生命科学前沿讲座课件

1、2022-4-18Wuhan University of Technology Xie H.FLS 13 - 1FLS Lecture 13RNAi 2006年诺贝尔奖成果主讲教师：谢浩主讲教师：谢浩2022-4-18Wuhan University of Technology Xie H.FLS 13 - 22022-4-18Wuhan University of Technology Xie H.FLS 13 - 3安德鲁法尔 克雷格梅洛 2006年诺贝尔生理学或医学奖得主 2022-4-18Wuhan University of Technology Xie H.FLS 13 - 420

2、22-4-18Wuhan University of Technology Xie H.FLS 13 - 5Powerful new research tool for gene silencingPursued by biotech industry as therapeutic for treating diseases: (e.g. Parkinsons, AIDS, cancer, hepatitis)First human clinical trial recently approved by FDARNA interference (RNAi)2022-4-18Wuhan Univ

3、ersity of Technology Xie H.FLS 13 - 61. Background on gene silencing2. Discovery of RNA interference3. Mechanism4. Applications: a few examplesC. elegansMammalian cell cultureMice5. Guidlines for designing siRNAs6. Latest developments in biotech industryRNAi-related therapeuticsOutline2022-4-18Wuhan

4、 University of Technology Xie H.FLS 13 - 7FLS 13-1Background on gene silencing2022-4-18Wuhan University of Technology Xie H.FLS 13 - 8To study the function of unknown genes:30,000+ genes in human genome, most are uncharacterizedKnock out the gene and examine which processes are affectedTo characteri

5、ze biological pathways:Study functional interactions between proteinsTo treat diseaseSilence defective proteinsSilence viral proteinsWhy silence a gene?2022-4-18Wuhan University of Technology Xie H.FLS 13 - 9Approaches for reducing or eliminating gene function in vivoDNAReduce transcription of the g

6、eneTranscriptional inhibitorMutate gene or remove from genomeMutagenesis; targeted gene knockout (e.g. knockout mice) mRNADegrade the genes mRNARNA interferenceInhibit translation of the geneantisense technologies (e.g. antisense Morpholinos)ProteinInhibit protein activitySmall molecule inhibitors,

7、substrate analogs, antibodies, sequestration, etc.2022-4-18Wuhan University of Technology Xie H.FLS 13 - 10FLS 13-2Discovery of RNA interference2022-4-18Wuhan University of Technology Xie H.FLS 13 - 11Discovery of RNA interferenceGuo et al. 1995 Cell 81: 611-6202022-4-18Wuhan University of Technolog

8、y Xie H.FLS 13 - 12Discovery of RNA interference2022-4-18Wuhan University of Technology Xie H.FLS 13 - 13FLS 13-3Mechanism of RNAi2022-4-18Wuhan University of Technology Xie H.FLS 13 - 14Dicer belongs to the RNase III ribonuclease family. These nucleases cut specificly dsRNA and generate dsRNA produ

9、cts with 5-phosphate and 3-hydroxyl groups, and two nucleotide 3 overhang termini. This enzyme family can be subdivided to three classes, based upon domain structure.Dicer (RNase III)2022-4-18Wuhan University of Technology Xie H.FLS 13 - 15Schemes of the RNase III superfamily proteins. Three classes

10、 of RNase III family proteins represented by human Dicer, human Drosha and eubacterial RNase III. Individual protein domains are indicated in different colors.2022-4-18Wuhan University of Technology Xie H.FLS 13 - 16RISC (RNA Induced Silencing Complex)RISC contains two signature components, small in

11、terfering RNAs (siRNAs) and Argonaute family proteins.2022-4-18Wuhan University of Technology Xie H.FLS 13 - 17Crystal structure of P. furiosus Argonaute. 2022-4-18Wuhan University of Technology Xie H.FLS 13 - 18A model for siRNA-guided mRNA cleavage by Argonaute.(A)View of the electrostatic surface

12、 potential of PfAgo indicating a positively charged groove (blue). (B)(B) A 3 portion of the siRNA (purple) was placed by superposition of the PAZ domain of the hAgo1-PAZ domain RNA complex on the PAZ domain of PfAgo. (C) Schematic depiction of the model for siRNA-guided mRNA cleavage. 2022-4-18Wuha

13、n University of Technology Xie H.FLS 13 - 19The RNAi Movie2022-4-18Wuhan University of Technology Xie H.FLS 13 - 20Mechanism of RNAi: Step 12022-4-18Wuhan University of Technology Xie H.FLS 13 - 21Mechanism of RNAi: Step 22022-4-18Wuhan University of Technology Xie H.FLS 13 - 22Mechanism of RNAi: St

14、ep 32022-4-18Wuhan University of Technology Xie H.FLS 13 - 23The systemic spread of gene silencing from one tissue to another has been well established in C. elegans and plants. A systemic RNA interference-deficient (sid) locus was demontrated required to transmit gene silencing between cells using

15、the green fluorescent protein (GFP) as a marker. Based on the structure of SID1, it was suggested that it might act as a channel for the import or export of a systemic RNAi signal or might be necessary for endocytosis of the systemic RNAi signal, perhaps functioning as a receptor. No homologue of si

16、d1 was detected in Drosophila, which is consistent with the apparent lack of systemic RNAi in this organism. Transmembrane Protein (Channel or Receptor)2022-4-18Wuhan University of Technology Xie H.FLS 13 - 24FLS 13-4Application of RNAi2022-4-18Wuhan University of Technology Xie H.FLS 13 - 25Applica

