Inoue Taketo, Inoue Shintaro, Nogi Yuhei, Tsukimoto Jun, Saito-Tarashima Noriko, Noji Sumihare, Mito Taro, Minakawa Noriaki
Graduate School of Pharmaceutical Sciences, Tokushima University, 1-78-1 Shomachi, Tokushima 770-8505, Japan.
Bio-Innovation Research Center, Tokushima University, 2272-2 Ishii, Ishii-cho, Myozai-gun, Tokushima 779-3233, Japan.
Biol Pharm Bull. 2025;48(6):941-950. doi: 10.1248/bpb.b25-00289.
Small interfering RNAs (siRNAs) hold great therapeutic promise due to their ability to selectively silence disease-associated genes. Although chemically modified siRNAs have demonstrated clinical efficacy, their development remains hindered by challenges such as stability and delivery under physiological conditions. Furthermore, in vitro screening of chemically modified siRNAs using cultured cells is cost-effective but often fails to recapitulate in vivo complexity, limiting predictive accuracy. To address this, we have developed a transfection-free siRNA evaluation platform using Gryllus bimaculatus (Gb), an insect model with natural RNA uptake capacity. We first demonstrated that 21-nucleotide siRNAs induced RNA interference upon abdominal injection under conditions of free uptake. We then generated transgenic lines harboring an EGFP reporter fused to the therapeutic siRNA target sequence and integrated into the β-actin locus via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9-mediated knock-in. Two transgenic strains (s1 and d1) were established and validated. We compared unmodified and chemically modified siRNAs designed using enhanced stabilization chemistry (ESC), a clinically validated modification pattern incorporating 2'-O-methyl, 2'-fluoro, and phosphorothioate modifications. While ESC-modified siRNAs showed reduced activity compared to unmodified natural siRNAs in conventional cell-based assays requiring transfection reagents, they exhibited consistent gene silencing in Gb, reflecting their enhanced biochemical stability under free uptake conditions at picomole-scale doses. These results establish Gb as a scalable, cost-effective, and biologically relevant platform for evaluating therapeutic siRNAs, particularly those incorporating chemical modifications.
小干扰RNA（siRNAs）因其能够选择性沉默疾病相关基因而具有巨大的治疗潜力。尽管化学修饰的siRNAs已显示出临床疗效，但其开发仍受到诸如生理条件下的稳定性和递送等挑战的阻碍。此外，使用培养细胞对化学修饰的siRNAs进行体外筛选具有成本效益，但往往无法重现体内的复杂性，从而限制了预测准确性。为了解决这一问题，我们开发了一种无需转染的siRNA评估平台，该平台使用具有天然RNA摄取能力的昆虫模型双斑蟋蟀（Gb）。我们首先证明，21个核苷酸的siRNAs在自由摄取条件下腹腔注射后可诱导RNA干扰。然后，我们生成了转基因品系，其携带与治疗性siRNA靶序列融合的增强绿色荧光蛋白（EGFP）报告基因，并通过成簇规律间隔短回文重复序列（CRISPR）-CRISPR相关蛋白9介导的敲入整合到β-肌动蛋白基因座中。建立并验证了两个转基因品系（s1和d1）。我们比较了使用增强稳定化学（ESC）设计的未修饰和化学修饰的siRNAs，ESC是一种经过临床验证的修饰模式，包含2'-O-甲基、2'-氟和硫代磷酸酯修饰。虽然在需要转染试剂的传统细胞检测中，与未修饰的天然siRNAs相比，ESC修饰的siRNAs活性降低，但它们在Gb中表现出一致的基因沉默，这反映了它们在皮摩尔剂量的自由摄取条件下增强的生化稳定性。这些结果确立了Gb作为评估治疗性siRNAs，特别是那些包含化学修饰的siRNAs的可扩展、经济高效且与生物学相关的平台。
