REVERSIR platform for the tailored control of GalNAc-siRNA conjugate pharmacology

Primary mouse hepatocytes were treated with 1 nM TTR-siRNA and 10 nM scrambled or TTR-REVERSIR by in vitro free uptake for 24 h. RNA extracted with the Purelink RNA kit (ThermoFisher) was used for cDNA library preparation with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the NextSeq500 desktop sequencer (Illumina), all according to manufacturers’ instructions. Raw RNA-seq reads were filtered with minimal mean quality scores of 25 and minimal remaining length of 36, using fastq-mcf. Filtered reads were aligned to the Mus musculus genome (NCBIM37) using STAR with default parameters. Uniquely aligned reads were counted by featureCounts. Differential gene expression (DEG) analysis was performed by the R package DESeq2. Here, we report the rapid and potent reversal of GalNAc-siRNA-mediated RNAi activity in vivo with REVERSIR technology, which is based on short, synthetic high-affinity oligonucleotide complementary to the siRNA guide strand. The modular, sequence-specific nature of the REVERSIR platform has the potential to enhance the therapeutic profile of any long-acting GalNAc-siRNA conjugate to enable fine-tuned control of RNAi pharmacology RNASeq analysis of REVERSIR in primary mouse hepatocytes

以体外自由摄取方式，将1 nM TTR-siRNA与10 nM 随机序列对照siRNA（scrambled siRNA）或TTR-REVERSIR处理原代小鼠肝细胞（primary mouse hepatocytes），孵育时长为24小时。使用Purelink RNA提取试剂盒（ThermoFisher）提取总RNA，严格按照制造商操作规程，采用TruSeq链特异性总RNA文库制备试剂盒（Illumina）构建cDNA文库，并在NextSeq500台式测序仪（Illumina）上完成测序。使用fastq-mcf工具对原始RNA-seq读段进行质量过滤，设置最低平均质量分值为25，保留序列的最小长度为36 bp。将过滤后的有效读段比对至小鼠（Mus musculus）基因组NCBIM37版本，比对工具为STAR，参数采用默认设置。通过featureCounts工具统计唯一比对读段的计数结果。使用R语言包DESeq2完成差异基因表达（differential gene expression, DEG）分析。本研究报道了利用REVERSIR技术在体内快速且高效地逆转GalNAc修饰siRNA介导的RNA干扰（RNA interference, RNAi）活性，该技术基于与siRNA向导链互补的短链合成高亲和力寡核苷酸。REVERSIR技术平台具备模块化与序列特异性的特性，有望提升所有长效GalNAc修饰siRNA偶联物的治疗属性，从而实现对RNA干扰药理学特性的精准调控。本数据集为原代小鼠肝细胞中REVERSIR的RNA测序分析数据。