Human pluripotent stem cell cardiomyocytes and hepatocytes with engineered genotypes for drug safety evaluation

Lead Research Organisation: University of Nottingham
Department Name: School of Medicine

Abstract

An essential part of drug development by pharmaceutical companies is safety assessment before the drugs are given to the general patient population. Current regulation necessitates that much of this testing is done in cells or organs derived from animals, or in living animals, which primarily comprise mice, rats, dogs and monkeys. New EU regulations on toxicity testing (known as Registration, Evaluation, Authorisation and restriction of Chemicals or 'REACH') mean that up to 54 million animals will be needed over the next 10 years to evaluate 30,000 compounds, which equates to 1800 animals per drug. Even with this level of animal use, published information suggests that preclinical testing is only 71% predictive of whether a drug will be toxic in humans. Poor predictability is due to species differences, where the physiology of humans is very different to the various animals used for the tests. In addition, current systems cannot mirror the genetic diversity of human populations, whereby one person can tolerate a very high dose of drug, while someone else will have an adverse reaction and end up in hospital. Indeed, adverse drug reactions account for 100,000 deaths per year in the US alone and this is mainly due to problems that develop with the person's heart or liver.

To try to reduce or replace animal use and to increase predictability to human toxicity, this proposal will form a new collaboration between scientists with skills in genetic engineering (Skarnes, Rosen; Wellcome Trust Sanger Centre), human pluripotent stem cell biology and robotic automation (hPSC; Denning, Young; University of Nottingham) and toxicity testing (Goldring, Park; University of Liverpool). The aim is to genetically engineer human stem cells that are healthy except for the newly-added mutation, which has been selected because it is believed to cause susceptibility or resistance to certain drugs. In the lab, we will then coax the stem cells into becoming liver or heart cells. By investigating the function and survival of these cells after treatment with a range of drugs, we will be able to quantitate the amount of positive or negative change associated with a particular mutation. This has not previously been possible because human heart or liver cells with these mutations have not been available, or because the background genetics vary so much from one patient to another that accurately determining the impact of a single mutation on drug response is difficult. Keeping background genetics the same but varying the mutation overcomes these problems and will provide the pharmaceutical industry with a new tool to evaluate drug response in healthy cells and those carrying mutations that confer drug susceptibility or resistance. This will not only reduce animal use but also increase the safety of drugs taken by people, including those who are predisposed to adverse drug reactions. If this technology only led to a 0.1% reduction in animal-based testing, it would have the potential to save up to 54,000 animals in Europe alone.

Technical Summary

Safety assessment of each new drug developed requires up to 1800 animals, which typically comprise non-rodent species (monkeys, dogs) and rodents (rats and mice). New EU regulations on toxicity testing (termed 'REACH') will use up to 54 million animals over the next 10 years to evaluate 30,000 compounds. