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Gene Silencing Strategies for Dissecting Disease Pathways Victoria Rusakova Senior Scientist Sigma-Aldrich Corporation.

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Presentation on theme: "Gene Silencing Strategies for Dissecting Disease Pathways Victoria Rusakova Senior Scientist Sigma-Aldrich Corporation."— Presentation transcript:

1 Gene Silencing Strategies for Dissecting Disease Pathways Victoria Rusakova Senior Scientist Sigma-Aldrich Corporation

2 Agenda Introduction to RNAi shRNA Lentiviral Transduction System Arrayed Kinome shRNA Library –Identifying gene targets contributing to androgen independent prostate cancer cell growth –Identifying novel human kinases essential for osteosarcoma cell survival siRNA Endoribonuclease-prepared siRNA (esiRNA) Screening Library –Discovering modulators of embryonic stem cell identity 2

3 33 Modulation of Gene Expression Small molecules mAbs Aptamers Central Dogma of Molecular Biology siRNA shRNA RNAProtein Zinc finger nucleases DNA

4 4 Areas Using RNAi Technology Gene function analysis Testing or verifying predicted gene function Pathway analysis Target the expression of a given gene in a pathway and monitor the expression of other genes to identify those genes associated with the target gene Target identification and validation Identification of potential drug targets, at the gene or protein level Drug discovery Develop potential therapeutic compounds based on identified targets 2006: The Nobel Prize in Physiology and Medicine awarded to Andrew Z. Fire and Craig C. Mello

5 RNAi: Types of Interfering RNAs Synthetic based Small or short interfering RNAs (siRNA) –Transfected directly into cells as oligonucleotides –Do not perpetuate as vectors dsRNA molecules (duplexes) shorter than 30bp Silencing duration and effectiveness mainly regulated by transfection efficiency Clone based Short hairpin RNAs (shRNA) –Give rise to siRNA after processing by Dicer protein Encoded by DNA vectors allowing multiple delivery methods –Standard transient transfection –Stable transfections –Delivery by virus 5

6 6 RNAi Delivery to the Cell

7 Agenda Introduction to RNAi shRNA Lentiviral Transduction System Arrayed Kinome shRNA Library –Identifying gene targets contributing to androgen independent prostate cancer cell growth –Identifying novel human kinases essential for osteosarcoma cell survival siRNA Endoribonuclease-prepared siRNA (esiRNA) Screening Library –Discovering modulators of embryonic stem cell identity 7

8 8 Recombinant Lentiviral Life Cycle

9 Viral Transduction Laboratory Workflow 9

10 Viral Titer and MOI (Multiplicity of Infection) Viral titer is a very important factor Allows determination of the correct experimental conditions using MOI –MOI (Multiplicity of Infection) used for desired transduction efficiency –The number of transducing lentiviral particles per cell When transducing a cell line for the first time, a range of MOI should be tested –Most successful screen require an MOI of 0.5 to

11 Lentiviral-mediated Gene Transfer in Different Cell Lines Significance of controlled conditions in lentiviral vector titration Use MOI for predicting gene transfer events Efficiency of lentiviral-mediated gene transfer to commonly used cell lines under different MOI Genet. Vaccines Ther. 2(1):6 (2004) Zhang B., et al., Department of Medicine, University of Queensland, Prince Charles Hospital, Brisbane, Australia 11

12 Enhancing Transduction Efficiency Magnetic transduction Applying magnetic fields during transduction to potentiate cell targeting and binding Serial transductions Allow the cells to recover for 1 day after initial transduction and follow with a second round Infecting cells with a higher titer virus VSV-G envelope protein allows for concentration via ultracentrifugation and ultrafiltration VSV-G envelope protein 12

13 Enhancing Transduction of Primary Cells TurboGFP particles + polybreneTurboGFP particles + ExpressMag Human keratinocytes transduced at a MOI of 1, incubated for 45 hours 13

14 Viral Transduction Laboratory Workflow 14

15 Transient versus Stable Transduction Time and cell division affects gene expression Gives immediate assessment of the system’s efficiency MOI 5 HT-29 cells Allow to establish clonal stable cell lines Provides a system for long-term gene silencing and phenotypic observation CHO-K1 cells MOI 1 15

16 Agenda Introduction to RNAi shRNA Lentiviral Transduction System Arrayed Kinome shRNA Library –Identifying gene targets contributing to androgen independent prostate cancer cell growth –Identifying novel human kinases essential for osteosarcoma cell survival siRNA Endoribonuclease-prepared siRNA (esiRNA) Screening Library –Discovering modulators of embryonic stem cell identity 16

