Engineering, delivery, and biological validation of artificial microRNA clusters for gene therapy applications.,Nature Protocols

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Engineering, delivery, and biological validation of artificial microRNA clusters for gene therapy applications.
Nature Protocols ( IF 13.1 ) Pub Date : 2019-11-20 , DOI: 10.1038/s41596-019-0241-8
Vivek Bhaskaran ₁ , Yizheng Yao ₁ , Fengfeng Bei ₁ , Pierpaolo Peruzzi ₁

Affiliation

The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4-5 h. Synthesis of the resulting DNA sequence requires 4-6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.

中文翻译：

用于基因治疗应用的人工 microRNA 簇的工程、交付和生物学验证。

调节 microRNA 生物发生和成熟的细胞机制依赖于少量的简单步骤和最低的生物学要求，并且在所有真核细胞中广泛保守。疾病也是如此。这使得能够同时表达多种所选 microRNA 的转基因工程具有很大的自由度，从而可以更全面地调节 microRNA 景观、研究它们的功能相互作用，以及将这种协同作用用于基因治疗应用的可能性。我们之前已经设计了功能相关的 microRNA 的转基因簇，以在脑癌中表达受抑制的 microRNA 模块，用于治疗目的。在这里，我们提供了用于基因治疗目的的此类人工 microRNA 簇的设计、克隆、递送和利用的详细方案。与其他方案相比，我们的策略有效地降低了分子克隆的要求，因为编码所需 microRNA 组合的核酸序列是在计算机中设计和验证的，然后直接合成为 DNA，准备好亚克隆到适当的递送载体中，用于体外和体内。序列设计和工程需要4-5小时。合成所得 DNA 序列需要 4-6 小时。该方案快速灵活，不需要特殊的实验室设备或技术，也不需要多个克隆步骤。任何具有基本分子生物学知识的研究生或技术人员都可以轻松执行。

更新日期：2019-11-21

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