Sioud Mouldy
Division of Cancer Medicine, Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway.
Methods Mol Biol. 2025;2965:179-187. doi: 10.1007/978-1-0716-4742-4_6.
The innate immune system is equipped with pattern recognition receptors (PRRs) that detect foreign nucleic acids, triggering signaling pathways that drive pro-inflammatory and interferon responses. While these immune reactions are crucial for antiviral defense, they can also present significant challenges for RNA-based therapeutics, leading to immunogenicity, toxicity, and/or reduced efficacy. Therefore, evaluating the immunogenicity of therapeutic RNAs is essential. This chapter provides detailed protocols for assessing the immunostimulatory potency of exogenous RNA using peripheral blood mononuclear cells (PBMCs) or whole blood, two widely used models for studying innate immune activation. The methods outlined include PBMC isolation from human blood, stimulation with synthetic or in vitro-transcribed mRNA, and subsequent cytokine measurement using enzyme-linked immunosorbent assays (ELISA).
先天免疫系统配备有模式识别受体（PRR），可检测外来核酸，触发驱动促炎和干扰素反应的信号通路。虽然这些免疫反应对于抗病毒防御至关重要，但它们也可能给基于RNA的疗法带来重大挑战，导致免疫原性、毒性和/或疗效降低。因此，评估治疗性RNA的免疫原性至关重要。本章提供了详细的方案，用于使用外周血单核细胞（PBMC）或全血评估外源性RNA的免疫刺激效力，这是两种广泛用于研究先天免疫激活的模型。所述方法包括从人血中分离PBMC、用合成或体外转录的mRNA进行刺激，以及随后使用酶联免疫吸附测定（ELISA）测量细胞因子。
