Presentation on theme: "Cellecta, Inc. Targets & Tools March 20/21, 2012 Cellecta, Inc. Targets & Tools March 20/21, 2012 Paul Diehl, Ph.D. Director of Business Development RNAi."— Presentation transcript:
Cellecta, Inc. Targets & Tools March 20/21, 2012 Cellecta, Inc. Targets & Tools March 20/21, 2012 Paul Diehl, Ph.D. Director of Business Development RNAi Genetic Screening for Drug Target Discovery
Cellecta Overview Founded: April 2006 Located: Mountain View, CA 13 NIH grants (10 direct SBIR and 3 sub-contractor) Network of collaborators (primarily cancer-related) Focus: Flexible and scalable broad-based screening and analysis assays to expedite the discovery and characterization of novel therapeutic targets and drugs. –Genome-scale RNAi-based genetic screening –High-throughput functional-peptide screening Also provide supporting services, including customized lentiviral-based shRNA libraries and related shRNA and lentiviral services
Main Applications for RNAi Screens Find and prioritize drug targets Understand disease progression and gene-disease associations Connect expression profiling data with functionality Analyze signal transduction pathways Characterize the mechanisms for compounds Find synergistic targets Research areas… Oncology and Cancer Biology Infectious Diseases Inflammation Other diseases with cell-culture model systems
RNAi Screening Formats 123 123 Target Cells Functional Assay Functional Assay / Selection Pooled Format shRNA Library shRNA constructs Arrayed Format Collection of siRNAs/shRNA Time and Resource Intense Assay to assay variability Fixed Routine Cell Culture One Assay All Results Flexible
shRNA Expression Library Construction Oligo Pool Synthesis –Agilent Arrays (unique) Defined complex pools High yield long oligos Very low mutation rate Cloning –Optimized Cloning High complexity >99% representation Packaging –Optimized Large Scale Maintain representation High Titer
Types of Screens Rescue Screens (Positive Selection): Find gene required to produce a response to added factors or compound, for example, genes necessary for trigging apoptosis or cell death in response to FAS, PUMA or other effectors. Viability Screens (Negative Selection): Identify essential genes in a specific population of cells (e.g., prostate, blood, mammary cancer cells) Selected Populations (e.g., FACS): Look for modulators of NF-κB, p53, c-myc, HSF-1, HIF-1α using fluorescent reporter cell line, or cells expressing specific Ab- detectable marker, such as specific receptors.
TGF-β Screen TGF-β is an extracellular cytokine that inhibits cell proliferation by arresting cell cycle progression and inducing apoptosis. Library: Pooled 27K shRNA library targeting 8,000 genes with 3-4 shRNAs per gene. Screening: In duplicate, 2,500,000 Hep3B cells (approximately 100- times the number of constructs in the library) were transduced at a multiplicity of infection (MOI) of 0.3.