17 Objective: Identify genes that, when silenced, can either enhance or suppress a given phenotype Modifier Screen 17

18 Optimization Plate Pre-arrayed aliquots of TurboGFP particles and controls Ideal for determination of optimal cell number and MOI for LentiExpress assays Human Kinase Plate A quick method for carrying out kinase screens 3109 pre-arrayed lentiviruses – shRNAs targeting 673 human kinase genes and controls –A total of well plates –Up to 80 shRNAs per plate LentiExpress Plates 18

19 Cancer Incidence (per 100K) Year Prostate cancer Prostate Cancer is the Most Frequently Diagnosed Cancer in American Men 19

20 normalPIN cancer metastases androgen death independence Progression Transition to Metastatic Disease 20

21 LNCaP cells Gene knockdown + - Experiment – Knockdown Genes in an Androgen-dependent Cell Line 21

22 Percent Expression UntreatedH2H3H5H6 Androgen Receptor Knockdown Normalized to Untreated Cells and Cyclophilin Validation of shRNA Clones in LNCaP Cells 22

23 LNCaP cells transduced with non-targeting shRNA LNCaP cells transduced with androgen receptor shRNA LNCaP Cells Treated with AR shRNA 23

24 Androgen Receptor Non-Target Androgen Receptor Knockdown Androgen receptor knockdown is stable under experimental conditions of the assay 24

25 Lentiviral shRNA particles targeting kinases LNCaP Cells Puromycin selection Split 1:2 shRNAshRNA + androgen Viability assay Modifier Screen 25

26 % Growth Relative to Control -- Vehicle % Growth Relative to Control – Androgen shRNA Kinome Screen – LNCaP 26

27 Agenda Introduction to RNAi shRNA Lentiviral transduction system Arrayed Kinome shRNA Library –Identifying gene targets contributing to androgen independent prostate cancer cell growth –Identifying novel human kinases essential for osteosarcoma cell survival siRNA Endoribonuclease-prepared siRNA (esiRNA) Screening Library –Discovering modulators of embryonic stem cell identity 27

28 28 Hypothesis Overexpression and activation of specific kinases occurs during growth of osteosarcoma cells Disruption of specific kinases will cause osteosarcoma cell death or apoptosis These kinases have the potential to be drug targets for sarcoma 28

29 29 10,00040,000 20,000 80,000160,000 Various seeding densities (cells/mL) were plated in wells containing tGFP positive control particles Courtesy of Zhenfeng Duan, M.D. Determining Optimal Transduction Conditions in KHOS 29

30 30 pLKO.1 Control Particles (C) Non-Target shRNA Control Particles (N) Control Media (M) 1 µg/ml of puromycin causes complete cell death of KHOS, U-2OS and UCH1 in 5 days Courtesy of Zhenfeng Duan, M.D. Negative Controls Used in the Optimization Plate 30

31 Protocol for shRNA Kinase Screen in Human Osteosarcoma Cells 31 Replace wells with fresh media Incubate plate at 37 °C, 5% CO2 overnight Add puromycin- supplemented media at 1µg/mL Dispense KHOS cells into 96 well lentiviral shRNA kinase plates Remove plates from incubator Analyze results with a cell proliferation assay kit Courtesy of Zhenfeng Duan, M.D. overnight Incubate plate at 37 °C, 5% CO2 7 days Change media every 2 days with puromycin Incubate plate at 37 °C, 5% CO2

32 32 C*C* C C C N N N N M M M M M M M M A7A8A9A10A11 B11 C2C3C4C5 M Courtesy of Zhenfeng Duan, M.D. Positive Hits from Screen 32

33 Positive Hit 1: PLK1 Reduced Viability Upon Silencing pLKO.1 particles Non target particles Media control 33

34 Positive Hit 2: ROCK1 Reduced Viability Upon Silencing pLKO.1 particles Non target particles Media control 34

35 35 LentiExpress Kinase Screen Summary Identified 4 gene candidates as potential therapeutic targets in osteosarcoma cells, including PLK1 and ROCK1 KHOS osteosarcoma cells exhibited decreased cell proliferation upon knockdown of these genes 35

36 Agenda Introduction to RNAi shRNA Lentiviral transduction system Arrayed Kinome shRNA Library –Identifying gene targets contributing to androgen independent prostate cancer cell growth –Identifying novel human kinases essential for osteosarcoma cell survival siRNA Endoribonuclease-prepared siRNA (esiRNA) Screening Library –Discovering modulators of embryonic stem cell identity 36

37 RNAi: Types of Interfering RNAs Synthetic based Small or short interfering RNAs (siRNA) –Transfected directly into cells as oligonucleotides –Do not perpetuate as vectors dsRNA molecules (duplexes) shorter than 30bp Silencing duration and effectiveness mainly regulated by transfection efficiency Clone based Short hairpin RNAs (shRNA) –Give rise to siRNA after processing by Dicer protein Encoded by DNA vectors allowing multiple delivery methods –Standard transient transfection –Stable transfections –Delivery by virus 37

