A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics
A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics
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DOI：
10.1093/nar/gkaa576
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发表时间：
2020-08-20
影响因子：
14.9
通讯作者：
Kritzer, Joshua A.
中科院分区：
文献类型：
--
作者：
Deprey, Kirsten;Batistatou, Nefeli;Kritzer, Joshua A.
RNA疗法是治疗由特定蛋白质的过表达或异常剪接引起的遗传疾病的有希望的策略。该领域在这类分子的临床疗效方面取得了重大进展，这主要是由于化学修饰和递送策略提高了核酸酶抗性并增强了细胞渗透。然而，RNA疗法发展的主要障碍仍然是用于测量细胞渗透的大多数方法的不精确、困难和经常有问题的性质。在这里，我们回顾了这些方法，并明确区分那些测量RNA治疗剂的总细胞摄取，其中包括生产性和非生产性摄取，以及那些测量胞质/核渗透，仅代表生产性摄取。我们批判性地分析了每种方法的优点和缺点。最后，我们使用关键的例子来说明，尽管有严格的实验和适当的控制，我们对RNA治疗剂的裸体摄取机制的理解仍然受到通常用于分析RNA递送的方法的限制。
RNA therapeutics are a promising strategy to treat genetic diseases caused by the overexpression or aberrant splicing of a specific protein. The field has seen major strides in the clinical efficacy of this class of molecules, largely due to chemical modifications and delivery strategies that improve nuclease resistance and enhance cell penetration. However, a major obstacle in the development of RNA therapeutics continues to be the imprecise, difficult, and often problematic nature of most methods used to measure cell penetration. Here, we review these methods and clearly distinguish between those that measure total cellular uptake of RNA therapeutics, which includes both productive and non-productive uptake, and those that measure cytosolic/nuclear penetration, which represents only productive uptake. We critically analyze the benefits and drawbacks of each method. Finally, we use key examples to illustrate how, despite rigorous experimentation and proper controls, our understanding of the mechanism of gymnotic uptake of RNA therapeutics remains limited by the methods commonly used to analyze RNA delivery.