Gene Silencing Strategies for Dissecting Disease Pathways

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

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

3. Modulation of Gene Expression DNA • Small molecules • mAbs • Aptamers • Zinc finger nucleases • siRNA • shRNA Central Dogma of Molecular Biology RNA Protein 3

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

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

8. Recombinant Lentiviral Life Cycle

9. Viral Transduction Laboratory Workflow

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 5.0

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

12. VSV-G envelope protein 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

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

14. Viral Transduction Laboratory Workflow

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

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

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

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 41 96-well plates Up to 80 shRNAs per plate LentiExpress Plates

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

20. normal PIN cancer metastases androgen death independence Transition to Metastatic Disease Progression

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

22. Androgen Receptor Knockdown Normalized to Untreated Cells and Cyclophilin 120 100 80 60 40 Percent Expression 20 0 Untreated H2 H3 H5 H6 Validation of shRNA Clones in LNCaP Cells

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

24. 120 100 80 60 % Expression 40 Androgen Receptor 20 Non-Target 0 4 days 5 days 7 days Androgen receptor knockdown is stable under experimental conditions of the assay Time Androgen Receptor Knockdown

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

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

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

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. Determining Optimal TransductionConditions in KHOS 10,000 40,000 80,000 160,000 20,000 Various seeding densities (cells/mL) were plated in wells containing tGFP positive control particles 29 Courtesy of Zhenfeng Duan, M.D.

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

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

32. Positive Hits from Screen C* A7 A8 A9 A10 A11 C C C B11 N N C2 C3 C4 C5 N N M M M M M M M M M 32 Courtesy of Zhenfeng Duan, M.D.

33. Positive Hit 1: PLK1Reduced Viability Upon Silencing pLKO.1 particles Non target particles Media control

34. Positive Hit 2: ROCK1Reduced Viability Upon Silencing pLKO.1 particles Non target particles Media control

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

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

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

39. Generation of esiRNA

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

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. 9:403-15 (2009)

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. 9:403-15 (2009)

43. Oct4 Assay: Proof of Principle • Individual wells transfected with • Control luciferase esiRNA • esiRNA to known pluripotency regulators • Sox2 • Oct4 • Stat3 • Visualized GFP by microscopy or FACS analysis GFP Expression Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)

44. High-throughput GFP fluorescence readout to identify primary hits Transfect Oct4-Gip ESC with control or genome-scale esiRNA library Readout Primary hit or positive control (cells have reduced GFP Expression) No hit or negative control (cells have high GFP Expression) Primary hit (cells have reduced GFP) Negative control (Luciferase esiRNA) Overview of Oct4 High-throughput Assay Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)

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. 9:403-15 (2009)

46. Paf1C Affects the Expression of Pluripotency and Lineage-marker Genes Ding, L. et al., Cell Stem Cell. 9:403-15 (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

49. Benefits Simple Titratable Modifications available Pooling is straightforward Efficiently transfected Easy to transfect cell lines Disadvantages Hard to transfect cells Transient knockdown Non-renewable Review of RNAi Effectors siRNA shRNA • 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. The RNAi Consortium (TRC) • 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