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RNA 干扰技术

RNA 干扰技术. 董莹 董翠玲 陈崇波. 2006 年诺贝尔生理学或医学奖： 美国斯坦福大学 Andrew Z ． Fire 美国马萨诸塞大学 Craig C ． Mello ， 以表彰他们发现了 RNAi 现象. 什么是 RNAi ？. 一、 RNAi 的发现 二、 RNAi 的分子机制 三、 RNAi 的生物学功能与意义 四、 RNAi 试验方法. 一、 RNAi 的发现. RNAi 的发现. Post Transcriptional Gene Silencing in Petunia. WT.

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RNA 干扰技术

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  1. RNA干扰技术 董莹 董翠玲 陈崇波

  2. 2006年诺贝尔生理学或医学奖： 美国斯坦福大学Andrew Z．Fire 美国马萨诸塞大学 Craig C．Mello， 以表彰他们发现了RNAi现象

  3. 什么是RNAi？ 一、RNAi的发现 二、RNAi的分子机制 三、RNAi的生物学功能与意义 四、RNAi试验方法

  4. 一、RNAi的发现

  5. RNAi的发现 Post Transcriptional Gene Silencing in Petunia WT Plant with a pigment transgene

  6. 1995年，Guo S等试图阻断秀丽线虫（C.elegans）中的par-1基因的表达 • 设计： 反义RNA特异性地阻断par-1基因的表达 正义RNA 以期观察到基因表达的增强 • 结果: 二者都同样地切断了par-1基因的表达途径。这是与传统上对反义RNA技术的解释不相符合。该研究小组一直没能给这个意外以合理解释

  7. 直到1998年2月，Fire A和Mello C才首次揭开这个悬疑之谜。 • 他们将体外转录得到的单链RNA纯化后注射线虫时发现，基因抑制效应变得十分微弱；而经过纯化的双链RNA却正好相反，能够高效特异性阻断相应基因的表达。 • 他们证实，Guo S博士遇到的正义RNA抑制基因表达的现象，以及过去的反义RNA技术对基因表达的阻断，都是由于体外转录所得RNA中污染了微量双链RNA而引起。 • 该小组将这一现象称为RNA干扰（简称RNAi）。

  8. RNAi概念 RNA干扰(RNAi)是指dsRNA在细胞内特异性地诱导与之同源互补的mRNA的降解，使相应基因的表达关闭，从而引发基因转录后水平沉默的现象。

  9. RNAi特点 • 转录后水平的基因沉默机制 • 较高的特异性 • 抑制基因表达具有较高的效率 • 有浓度、时间双重依赖性 • 基因表达的效应可以突破细胞界限

  10. 二、RNAi的分子机制 1.起始阶段(the initiation phase) 2.效应阶段(the subsequent effector phase)

  11. Dicer dsRNA通过外源导入、转基因或者病毒感染等方式进入细胞后，专一性的双链RNA内切酶Dicer识别dsRNA，在ATP的参与下逐步把dsRNA切割成长约21～23 nt的片段。

  12. siRNA参与形成RNA诱导的沉默复合物(RISC)，ATP激活的RISC在双链siRNA的反义链指导下，寻找与siRNA具有同源序列的内源靶mRNA，并在距离siRNA3’端12个碱基的位置切割靶mRNA，导致转录后基因沉默。

  13. 三、RNAi的生物学功能与意义

  14. RNAi的生物学功能 • 抵抗病毒入侵 • 抑制转座子活动，保护基因组 的完整性 • 调控基因表达 • 清除畸变的RNA • 参与基因组重排

  15. RNAi的生物学意义 1. 研究功能基因组学的新工具 2. 研究信号传导通路的新途径 3. 研究发育过程中起作用的基因 4. 开展基因治疗的新策略

  16. 四、RNAi试验方法

  17. 1.筛选RNAi探针 2.制备siRNA 化学合成、体外转录 、用RNaseⅢ消化长片断双链RNA、siRNA表达载体、siRNA表达框架 3.导入细胞 电穿孔法、磷酸钙共沉淀、DEAE一葡聚糖、 阳离子脂质体试剂 4.RNAi转染效率 荧光蛋白(如GFP)或荧光素酶等报告基因与其共表达法

  18. Abstract：In vitro, RNAi is an almost-standard method to knockdown any target gene. In vivo, its effcient delivery and specificity to the target gene challenge its therapeutic application. Now, many efforts have made to enhance siRNA delivery and target organ specificity.

  19. In vitro several transfection reagents allow the delivery of siRNAs in mammalian cells in the presence or absence of serum • Invivo require the development of more sophisticated formulations and/or the identification of optimal modes of administration

  20. Therapeutic siRNA molecules • Cancer : Target molecules usually represent genes that have been shown previously to be relevant or rate-limiting for tumor growth • Antiviral treatment: novel RNAi-based approaches to battle viral infections rely on the specific siRNA-mediated knockdown of virus-specific genes

  21. This studies provide valuable insights into the delivery and efficacy of for the induction of RNAi.

  22. Clinical trials • ALN-RSV01: target the human RSV after viral infection, which is the first example of an antivirus siRNA-based therapeutic in a phaseⅠclinical study. • Sirna-027: To treat AMD,after 24-month phase II study, It has already used for recruiting patients with AMD. • Cand5: which is now named Bevasiranib,was the first siRNA to enter both phase I and II clinical trials.

  23. Strategies for in vivo siRNA delivery The advantages of the direct application of siRNAs: • the lower probability on specific side effects • the safety of siRNA delivery is based on nonviral transfer strategies • siRNAs can not integrate into the genome

  24. Successful siRNA based gene targeting relies on several preconditions: • Protect the instable siRNA molecules • Efficient cellular uptake and intracellular release into the cytoplasm • The absence of intracellular immune responses

  25. It can be considered as examples of nonviral envelopes that protect siRNAs, thus increasing serum stability, reducing renal excretion and mediating siRNA uptake into the cells through endocytosis nanoparticles with positively charged macromolecules. Based on electrostatic interactions, complexes are formed with atelocollagen chitosan or PEI • The formulation of siRNAs in carrier systems： • Liposomal formulations • Nanoparticles • polyethylenimine (PEI) PEI/siRNA complexes are good for enhancing tissue specificity and in vivo biocompatibility, reducing immunogenicity and toxicity and increasing siRNA delivery

  26. Conclusion and outlook • A very efficient and specific method for the knockdown • The success of siRNAs will depend on their efficient and safe in vivo delivery • siRNAs for gene knockdown will be their “double specificity”, optimal siRNA sequences and increased target organ specificity

  27. Thank you for your attention！

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