Metagenomics, European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory, Wellcome Trust Genome Campus, Hinxton, UK.
Department of Molecular and Applied Biosciences, Faculty of Science and Technology, University of Westminster, London, UK.
Mol Ther Nucleic Acids. 2014 Feb 4;3(2):e145. doi: 10.1038/mtna.2013.73.
TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.
TT-034（PF-05095808）是一种表达三种短发夹 RNA（shRNA）前药的重组腺相关病毒血清型 8（AAV8）制剂，可靶向丙型肝炎病毒（HCV）RNA 基因组。细胞质酶 Dicer 将每个 shRNA 切割成多个潜在的活性小干扰 RNA（siRNA）药物。使用下一代测序（NGS）来鉴定和描述活性 shRNA 成熟产物，我们观察到每个 TT-034 编码的 shRNA 可以被加工成多达 95 个单独的 siRNA 链。通过 Sanger 5' RNA 连接酶介导的 cDNA 末端快速扩增（5-RACE）以及通过合成 shRNA 和 siRNA 类似物研究，很少有这些被认为是活跃的。此外，NGS 对 5-RACE 产物（RACE-seq）的检查表明，合成的 siRNA 可以在靶 RNA 上的多达五个不同位置以序列依赖性的方式指导切割，而不是一个位置。这些数据支持 TT-034 的作用机制是针对靶标而没有细胞毒性，并质疑关键 RNA 干扰（RNAi）酶 Dicer 和 siRNA 诱导的沉默复合物（siRISC）对底物加工的公认精度。分子治疗-核酸（2014 年）3，e145；doi:10.1038/mtna.2013.73；在线发表于 2014 年 2 月 4 日。
Zotero 插件
