Division of Arthritis and Rheumatic Diseases, Oregon Health & Science University and Portland VA Medical Center, Portland, OR 97239, United States.
Neuroimmunology Research, Portland VA Medical Center, Portland, OR 97239, United States.
Biochem Biophys Res Commun. 2014 Oct 3;452(4):1040-5. doi: 10.1016/j.bbrc.2014.09.037. Epub 2014 Sep 18.
Cell type specific delivery of RNAi to T cells has remained to be a challenge. Here we describe an aptamer mediated delivery of shRNA to CD4(+) T cells targeting RORγt to suppress Th17 cells. A cDNA encoding CD4 aptamer and RORγt shRNA was constructed and the chimeric CD4 aptamer-RORγt shRNA (CD4-AshR-RORγt) was generated using in vitro T7 RNA transcription. 2'-F-dCTP and 2'-F-dUTP were incorporated into CD4-AshR-RORγt for RNase resistance. CD4-AshR-RORγt was specifically uptaken by CD4(+) Karpas 299 cells and primary human CD4(+) T cells. The RORγt shRNA moiety of CD4-AshR-RORγt chimera was cleaved and released by Dicer. Furthermore, CD4-AshR-RORγt suppressed RORγt gene expression in Karpas 299 cells and CD4(+) T cells and consequently inhibited Th17 cell differentiation and IL-17 production. These results demonstrate that aptamer-facilitated cell specific delivery of shRNA represents a novel approach for efficient RNAi delivery and is potentially to be developed for therapeutics targeting specific T cells subtypes.
细胞类型特异性的 RNAi 递送至 T 细胞一直是一个挑战。在这里，我们描述了一种适体介导的靶向 RORγt 的 shRNA 递送至 CD4(+) T 细胞，以抑制 Th17 细胞。构建了编码 CD4 适体和 RORγt shRNA 的 cDNA，并用体外 T7 RNA 转录生成嵌合 CD4 适体-RORγt shRNA (CD4-AshR-RORγt)。2'-F-dCTP 和 2'-F-dUTP 被掺入 CD4-AshR-RORγt 中以增加对核酸酶的抗性。CD4-AshR-RORγt 被 CD4(+) Karpas 299 细胞和原代人 CD4(+) T 细胞特异性摄取。CD4-AshR-RORγt 嵌合体的 RORγt shRNA 部分被 Dicer 切割和释放。此外，CD4-AshR-RORγt 抑制了 Karpas 299 细胞和 CD4(+) T 细胞中 RORγt 基因的表达，从而抑制了 Th17 细胞的分化和 IL-17 的产生。这些结果表明，适体介导的细胞特异性 shRNA 递送代表了一种有效的 RNAi 递送的新方法，并且有可能针对特定的 T 细胞亚型开发用于治疗的方法。
