Case studies: Applying CRISPR to the drug discovery pipeline

CRISPR-Cas9 is being used in a variety of assays to increase accuracy and speed in drug discovery and development. 

The CRISPR revolution has fueled a rapid expansion of the use of gene-edited cells in a variety of drug development assays. CRISPR-Cas9 gene editing has increased the accuracy and speed of the pre-clinical drug development process. This technology has allowed precise target modulation for better hits, enhanced assays for improved disease recapitulation and the ability to respond rapidly to clinical outcomes ^1,2.

Overview

• Measuring the CRISPR effect
• Case Study 1: HTS of next-generation drug candidates with CRISPR
• Case Study 2: Gene target ID via CRISPR-edited iPSC differentiation
• Case Study 3: Testing novel oncology targets with CRISPR-edited cells

Measuring the CRISPR effect

Three key elements are required for success with functional genomic screening:

• The ability to specifically perturb gene function
• An assay to measure phenotypic responses
• A mechanism to couple the observations together for gene identification

Each of these elements must be tailored to the biology being studied ¹. CRISPR offers precisely targeted gene-editing, allowing full gene knockout, targeted gene knock-in or the activation or partial inhibition of genes through CRISPRa/i. Selecting the right assay to measure cellular responses to gene perturbation and any compounds to treat such disease states is vital ². This ensures the right data is being collected and that any interpretations being made regarding gene function or hit ID from these assays are accurate.

CRISPR-edited cells were first applied to cancer biology. For these studies, knockout cell line responses to a simple cell survival assay were sufficient to predict drug responses in tumors over-expressing potential oncolytic genes ¹. However, for more complex diseases or to present a complete picture of cell behavior for accurate hit ID, measurements such as cell health may not offer the level of information needed. In such cases, more advanced phenotypic analyses must be coupled to CRISPR-based screening efforts.

CRISPR is now being used in a range of assays, both in vitro and in vivo, throughout the preclinical phase to deliver improved results and speed processes. Research has shown that the most commonly measured cellular endpoint in CRISPR screening studies was reporter assays, followed by cell proliferation and protein secretion. Other desirable endpoints measured in assays using CRISPR-edited cells were apoptosis, cell viability (membrane integrity), high content imaging for phenotypic changes and cell cycle analysis ³. These assays are typically performed using the CRISPR-edited cell line alongside the parental wild-type (WT) cell line for the comparison of any response ².

The following case studies show how CRISPR has been applied to increase accuracy and speed the drug discovery process, providing answers that were unavailable with previous technologies.

Case study 1: High throughput screening of next-generation drug candidates with CRISPR

Gefitinb and erlotinib target the epidermal growth factor receptor (EGFR) in the treatment of non-small cell lung cancer  ⁴. Following observed patient resistance to these treatments, it was determined that the T790M oncogenic mutation in EGFR was responsible for this effect, preventing the drugs from interacting with the EGFR tyrosine kinase domain target site ^5,6. Using structure-based design Astra Zeneca developed osimertinib to target the T790M mutation. However, in clinical trials resistance was again observed from a second acquired mutation in EGFR, C797S ⁷.  

Chemical structures of gefitinib, erlotinib and osimertinib. As patient resistance was acquired to each drug CRISPR engineering helped speed the identification of new next-generation therapeutics to treat NSCLC patients.

To avoid unnecessary development costs and allow rapid progress in this field, they looked for a way to quickly feed this information back into their pre-clinical drug discovery program. Using CRISPR-Cas9 the exact C797S mutation was created in cancer cell lines for use in vitro and in vivo. Application of the relevant C797S cell line model enabled drug screening to identify next-generation compounds targeting this new mutation. This patient in a dish strategy was used to expose the CRISPR-edited cell line model to candidate drug compounds in screening and the results were analyzed via high throughput ELISAs for the identification of precision medicines ⁷.

Case study 2: Gene target ID via CRISPR-edited iPSC differentiation

Macrophages are part of the innate immune response and change their physiology and cytokine profile to promote inflammation and tissue repair following injury. In patients with chronic obstructive pulmonary disease (COPD), there is a decreased anti-inflammatory macrophage activity in lung cells ⁸.