38 MISSION esiRNA Technology Transfect into cell “ Super-pool” of hundreds of siRNAs against 1 target gene Assembly into RISC Targeting of single mRNA mRNA cleavage and degradation 38

39 Generation of esiRNA 39

40 MISSION esiRNA esiRNA Gene #1 esiRNA Gene #2 esiRNA Gene #3 esiRNA Gene #4 etc. 1 esiRNA super-pool targeting one gene per well 40

41 41 Discovering Modulators of Embryonic Stem Cell Identity Objective Obtain a systematic understanding of the genes associated with ESC identity Approach Perform a genome-scale RNAi screen to identify genes regulating ESC identity using an Oct4 reporter assay Ding, L. et al., Cell Stem Cell. 9: (2009)

42 42 Oct4 Assay Oct4 expression can be used to monitor the differentiation status of ESC Screen performed in an Oct4 reporter mouse embryonic stem cell line (Oct4-Gip) GFP expression is controlled by Oct4 regulatory elements Transfect cells with esiRNA and monitor changes in GFP expression Quantification of GFP fluorescence faithfully reflects the self-renewal and differentiation status in individual cells Ding, L. et al., Cell Stem Cell. 9: (2009)

43 43 Oct4 Assay: Proof of Principle GFP Expression Individual wells transfected with Control luciferase esiRNA esiRNA to known pluripotency regulators –Sox2 –Oct4 –Stat3 Visualized GFP by microscopy or FACS analysis Ding, L. et al., Cell Stem Cell. 9: (2009)

44 44 Overview of Oct4 High-throughput Assay Transfect Oct4-Gip ESC with control or genome- scale esiRNA library High-throughput GFP fluorescence readout to identify primary hits Negative control (Luciferase esiRNA) Primary hit (cells have reduced GFP) No hit or negative control (cells have high GFP Expression) Primary hit or positive control (cells have reduced GFP Expression) Readout Ding, L. et al., Cell Stem Cell. 9: (2009)

45 45 Summary of Oct4 High-throughput Assay 259 known and novel candidate pluripotency genes identified Secondary screen performed using individual esiRNAs synthesized for the 21 strongest candidates 16 genes were confirmed Validated targets included components of the of the Pol II-associating factor 1 complex (Paf1C) Paf1C contains Paf1, Ctr9, Cdc73, Rtf1, and Leo1 Regulates transcription initiation, elongation, and start site selection Ding, L. et al., Cell Stem Cell. 9: (2009)

46 Paf1C Affects the Expression of Pluripotency and Lineage-marker Genes 46 Ding, L. et al., Cell Stem Cell. 9: (2009)

47 Summary of Study siRNA (esiRNA) is an effective tool for modulating gene function in stem cells A screen using esiRNA identified 259 known and novel candidate pluripotency genes Validated targets included components of the of the Pol II-associating factor 1 complex (Paf1C) Paf1C affects the expression of pluripotency and lineage-marker genes 47

48 48

49 49 Review of RNAi Effectors siRNAshRNA Benefits Simple Titratable Modifications available Pooling is straightforward Efficiently transfected –Easy to transfect cell lines Disadvantages Hard to transfect cells Transient knockdown Non-renewable Benefits Renewable resource Transient or stable knockdown Transfection or viral delivery –Viral delivery to most cells In vivo use potential –Knockdown mice Disadvantages Design rules less understood Transfection less efficient

50 50 Goals Create a lentiviral based shRNA libraries targeting human and mouse genes Make clones available to researchers worldwide for the study of disease and gene function Academic Laboratories Broad Institute, MIT/Harvard, Massachusetts General Hospital, Dana Farber Cancer Institute, Whitehead Institute, Washington University and Columbia University Life Science Organizations Sigma-Aldrich, Novartis, Eli Lilly, Bristol-Myers Squibb and Academia Sinica in Taiwan The RNAi Consortium (TRC)

51 51 TRC1 shRNA Transfer Vector Transfer vector pLKO.1-puro Lentiviral-based (HIV derived) Vector shRNA Promoter U6 (human) Design Broad Institute algorithm 21 bp stem 6 bp loop 5 clones per target gene High gene coverage Multiple knockdown levels Verification of phenotype –Different shRNA produces same result 3' UTR clone for cDNA rescue

52 52 TRC2 TRC2 Goals KD evaluation for 150,000 clones by qRT-PCR Optimize vector elements Consider and evaluate special purpose vectors Develop new and improved screening methods –Pooled libraries

53 53 TRC2 shRNA Transfer Vector Woodchuck hepatitis post-transcriptional regulatory element (WPRE) Sigma uses 3 rd generation safety & design SIN vector (self inactivating vector) Replication incompetent lentiviral particles Recommended biosafety level: BSL-2


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