TGF-β HT Sequencing Results The figure plots the abundance of each shRNA in each of the populations x-axis: after transduction y-axis: after selection If shRNA does not affect a critical genes for the response… shRNA disappears from population from apoptosis. If shRNA does target a critical gene for apoptosis signaling… shRNA is represented in the treated population (cell survives). shRNA to about 100 genes are significantly enriched shRNAs highly represented in the treated population presumably target genes involved in TGF-beta apoptosis signaling
TGF-β Findings Hits around PI3K and TGF-β signaling pathways Genes identified with the TGF-β screen starred Validation: 18 of 19 selected TGF-β hits confirmed to knockdown target and conferred TGF-β resistance. TGF R1 OASL ID3G6PD DTX1LTB4DH CASP1TRIM59 GABPB2MMP9 TCN1EYA2 MAFTGFB3 DMRTA1RHOBTB1 DAPSMAD4
shRNA Design ~70% of shRNA exhibit >70% knockdown
Optimized Vector Design Optimized, validated shRNA structure for pooled format screens shRNA-specific 18-nt bar-codes compatible with Illumina-Solexa HT sequencing High knockdown level by U6-constitutive or U6Tet-regulated expression of shRNAs RFP reporter for monitoring transduced cells Puro R for selection transduced cells
HT Sequencing of shRNA Libraries Good Library (DECIPHER) Poor Libraries ~90% inserts within 10-fold abundance range (i.e. narrow distribution) ~95% of shRNA constructs without mutations/deletions ~98% shRNA insert rate >20-30% of shRNA constructs are poorly represented (i.e. wide distribution) >30% of shRNA constructs have mutations/deletions Plasmid library 27K shRNAs Library QC … Virtually all shRNA inserts are present in relatively equal abundance Clones are randomly pulled and sequenced to check for mutation and insert rate
Viability Screen for Blood Neoplasia Biological Triplicates K562 Cells Cellecta Human 27K Library Targeting Signaling Pathway Genes
Viability Hits Associated with K562 Cells * Overlap with previous study: Luo B, et al. Highly parallel identification of essential genes in cancer cells. Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20380-5. * *
Validation of Predicted Essential Genes in K562 cells with Small Molecule Drugs R115777 ZM33637 PD198306 U0126 SX011 SB203580 SB302190 C401 NSC 625987 Top Canonical Pathway SIGNALLING TO RAS E2F REGULATION OF DNA REPLICATION PYRUVATE METABOLISM AND TCA CYCLE REGULATION OF RHEB GTPASE ACTIVITY BY AMPK ACTIVATION OF ATR IN RESPONSE TO REPLICATION STRESS MTOR SIGNALLING SIGNALLING TO ERKS SHC MEDIATED SIGNALLING GRB2 EVENTS IN EGFR SIGNALING SOS MEDIATED SIGNALLING SHC RELATED EVENTS G PROTEIN BETA GAMMA SIGNALLING GLUCOSE REGULATION OF INSULIN SECRETION
Viability Gene Signatures in Cancer Cells K562 HL60 BJ-TERT Jurkat Raji HeLa DU145 A549 H1437 P-value 01 27K Decipher module 1 General Viability Genotype-specific Disease-specific
Mouse Rescue from FAS Hepatitis 6 Mice injected with siRNAs targeting mouse paralogs to targets found with FAS screen: CCR4, APCS, WASF1, HNRNP, CASR, DEGS. siRNA to BCME was used as a control. At 72 hours after siRNA, mice were injected with FAS Jo2 antibody (induces fulminant hepatic failure) siRNA Protects Mice from Fas-induced Fulminant Hepatic Failure 3 siRNA pools single siRNAs
Small Molecule Inhibitors 30 targets selected with known inhibitory pharmaceuticals and tested in HeLa, A549 and HT1080 cells. The 3 best candidates from in vitro screen were followed up in vivo. (i.e., good dosages, low toxicity, correct tissue specificity) Protection Concentrations for FAS-Induced Hela Cells BIM-peptide
Small Molecules in Mice N-Ethylmaleimide (NEM) KCNQ inhibitor BIM-peptide SSTR5 inhibitor Cantharidine PP2A inhibitor Protection from Acute Fulminant Hepatitis Intraperitoneal dose with of selected compounds (PBS for controls) Lethal challenge with 0.6 µg/g of anti-Fas Jo2 antibody (kills 100% of mice within 20 h)
Cellecta, Inc. 320 Logue Ave. Mountain View, CA, USA 1-877-938-3910 email@example.com www.cellecta.com Cellecta, Inc. 320 Logue Ave. Mountain View, CA, USA 1-877-938-3910 firstname.lastname@example.org www.cellecta.com Thank You! Questions? Paul Diehl Director of Business Development Tel: 650-938-4050 E-mail: email@example.com Paul Diehl Director of Business Development Tel: 650-938-4050 E-mail: firstname.lastname@example.org
Your consent to our cookies if you continue to use this website.