Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany.
PLoS One. 2013 Aug 5;8(8):e71057. doi: 10.1371/journal.pone.0071057. Print 2013.
Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.
治疗性寡核苷酸包括 siRNA 和 Toll 样受体 (TLR) 或 RIG-I 样螺旋酶 (RLH) 的免疫刺激配体，是一类很有前途的新型药物。它们正在广泛的应用领域进行临床开发，例如作为疫苗佐剂和癌症的免疫疗法。治疗性寡核苷酸的特征是具有物种特异性的免疫激活，导致细胞因子释放，这既是一种不想要的副作用，也是预期的药理学作用。因此，迫切需要设计用于治疗性寡核苷酸的可靠体外测试，以便预测临床疗效并防止在临床开发中出现意外的有害影响。为了达到这个目的，我们在这里建立了一种快速且易于操作的人全血测定法（WBA）。它对合成 TLR 配体（R848：TLR7/8，LPS：TLR4）的反应与更耗时的外周血单核细胞（PBMC）基于测定法相当。相比之下，CpG-DNA（TLR9 配体）在体内静脉注射引起的 I 型 IFN 谱在 WBA 中比在刺激的 PBMC 中更精确地复制。由于肝素和 EDTA 但不是水蛭素会将寡核苷酸从其递送剂中置换出来，因此只有水蛭素有资格成为 WBA 中使用的抗凝剂。与 PBMC 测定法相比，水蛭素 WBA 具有类似的区分 TLR7 激活和修饰的非刺激 siRNA 序列的能力。基于 RNA 的免疫激活 TLR7/8 和 RIG-I 配体在依赖于所使用的递送剂的 Hirudin-WBA 中诱导大量 IFN-α。总之，我们提出了一种人 Hirudin WBA 来确定临床前开发中治疗性寡核苷酸诱导的细胞因子释放，该方法易于进行，并能很好地反映体内人类细胞因子反应。
