Studying gene expression: An improved - PowerPoint Presentation

Studying gene expression: An improved high-content RNAi screening technologyDiscovering more, faster, cost-effectively Dr Asongwe L. A. Tantoh

IntroductionThis presentation is intended to give an overview on:a revolutionised high-content/throughput screening (HCS/HTS) platform brief history on HCS/HTS inception the different HTS platforms and their limitations comparison between the different HTS platforms our technology description and proof of concept of the platform validation data from our high density microarray platform other potential application(s) of the platform the long term goal(s) behind the technology

Revolutionary events in functional genomicsWhere it all started Discovery of the RNAi mechanism (  Drs Andrew Fire and Craig Mello:2006 ) RNAi mechanism 1 The human genome Sequencing of the Human genome ( 2000) 2

RNADNA DNA replication RNA Transcription Reverse transcription Physiology Physiology Folded with function Protein Translation Protein siRNA RNAi mechanism Central Dogma of Molecular Biology RNAi mechanism

Human genome sequencingsiRNAmiRNAshRNA CRISPR/Cas9; used for genome editing Vast number of small molecule libraries encoding for the human genome Enable our understanding of how genes are regulated; d uring biological processes d uring pathogen-host interaction

How do we screen through hundreds of thousands of chemical libraries with relative ease and in a short time?

96 well plate (100 µl) 1536 well plate (4µL ) Well plate formats in high-density screening Traditional HT screening format: Limitations e xpensive  automated liquid handling devices required f ully automated integrated systems h uge infrastructure h ighly trained workers required/ Experienced scientist c ost ineffective 384 well plate(25µl )

Conventional HT arrayers - like the one to the left: are slow : 100 arrays in 17 hours - pins are washed, blotted and carry only 100nL reservoir are 95% wasteful of library print mixes mix viscosity leads to poor repeat prints (10-20 spots in one pass maximum ) use print pins that are reused to print the array p rint pins are expensiveCan not scale up Conventional HT microarray format: Limitations

cost effective e asy to operate we measured printing quality using a single continuous print of siRNA samples o ur arrayer prints >95% spots in one contact prints 500-750 essentially identical arrays in 1.5 hours with 2 FTE CSIR arrayer and its performance

low volume very expensive (750$ each ) not suited to transfection mix jams very slow wasteful of mix Our approach capillary print element conventional approach The microarrayer pin used in current technology is not a solution for HCS Our array technology

Why capillaries?c apillaries deposit accurate spots t he reservoir volume of a capillary is 40X greater than a print pin: 4000nL Vs. 100nL c apillaries don’t need refilling or washing t hey fill automatically by capillary action t hey exist in different sizes (from 150 to 600µm) i nexpensive t hey are readily commercially available

Our platform: How we print Encapsulated siRNAs Step 1: load the filler plate with encapsulated siRNAs Step 2: capillary loading Step 3: stack the printing plates in the print head : assembling the print array Step 4: contact print >1000 arrays 1 2 3 4 entire array 3 150 spots printed in one contact 2.5 Sec scale: thousand plus copies

array one shot 3 150 spots/2.5Sec 24 speed 1array in 2.5 Sec 1 array/hour output 1000-1500 arrays 100 arrays time 1.5 hours 15 hours cost <20 000$ >200 000$ waste 20% 95% missing spots <5% 6-10% precision 25µm similar ease of use **** * CSIR arrayer Conventional arrayer CSIR arrayer vs conventional arrayer

Our microarray plate vs. 96, 384, 1 536 well platesNumber of 384 well platesrequired/Persomics plateVol/well= 25ul (384 well plate ) Number of 96 well plates required/Persomics plate Vol/well= 100ul (96 well plate) 2.05 Number of 1536 well plates required/Persomics plate Vol/well= 4ul (1536 well plate) High - density microarray plate comprising of 3150 siRNA spots 8.2 32.8 High - density well plate s

15 R educing variation less variation HDS-plate 3 150 individual experiments 96-well plates required

16 Ease of use Less handling HDS-plate 3 150 individual experiments 96-well plates required

Our research

Capillary printedsiRNA spot B C 400 350 300 250 200 150 100 50 0 Spot diameter ( μm ) Capillary contact printing of encapsulated siRNA Capillary contact printed optically addressable siRNA spot (A), Confocal image, average diameter (B) and Spot width distribution (C). 400 350 300 Spot diameter ( μm ) 35 25 15 5 Frequency (%) A siRNA spot characterization

siRNA spot cell cultivation silencing siRNA spot each spot contains an siRNA targeting a specific protein each spot silences production of one protein T he HCS array principle

Suppresses NF-kB/p65 expression p65 siRNA spots p65 siRNA control siRNA > 70% knockdown in p65 expression Creating wells without walls Suppression of NF- κB /p65 expression p65 siRNA Verification of gene silencing m ean intensity/pixel .10 3 Control p56 siRNA

Creating wells without walls Control siRNA spot Incenp siRNA spot cells with multiple nuclei Arrest of cytokinesis by INCENP siRNA Verification of gene silencing

Array data validation

TNF induced p65 translocation + TNF Assay conditions designed to give maximal p65 transport into the nucleus p65 was detected in HeLa cells using indirect immuno-labelling after TNF stimulation unstimulated control cells translocation into the nucleus

Arrays printed containing printed siRNA covering: all kinases, phosphatases, ubiquitinylases , proteases printed controls: control, XPO1, & p65 siRNA control siRNA is a non targeting siRNA silencing no genes XPO1 is the nuclear export factor for p65 & drives its recycling back out of the nucleus p65 siRNA directly silences p65 these controls enable understanding of the array quality in screening & XPO1 /p65 siRNA will give different phenotype to control XPO1 will accumulate p65 in the nucleus p65 will reduce p65 levels in the cells Arrays comprise 3 150 individual siRNA spots Array based screening

Knowledge directed pathway exploration we used two genes known to influence p65 nuclear transport to define where to find hits CHUK IKBKB both are kinases regulating signalling from TNF => p65/ NFkB loss of these kinases stop nuclear import of p65 both are in the printed library, as single spots on each array 32 replicates enable robust screening controls are well defined Principal Component analysis of the data gives excellent separation of controls, toxic siRNA and CHUK/IKBKB

Basis used for hit selection

AGenes connectivity results using pathway studio > 50% connectivity Novel therapeutic targets Expressed phenotypes XPO1 Non-targeting RELA IKBKB CHUK B

Summary w e screened the TNF alpha pathway at the highest density ever performed (to our knowledge) 100 800 individual siRNA cell based experiments were performed in 4 days, with 2 extra days for analysis high replicate, highly robust data using conventional technology, this would require 3 months CSIR/Persomics array technology...applied to cell screening

Commercialised platform Boston Persomics array printing lab Persomics preparation lab

Arrays, opportunities

siRNA is the most challenging format for HCS arrays we want to move on to: compound arrays novel array architecture c ompound arrays have great potential s ince Bailey showed the principle in 2004, we are not aware of any uptake of the method Beyond siRNA

compound arrays have not been adopted because: technical complexity of making them with arrayers CSIR/Persomics arrayer was designed for mass production of compound arrays C ompound arrays

Proof of conceptPAO spotPAO spot 2004 PNAS POLYMER DRUG CELLS ON DRUGS Bailey’s result Bailey’s printing approach Layer printing

Single spot - localized drug effectSuperior production method and higher effect and reproducibility DRUG Cells on drug drug impregnated in polymer POLYMER CSIR compound printing approach Phenylarsine oxide (PAO: toxic) + Ctrl spots PAO spot PAO spot Bailey’s result reproduced at CSIR

Conclusion CREATING A MINIATURIZED HIGH THROUGHPUT SCREENING PLATFORM 3150 experiments on one plate MINIATURIZATION

To offer South African and African researchers a high throughput screening product that is;miniaturi s ed and standardi s ed for screening commoditis e the technology: make it portable, easy to manufacture easy to use ‘out of the box ’exceed current technology standards create outstanding products Conclusion: Our Goals

Conclusion: Our GoalsTo enable researchers access to rapidly test thousands of molecules in various bioassays in an easy and timeous manner; i.e. screening through hundreds of; s mall interfering RNAs ( siRNAs , miRNA,shRNA ) S mall -m olecule compound drug Repurposing (alternative uses): FDA approved Drugs “ cDNAs” Using the current platform to screen for biological targets that will serve as lead s chemicals for new therapeutic drugs

Thank you for your attentionTeam Members:Dr Dalu MancamaDr Lindiwe Thete Dr Natasha Kolesnikova Dr Alex Alexandre Dr Hazel Mufhandu Dr Asongwe Tantoh