MicroRNA-dependent suppression of biological pacemaker induced by chemically modified messenger RNA. Sanchez et al

Chemically-modified messenger RNA (CMmRNA) with selectively altered nucleotides are used to deliver transgenes, but translation efficiency is variable. We have transfected CMmRNA encoding human T-box transcription factor 18 (CMmTBX18) into heart cells, or the left ventricle of rats with atrioventricular block. TBX18 protein expression from CMmTBX18 is weak and transient, but Acriflavine, an Argonaute 2 inhibitor, boosts TBX18 levels. Small RNA sequencing identified two upregulated microRNAs (miRs) in CMmTBX18 transfected cells. Co-administration of miR-1-3p and miR-1b antagomiRs with CMmTBX18 prolongs TBX18 expression in vitro and in vivo, and is sufficient to generate electrical stimuli capable of pacing the heart. Different suppressive miRs likewise limit the expression of VEGF-A CMmRNA. Cells therefore resist translation of CMmRNA therapeutic transgenes by upregulating suppressive miRs. Blockade of suppressive miRs enhances CMmRNA expression of genes driving biological pacing or angiogenesis. Such counterstrategies constitute an approach to boost the efficacy and efficiency of CMmRNA therapies.

携带经选择性修饰核苷酸的化学修饰信使RNA（Chemically-modified messenger RNA, CMmRNA）可用于递送治疗性转基因，但其翻译效率存在差异。本研究将编码人T-box转录因子18的CMmRNA（CMmTBX18）转染至心肌细胞，或直接注射至患有房室传导阻滞的大鼠左心室。CMmTBX18介导的TBX18蛋白表达微弱且具有瞬时性，但Argonaute 2抑制剂吖啶黄（Acriflavine）可提升TBX18的表达水平。通过小RNA测序，研究人员在转染CMmTBX18的细胞中鉴定出两种上调的微小RNA（microRNAs, miRs）。将miR-1-3p与miR-1b抗miRNA寡核苷酸与CMmTBX18联合给药，可在体外与体内延长TBX18的表达时长，且足以产生可实现心脏起搏的电刺激信号。其他类型的抑制性miRs同样会限制编码血管内皮生长因子A（VEGF-A）的CMmRNA的表达。由此可见，细胞可通过上调抑制性miRs来抵抗CMmRNA治疗性转基因的翻译过程。阻断抑制性miRs可增强驱动生物起搏或血管生成的基因的CMmRNA表达水平。此类对抗策略可为提升CMmRNA治疗的疗效与效率提供可行路径。