Validating CRISPR-engineered human pluripotent stem cell-derived cardiomyocytes as tools to assist with toxicity profiling, adverse drug reaction notices and black box warnings

Project background

Cardiotoxicity, arising from cardiac cell death or altered electrophysiology, is a major cause of drugs not progressing out of clinical trials. This poses a significant burden to the pharmaceutical industry. Current approaches to assess the cardiotoxic potential of compounds require large numbers of animals, primarily rodents and dogs, to generate primary cells and tissues for assays or as whole animal models. However, animal models of cardiotoxicity are poorly predictive of the human response due to substantial species differences, such as different heart rates and electrocardiogram durations, making translation of the results into humans difficult. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from healthy individuals are becoming established in better evaluating the impact of drugs and genetic disease on cell structure and function, but only a few disease models are available.

With previous NC3Rs funding, Professor Denning and his team developed 17 isogenic sets of hiPSC-CMs with patient-relevant mutations in genes associated with hypertrophic cardiomyopathy (HCM)1-5. This prevalent genetic disease of the heart can cause sudden cardiac death at a young age. Detailed phenotyping of the hiPSC-CMs produced showed altered force and arrhythmogenesis, and possibility for partial drug-based rescue, allowing evaluation of how genotype-drug interaction affects cell structure, function and viability. 

Why we funded it

This Technologies to Tools grant aims to replace the animals used in some cardiotoxicity studies, by developing an in vitro hiPSC-CM assay using healthy and HCM cells.  

Working with the Medicines Discovery Catapult, Professor Denning will further characterise the utility of hiPSC-CMs in predicting drug safety/toxicity against a larger set of compounds, whether this toxicity profile changes in healthy or diseased context, and whether administering more than one drug affects safety. The aim is to develop tools and robust assay technologies to assist in assigning whether a drug is cardiotoxic or poses enough of a health risk to carry an adverse drug reaction warning or a black box notice indicating the drug could pose a serious hazard to health.

Prior to receiving regulatory approval, every drug must progress through safety testing to assess cardiac electrical and structural toxicity. For cardiotoxicity testing, clinical research organisations and/or pharmaceutical companies typically assess each compound using primary cells (~10 animal hearts per 384-well plate to assess two to six drugs), ex vivo studies (~4 hearts per drug) and/or in vivo studies (24 mice and 24 dogs per drug). This equated to over 350,000 animals used for the 6,000 compounds at the preclinical stage of development in 2015 (The Pharmaceutical Industry and Global Health: Facts and Figures 2017).

The hiPSC-CMs could also have replacement potential in academia, where animal models are used for HCM modelling. Professor Denning estimates, based on work in laboratories of two of his collaborators, that 50-100 adult mice are used per year per lab. The methods used to induce HCM can be highly invasive, such as surgical interventions to constrict the ascending aorta, and are classified as “severe” under the Animals (Scientific Procedures) Act 1986. Based on literature and the experience of collaborators, validation of a human model of HCM has the potential to reduce this animal use by up to 50%, equating to a replacement of 50-100 animals per year in these two labs alone.

Research methods

Two healthy (wildtype) hiPSC-CM lines and their respective isogenic mutant heterozygote counterpart (HCM disease) lines will be used in this study. These will be developed into three formats with increasing complexity, from 2D monolayers to 3D co-cultures. Each format will be analysed using the MUSCLEMOTION algorithm, developed by the researchers to calculate contractility and identify arrhythmias (Sala et al., 2018). Drugs, from four categories 1) Cardio-safe; 2) cardio-toxic; 3) contraindicated by a secondary drug; 4) contraindicated by impaired heart function, will be provided by the MDC and screened in the model at human relevant concentrations. This study will determine whether healthy and mutant isogenic sets of hiPSC-CMs can unveil differential drug effects without using genetically engineered mice. Furthermore, the transition of the hiPSC-CMs from Nottingham to the MDC and the associated 2D and 3D screens will be achieved during this project.  

References

  1. Mosqueira D, et al. (2018). CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy. Eur Heart J. 39(43):3879-3892. doi: 10.1093/eurheartj/ehy249.
  2. Sala L, et al. (2018). MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo. Circ Res. 122(3):e5-e16. doi: 10.1161/CIRCRESAHA.117.312067.
  3. Kondrashov A, et al. (2018). Simplified Footprint-Free Cas9/CRISPR Editing of Cardiac-Associated Genes in Human Pluripotent Stem Cells. Stem Cells Dev. 27(6):391-404. doi: 10.1089/scd.2017.0268.
  4. Smith JGW, et al. (2018). Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits. Stem Cell Reports. 11(5):1226-1243. doi: 10.1016/j.stemcr.2018.10.006.
  5. Lam CK and Wu JC (2018) Disease modelling and drug discovery for hypertrophic cardiomyopathy using pluripotent stem cells: how far have we come? Eur Heart J. 39(43):3893-3895. doi: 10.1093/eurheartj/ehy388.
  6. The Pharmaceutical Industry and Global Health: Facts and Figures 2017 (accessed September 2019) https://www.ifpma.org/wp-content/uploads/2017/02/IFPMA-Facts-And-Figures-2017.pdf
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Technologies to Tools grant

Status:

In progress

Principal investigator

Professor Chris Denning

Institution

University of Nottingham

Co-Investigator

Medicines Discovery Catapult

Grant reference number

NC/T2T0119

Award date:

Aug 2019 - Jul 2020

Grant amount

£49,996