Chemical Engineering, Arizona State University, Tempe, AZ, USA.
Biological Design, Arizona State University, Tempe, AZ, USA.
J Control Release. 2018 Sep 28;286:210-223. doi: 10.1016/j.jconrel.2018.06.030. Epub 2018 Jun 28.
Effective transgene expression in mammalian cells relies on successful delivery, cytoplasmic trafficking, and nuclear translocation of the delivered vector, but delivery is impeded by several formidable physicochemical barriers on the surface of and within the target cell. Although methods to overcome cellular exclusion and endosomal entrapment have been studied extensively, strategies to overcome inefficient nuclear entry and subsequent intranuclear barriers to effective transient gene expression have only been sparsely explored. In particular, the role of nuclear packaging of DNA with histone proteins, which governs endogenous gene expression, has not been extensively elucidated in the case of exogenously delivered plasmids. In this work, a parallel screen of small molecule inhibitors of chromatin-modifying enzymes resulted in the identification of class I/II HDACs, sirtuins, LSD1, HATs, and the methyltransferases EZH2 and MLL as targets whose inhibition led to the enhancement of transgene expression following polymer-mediated delivery of plasmid DNA. Quantitative PCR studies revealed that HDAC inhibition enhances the amount of plasmid DNA delivered to the nucleus in UMUC3 human bladder cancer cells. Native chromatin immunoprecipitation (N-ChIP)-qPCR experiments in CHO-K1 cells indicated that plasmids indeed interact with intracellular core Histone H3, and inhibitors of HDAC and LSD1 proteins are able to modulate this interaction. Pair-wise treatments of effective inhibitors led to synergistic enhancement of transgene expression to varying extents in both cell types. Our results demonstrate that the ability to modulate enzymes that play a role in epigenetic processes can enhance the efficacy of non-viral gene delivery, resulting in significant implications for gene therapy and industrial biotechnology.
哺乳动物细胞中的有效转基因表达依赖于递送至细胞内的载体成功地进行胞质运输和核转位，但是，在靶细胞的表面和内部存在多种严峻的物理化学屏障，阻碍了载体的递送。尽管已经广泛研究了克服细胞排斥和内体捕获的方法，但克服低效核进入和随后的核内障碍以实现有效的瞬时基因表达的策略仅得到了稀疏的探索。特别是，在研究外源递送来的质粒的情况下，DNA 与组蛋白的核包装在调节内源性基因表达方面的作用尚未得到广泛阐明。在这项工作中，对染色质修饰酶的小分子抑制剂进行了平行筛选，结果鉴定出 I/II 类 HDACs、sirtuins、LSD1、HATs 以及甲基转移酶 EZH2 和 MLL 作为靶标，其抑制作用导致聚合物介导的质粒 DNA 递送至细胞核后转基因表达增强。定量 PCR 研究表明，HDAC 抑制增强了在 UMUC3 人膀胱癌细胞中递送至细胞核的质粒 DNA 的量。CHO-K1 细胞中的天然染色质免疫沉淀（N-ChIP）-qPCR 实验表明，质粒确实与细胞内核心组蛋白 H3 相互作用，并且 HDAC 和 LSD1 蛋白的抑制剂能够调节这种相互作用。两种有效抑制剂的两两处理在两种细胞类型中都以不同程度协同增强了转基因表达。我们的结果表明，调节在表观遗传过程中起作用的酶的能力可以增强非病毒基因递送的效果，这对基因治疗和工业生物技术具有重要意义。
