Kusumoto Kohshi, Sasaki Kiyomi, Uchida Yasunori, Utsumi Ayaka, Yoshida Tokuyuki, Obika Satoshi, Inoue Takao, Okuhira Keiichiro
Osaka Medical and Pharmaceutical University, 4-20-1, Nasahara, Takatsuki, 569-1094, Osaka, Japan.
Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, 210-9501, Kanagawa, Japan.
Sci Rep. 2025 Jan 2;15(1):586. doi: 10.1038/s41598-024-84310-6.
Recent advances in the clinical development of oligonucleotide therapeutics, such as antisense oligonucleotides (ASOs) and small interfering RNAs, have attracted attention as promising therapeutic modalities for genetic and intractable diseases. These oligonucleotide therapeutics exert their efficacy by binding to target RNAs present within cells; however, the mechanisms underlying their cellular uptake, especially their passage through membranes, remain largely unclear. In the nematode, Caenorhabditis elegans, the multi-pass transmembrane protein, SID-1, is involved in the cellular uptake of double-stranded RNAs. In mammals, SIDT1 and SIDT2 (SID-1 transmembrane family, members 1 and 2, respectively) are homologs of SID-1, yet their functional differences are not fully understood. In this study, we conducted a comparative analysis of the amino acid sequences of mammalian SIDT1 and SIDT2 to identify regions characteristic to each. By inducing SIDT1 or SIDT2 expression in human cell lines, we demonstrated that SIDT2 enhanced the knockdown activity of gapmer ASOs and potentially promoted their endosomal escape into the cytosol. Furthermore, by analyzing chimeric proteins of SIDT2 and SIDT1, we identified a region in SIDT2 that might be crucial for the enhancement of gapmer ASO activity. These findings elucidate the novel role of SIDT2 in the transport mechanism of gapmer ASOs and are expected to contribute to further development of oligonucleotide therapeutics.
寡核苷酸疗法（如反义寡核苷酸（ASO）和小干扰RNA）的临床开发取得的最新进展，作为治疗遗传性和难治性疾病的有前景的治疗方式已引起关注。这些寡核苷酸疗法通过与细胞内存在的靶RNA结合发挥其功效；然而，其细胞摄取的机制，尤其是它们穿过膜的机制，在很大程度上仍不清楚。在秀丽隐杆线虫中，多次跨膜蛋白SID-1参与双链RNA的细胞摄取。在哺乳动物中，SIDT1和SIDT2（分别为SID-1跨膜家族成员1和2）是SID-1的同源物，但其功能差异尚未完全了解。在本研究中，我们对哺乳动物SIDT1和SIDT2的氨基酸序列进行了比较分析，以确定各自的特征区域。通过在人细胞系中诱导SIDT1或SIDT2表达，我们证明SIDT2增强了间隙mer ASO的敲低活性，并可能促进其从内体逃逸到细胞质中。此外，通过分析SIDT2和SIDT1的嵌合蛋白，我们确定了SIDT2中一个可能对增强间隙mer ASO活性至关重要的区域。这些发现阐明了SIDT2在间隙mer ASO转运机制中的新作用，有望为寡核苷酸疗法的进一步发展做出贡献。
