调节该表达。含有最有效的突变组合的复制子在体内表现出增强的持续时间和货物基因表达。与野生型复制子相比，将表达IL-2的突变体注射到鼠B16 F10黑色素瘤中显示肿瘤内IL-2增加5.5倍，浸润性CD 8 T细胞增加2.1倍，导致肿瘤生长显著减缓。因此，这些突变复制子可用于改进用于疫苗接种、癌症免疫疗法和基因疗法的RNA疗法。
Self-replicating (replicon) RNA is a promising new platform for gene therapy, but applications are still limited by short persistence of expression in most cell types and low levels of transgene expression in vivo. To address these shortcomings, we developed an in vitro evolution strategy and identified six mutations in nonstructural proteins (nsPs) of Venezuelan equine encephalitis (VEE) replicon that promoted subgenome expression in cells. Two mutations in nsP2 and nsP3 enhanced transgene expression, while three mutations in nsP3 regulated this expression. Replicons containing the most effective mutation combinations showed enhanced duration and cargo gene expression in vivo. In comparison to wildtype replicon, mutants expressing IL-2 injected into murine B16F10 melanoma showed 5.5-fold increase in intratumoral IL-2 and 2.1-fold increase in infiltrating CD8T cells, resulting in significantly slowed tumor growth. Thus, these mutant replicons may be useful for improving RNA therapeutics for vaccination, cancer immunotherapy, and gene therapy.