Sue Pearson Ph.D. Freelance Writer GEN
New Tech Aids Drug Discovery
“Now is the best time to be in drug discovery because of the technical capabilities we have, which we simply didn’t have just five years ago” states Steve Rees, vice president, Discovery Biology, AstraZeneca at the recent ELRIG conference in Liverpool, UK. Rees qualified this statement by adding, “With new sequencing platforms, we can sequence any genome for $800 and by 2025, it is likely that the field may have sequenced 20 million genomes—so by then we may never need to sequence another one to understand the genetic basis of disease. Additionally, the advent of CRISPR means we can manipulate any gene or target, and that’s exciting.”
One new high-throughput screening method, which showcases what can be achieved when CRISPR is combined with sequencing techniques, was described at ELRIG by Paul Datlinger, a Ph.D. student at the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences. He detailed how his group has developed CRISPR droplet sequencing (CROP-seq), a method using pooled CRISPR/Cas9 screens combined with single-cell transcriptome sequencing.
Datlinger states, “Pooled genetic screens can edit cells in bulk, but are limited to a simple readout, such as cell survival. Arrayed screens provide more specific data, but they are difficult to do and are not very reproducible because each sample must be processed separately. Also, both pooled and arrayed screens cannot resolve the effects of a genetic perturbation at single-cell resolution, whereas CROP-seq can reveal the effects of a gene knockout in different cell types or developmental stages.”
CROP-seq involves using droplet-based single-cell RNA sequencing (RNA-seq) with specifically engineered guide RNA (gRNA) lentiviral vectors that include a gRNA cassette within a 3′ long terminal repeat, which is duplicated during viral integration. This expresses an RNA polymerase III transcript for genome editing and a polyadenylated RNA polymerase II transcript detected by single-cell RNA-seq to assign single-cell transcriptomes to unique gRNAs.
Datlinger described how CROP-seq was used to screen for T-cell receptor (TCR) pathway induction and showed that in anti-CD3/CD28-stimulated cells, gRNAs for target genes immediately downstream of the TCR (such as LCK and ZAP70 kinases and the adapter protein LAT) had a strong negative effect on TCR activation.
Datlinger concluded, “By combining guided-RNA expression with transcriptome responses in thousands of single cells, we have the benefits of both pooled and arrayed screens in a simple to automate workflow. Using the 10x Genomics Chromium™ System, we are able to process 80/000 cells per hour, and if we combine this with the Illumina NovaSeq®, we can screen gRNA libraries for entire gene families.”
Boosting Western Blots
Another interesting assay screening technique for protein profiling was presented by Gerrit Erdmann, Ph.D., lab head, at German-based contract research organization (CRO) NMI TT Pharmaservices. Erdmann says: “Protein profiling via Western blotting is too low-content. You can get higher content using mass spec, but this requires large sample amounts, expertise, and equipment. To boost the output of a Western blot, we have combined the Luminex bead-based multiplexing with Western blotting into a technique called DigiWest®, which enables us to analyze up to 800 total and phospho proteins using as little as 10–50 µg of total protein sample.”
In the DigiWest® method, proteins on a Western blot are biotinylated and each sample lane is cut into strips. The strips are placed in wells of a 96-well plate and bound proteins are solubilized. Then Neutravidin-coated Luminex beads are added to each well to immobilize the biotinylated proteins on the bead surfaces. A small aliquot of the bead-set is used in a direct immunoassay, and the sample is read on a Luminex FlexMAP 3D flow cytometer.
Erdmann presented a case study of how the DigiWest® method was used to detect biomarkers for resistance to platinum-based chemotherapy in ovarian cancer patients. He showed that from a set of 24 tumor specimens of relapsed versus sensitive platinum-treated ovarian cancer patients analyzed using 478 antibodies (total and phospho proteins), the DigiWest® protein signatures distinguished platinum-resistant versus sensitive patients, and revealed eight strong biomarker candidates.
According to Rees, there is a need for good human cell-based assays in preclinical drug discovery to reduce the use of animal testing. He states: “We are moving away from 2D cell culture and much more towards 3D cell culture and the organ-on-a-chip model. Currently, there are good lung, liver, and kidney models, but we need the body-on-a-chip to determine how a compound will interact with all the organs.”
Interesting work on one promising kidney model produced using the OrganoPlate® microfluidic 3D culture plates was presented by Linda Gijzen, scientist at Mimetas, a biotech firm based in the Netherlands.
