Nair Jayaprakash K, Attarwala Husain, Sehgal Alfica, Wang Qianfan, Aluri Krishna, Zhang Xuemei, Gao Minggeng, Liu Ju, Indrakanti Ramesh, Schofield Sally, Kretschmer Philip, Brown Christopher R, Gupta Swati, Willoughby Jennifer L S, Boshar Julie A, Jadhav Vasant, Charisse Klaus, Zimmermann Tracy, Fitzgerald Kevin, Manoharan Muthiah, Rajeev Kallanthottathil G, Akinc Akin, Hutabarat Renta, Maier Martin A
Alnylam Pharmaceuticals, Cambridge, MA 02142, USA.
Nucleic Acids Res. 2017 Nov 2;45(19):10969-10977. doi: 10.1093/nar/gkx818.
Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.
将合成的三分支N-乙酰半乳糖胺（GalNAc）配体共价连接到化学修饰的小干扰RNA（siRNA）上，能够实现去唾液酸糖蛋白（ASGPR）介导的治疗活性siRNA在体内向肝细胞的靶向递送。这种方法已成为将RNA干扰（RNAi）疗法以及其他类型的研究性寡核苷酸疗法递送至肝脏的变革性方法。然而，为了将完整药物有效地功能性递送至所需的亚细胞区室，至关重要的是使核酸稳定以抵抗核酸酶降解。在此，我们比较了具有相同序列但修饰模式不同的两种siRNA，其对核酸酶活性的保护程度不同。在不同生物基质中的体外稳定性研究表明，5'-核酸外切酶是内溶酶体区室中最普遍的核酸酶活性，并且两条siRNA链5'-区域的额外稳定化显著增强了GalNAc-siRNA缀合物的整体代谢稳定性。与体外研究结果高度一致，稳定性的增强转化为小鼠肝脏暴露、基因沉默效果和作用持续时间的显著改善。针对不同转录本的第二组缀合物的后续研究证实了先前的结果，并提供了关于RNA诱导沉默复合体（RISC）加载动力学的更多见解，且证明了在非人类灵长类动物中的良好转化效果。
