Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, People's Republic of China.
Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, People's Republic of China.
J Nanobiotechnology. 2021 Oct 2;19(1):303. doi: 10.1186/s12951-021-01044-7.
Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown.
We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs.
Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.
长非编码 RNA（lncRNA）在许多生理和病理过程中发挥重要作用，这表明 lncRNA 可以作为基因治疗的潜在靶点。稳定表达是 lncRNA 研究的基础技术。慢病毒是稳定表达最常用的递送系统之一。然而，它最初是为 mRNAs 设计的，慢病毒载体在 lncRNAs 中的适用性在很大程度上是未知的。
我们发现慢病毒载体产生具有不正确终止的 lncRNA，在 3'-末端附加了一个约 2kb 的额外片段。因此，二级结构发生改变，RNA-蛋白质相互作用受阻，某些 lncRNA 的功能受损，这表明慢病毒载体不是 lncRNA 的理想递送系统。在这里，我们开发了一种新的 lncRNA 递送方法，称为使用转座子系统的内源性特征表达长非编码 RNA（ELECTS）。通过在 lncRNA 序列后插入终止信号，ELECTS 产生没有 3'-侧翼序列的转录本，并保留 lncRNA 的天然特征和功能，这是慢病毒载体无法实现的。此外，ELECTS 对操作人员没有潜在的感染风险，并且所需时间更少。ELECTS 为 lncRNA 的稳定表达提供了一种可靠、方便、安全和高效的递送方法。
我们的研究表明，慢病毒载体的转录终止不当对 lncRNA 的分子作用和细胞功能有根本影响。本研究中开发的 ELECTS 系统将为 lncRNA 研究提供一种方便可靠的方法。
