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.

中文翻译：

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对用于研究RNA治疗药物的细胞摄取和亚细胞定位的方法的严格分析。

RNA疗法是一种治疗因特定蛋白质的过表达或异常剪接而引起的遗传疾病的有前途的策略。该领域在此类分子的临床功效方面已取得了长足的进步，这在很大程度上是由于化学修饰和递送策略可改善核酸酶抗性并增强细胞渗透性。然而，RNA疗法发展中的主要障碍仍然是大多数用于测量细胞渗透性的方法的不精确，困难且常常是有问题的性质。在这里，我们回顾了这些方法，并清楚地区分了测量RNA治疗药物的总细胞吸收（包括生产性和非生产性摄入）与测量胞质/核渗透（仅代表生产性摄入）的方法。我们严格分析每种方法的优缺点。最后，我们使用关键示例来说明，尽管进行了严格的实验和适当的控制，但我们对体操疗法对RNA疗法的摄取机理的理解仍然受到通常用于分析RNA传递的方法的限制。