17、tion of RNAi2022-4-18Wuhan University of Technology Xie H.FLS 13 - 26Simple model: C. elegans2022-4-18Wuhan University of Technology Xie H.FLS 13 - 27C. elegans genome-wide RNAi screen 16,757 genes targeted (86% of genome) 1,722 (10.3%) resulted in defects in viability, growth rate, development or f

18、ertility Estimated success rate of 63% based on existing mutants (Kamath et al., 2003. Nature. 421: 231-237) Why such a low percentage? Redundancy Some cell types refractive to RNAi Some genes less sensitive to RNAi2022-4-18Wuhan University of Technology Xie H.FLS 13 - 28 Cell lines derived from mam

19、malian cells e.g. tumor cells, embryonic stem (ES) cells, transformed “normal” cells, primary cells Highly amenable to biochemical studies Not very amenable to most genetic techniques RNA interference provides quick, effective tool for gene silencingMammalian cell culture2022-4-18Wuhan University of

20、 Technology Xie H.FLS 13 - 29RNAi in mammalian cell culture Size limitations:19-29 nt dsRNAsdsRNA 30 nt triggers interferon / antiviral / Protein Kinase R response resulting in global translation arrest Generating siRNAs for cell culture experiments Synthesize siRNA in vitro or order pre-synthesized

21、 siRNA Use expression vector to generate small hairpin RNA (shRNA) or complementary RNA fragments in vivo Delivery methods Transfection methods Lipofectamine, Oligofectamine, electroporation, Calcium Phosphate Viral entry vector Lentivirus, adenovirus, etc. For current list of RNAi product suppliers

22、, see: Nature 431: 607-6092022-4-18Wuhan University of Technology Xie H.FLS 13 - 30RNAi in mammalian cell cultureApproach 1: Transfection of dsRNA2022-4-18Wuhan University of Technology Xie H.FLS 13 - 31RNAi in mammalian cell cultureApproach 2: Transfection of vector expressing2022-4-18Wuhan Univers

23、ity of Technology Xie H.FLS 13 - 32RNA interference in miceApproaches for whole organism expression:Express short hairpin RNA (shRNA) in live mice:- Introduce into mouse embryonic stem cells and use standardtransgenic techniques to build transgenic miceOR- Inject directly into fertilized eggs and im

24、plant in mouse uterusApproaches for tissue-specific expressionInject siRNA directly into targeted organ or tissueORUse shRNA expression vector driven by tissue-specific promoter to build transgenic mice2022-4-18Wuhan University of Technology Xie H.FLS 13 - 33RNA interference in miceKunath et al., 20

25、03 Nature Biotech. 21: 559-5612022-4-18Wuhan University of Technology Xie H.FLS 13 - 34RNA interference in mice2022-4-18Wuhan University of Technology Xie H.FLS 13 - 35RNA interference in mice2022-4-18Wuhan University of Technology Xie H.FLS 13 - 36RNA interference in mice2022-4-18Wuhan University o

26、f Technology Xie H.FLS 13 - 37Additional effects of siRNA2022-4-18Wuhan University of Technology Xie H.FLS 13 - 38Additional effects of siRNAsiRNA mediated transcriptional silencing siRNA targeted against gene promoters can facilitate DNA methylation and heterochromatin-mediated silencing Previously

27、 observed in yeast, flies, and plants Recently described in human cells:Kawasaki & Taira 2004 Nature 431: 211-2172022-4-18Wuhan University of Technology Xie H.FLS 13 - 39FLS 13-5Guidlines for designing siRNAs2022-4-18Wuhan University of Technology Xie H.FLS 13 - 40Designing siRNAsGuidelines Test

28、 several siRNAs targeting different regions of gene Thermodynamic considerations in designing siRNAs: High internal stability at the 5 sense strand Low internal stability at the 5 antisense strand Absense of internal repeats or palindromes Overall low to medium G+C content (30-50%) Base preferences

29、Presence of A at positions 3 and 19 of sense strand Absence of G or C at position 19 of sense strand Presence of a U at position 10 of sense strand For more details, see: Mittal 2004 Nat. Rev. Gen. 5: 355-365 Avoid sequences with strong homology to other genes in the genome2022-4-18Wuhan University

30、of Technology Xie H.FLS 13 - 41Designing siRNAs Limitations Gene product levels often reduced rather than eliminated Some RNAi techniques are transient Some siRNAs cause non-specific silencing Additional resources Free web-based siRNA design tools:- http:/ /siRNAext/ (Whitehead

31、Institute at MIT)- http:/ Confirm siRNA sequence specificity using BLAST- /BLAST2022-4-18Wuhan University of Technology Xie H.FLS 13 - 42FLS 13-6Latest developments in biotech industry2022-4-18Wuhan University of Technology Xie H.FLS 13 - 43RNAi: First human clinical trial1.Acuit

32、y PharmaceuticalsDeveloping treatment for age-related macular degenerationUsing siRNA to target Vascular Endothelial Growth Factor (VEGF)siRNA injected into eyes of elderly patients2.Prospective future trialsAlnylam Pharmaceuticals & Merck - eye diseasesSirna Therapeutics and Eli Lilly - cancerBenitec (Australia) - hepatitis C3.Major hurdles for use as therapeuticTarget specificityDrug delivery to appropriate cells or tissues2022-4-18Wuhan University of Technology Xie H.FLS 13 - 44 中国医学科学院/协和医科大学（Chinese Aca