Salt-induced kinase (SIK) has been proposed as a drug target for COPD. Studies from Merck showed that inhibition of this target changes the macrophage cytokine profile to promote anti-inflammatory responses, with an increase in IL-10 and inhibition of TNFα ⁹.

SIK is widely expressed in the body as three different isoforms. To determine the isoform responsible for COPD the researchers turned to CRISPR-Cas9. Using CRISPR they generated biallelic point mutations of the different kinase isoforms in induced pluripotent stem cells (iPSC). These CRISPR iPSC cell lines with different combinations of kinase activity knocked out, were analyzed by differentiating through monocytes to macrophages. Experiments showed that the human iPSC-derived macrophages yield very similar cytokine profiles to human primary monocyte-derived macrophages, allowing these CRISPR-edited iPSC cell lines to serve as appropriate experimental model systems ¹⁰.

Using this system one combination of SIK isoforms was observed to give the desired reduction in IL-10 protein fold change in the cell activity assay when the cells were challenged with lipopolysaccharide ¹⁰. The results allowed the researchers to identify the right target and progress from hypothesis to human proof-of-concept.

Case study 3: Testing novel oncology targets with CRISPR-edited cells

In 2014, two research publications highlighted MutT Homolog 1 (MTH1) as a potential new target for oncology based on RNAi knockdown results ^{11, 12}. Following this, researchers at Astra Zeneca sought to repeat and extend this target validation work ¹³. Using CRISPR-Cas9 they introduced indels into exon 2 of the MTH1 gene to create MTH1 knockout cell lines, using western blots were used to show full protein knockdown. The resulting KO cell lines showed equivalent growth to the WT ¹³. Further treatment of the MTH1 KO cells with specific inhibitors showed no difference in the cell viability of the KO and WT cell lines ¹³. Using this CRISPR approach, the target was devalidated and resource saved pursuing false targets.

The precision offered by CRISPR-Cas9 has brought increased accuracy to speed drug discovery and development, and as this technology is increasingly incorporated into workflows in a variety of assay designs we can hope to see improved results through the preclinical drug pipeline.

References

1. Lawo S and le Sage C. How Phenotypic CRISPR Screening is Revolutionising Drug Discovery. DDW. Fall: 53-58. (2018)
2. Scott A. How CRISPR is transforming drug discovery. Nature 555, S10-S11 (2018)
3. Comley J. CRISPR Cas9: Transforming Gene Editing in Drug Discovery Labs. DDW. 17(4):33-48 (2016)
4. Nurwidya F, Takahashi F, Takahashi K. Gefitinib in the treatment of nonsmall cell lung cancer with activating epidermal growth factor receptor mutation. J Nat Sci Biol Med. 7(2): 119–123 (2016)
5. Arcila ME, Oxnard GR, Nafa K, Riely GJ, Solomon SB, Zakowski MF,  et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clin Cancer Res. 17(5):1169-80. (2011)
6. Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB, Fidias P,  et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 3(75):75ra26 (2011)
7. Thress KS, Paweletz CP, Felip E, Chul Cho B, Stetson D, Dougherty B,  et al. Acquired EGFR C797S mediates resistance to AZD9291 in advanced non-small cell lung cancer harboring EGFR T790M. 21(6): 560–562. (2015)
8. Barnes PJ. Inflammatory mechanisms in patients with chronic obstructive pulmonary disease. J Allergyl Clin Immunol. 138(1): 16-27 (2016)
9. Clark K, MacKenzie KF, Petkevicius K, Kristariyanto Y, Zhang J, Geun Choi H, et al. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages. Proc Nat Acad Sci. 109(42):16986-16991 (2012)
10. Mayr L. CRISPR/Cas9 in drug discovery: Applications in target discovery, validation, and hit screening. Science [Accessed: 27/-7/2020] (2016)
11. Ged H, Koolmeister T, Jemth AS, Eshtaf S, Jacques SA, Strӧm CE,  et al. MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. Nature. 508:215-221 (2014)
12. Huber KVM, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A,  et al. Stereospecific targeting of MTH1 by (S)-crizotinib as an anti-cancer strategy. Nature 508:222–227 (2014)
13. Kettle JG, ALwan H, Bista M, Breed J, Davies BL, Eckersley K, et al. Potent and Selective Inhibitors of MTH1 Probe Its Role in Cancer Cell Survival. J Med Chem. 59(6):2346-61 (2016)