OrganoPlates, based on the standard 384-well plate format, were seeded with 40 renal proximal tubule boundaries from Renal Proximal Tubule Epithelial Cells (RPTEC). These were cultured against an Extracellular Matrix (ECM) using Phaseguides™, a liquid-handling technology where no physical barriers are present between the lanes in the OrganoPlate, allowing co-cultured cells in adjacent lanes to interact and migrate freely. After the RPTECs adhered to the ECM, media perfusion was begun to mimic fluid flow, exchange of nutrients, oxygen, and metabolites. The cells formed a monolayer and then tube-like structures in the perfusion channel. Gijzen states: “Growing cells in a 3D tubular structure allows phase contrast or confocal imaging to assess epithelial barrier integrity and function.”
To demonstrate barrier integrity, Gijzen showed that when a medium containing a fluorescent dye was added to the RPTECs, the dye remained in the lumen of the RPTEC tubules and when a toxicant, Amphotericin B was added to the RPTECs, the fluorescent dye diffused out of the lumen into the ECM.
Gijzen also presented examples of how the RPTEC tubules could be used in epithelial transport assays. RPTEC tubules exposed to a medium containing a fluorescent dye and cyclosporine-A, transporter prevented efflux of calcein-AM out of the tubule and RPTEC tubules exposed to a medium containing a fluorescent dye and phlorizin, inhibitor prevented influx of a Glucose Analog 6 NBDG into the tubule measured by analyzing the fluorescent signal at the RPTEC/ECM interface. Gijzen adds: “Renal cells can be easily cultured in OrganoPlates to mimic a fully functional proximal kidney tubule enabling biology labs all the way up to high-throughput screening facilities to perform toxicity and transport studies by real-time imaging.”
Moving more towards the body-on-a-chip model, Olivier Frey, Ph.D., head of technology platforms a at Swiss-based, in vitro testing company InSphero, described an application of its 3D InSight™ Human Liver Microtissues in combination with microfluidics to determine how a compound would interact with tumor cells after it had been metabolized by the liver.
Frey explains: “With our 3D InSight Human Liver Microtissues we have cultured liver and tumor spheroids for eight days in different wells of our GravityFLOW microfluidic assay plate interconnected by a perfusion channel. To allow media perfusion between the liver and tumor spheroids the plate is tilted back and forth on an automated GravityFLOW platform inside an incubator and we have optimized the flow and set up so that there is minimal dead volume or bubbles which could cause problems with an assay.”
To demonstrate how this liver-tumor model could be used, Frey presented cell imaging and mass spectroscopy data, eight days after the pro-drug cyclophosphamide was added to perfused tumor spheroids only and to tumor spheroids linked to liver spheroids via a perfusion channel. The imaging results showed that the tumor size did not increase significantly in the wells where the tumor spheroids were linked to liver spheroids, yet it did grow in the wells containing tumor spheroids only. The MS data revealed that the liver spheroids successfully transformed cyclophosphamide to its active metabolite on the chip.
Frey summarizes: “Assessing liver-mediated pro-drug activation is a first important step that nicely illustrates the potential of the microfluidic multi-tissue platform. A broad range of our 3D InSight Microtissues, which are produced off chip from various cell types, can be loaded and differently combined in the perfusion system. This approach fully exploits the functionality of the spheroid model and opens the door to many advanced in vitro preclinical testing assays in a scalable body-on-a-chip automated plate format.”
Miniaturizing Drug Discovery
Alongside new developments in gene editing and cell-based assays, sample management and compound screening is also undergoing revolutionary changes. Rees states: “Working with LabCyte and Brooks, we have created the acoustic tube to miniaturize compound management. The use of these tubes, which store just 75µl of compound, with acoustic liquid handling systems such as the LabCyte Echo allows us to dispense 25nl of compound for screening. In addition, the flexibility created allows us to cherry-pick 80,000 compounds a day from our compound collection for screening.”
According to Rees, this has led AstraZeneca to look at other ways of improving high-throughput screening and has resulted in working in partnership with Waters and LabCyte to help them develop an acoustic mass spectrometry system. Rees comments: “Acoustic mass spec enables a biochemical screen, which can directly measure substrate to product conversion and significantly reduces the assay development cycle time. This technique reduces the cost of screening by more than 75% and results in an increase in assay quality. This technique is truly enabling and is changing the way we work.” Rees concludes, “Over the past decade, when you walked into compound screening labs, they have not really changed—but with the advent of so much new technology, when you go into a compound-screening facility in 10 years’ time, I predict it will look totally different.”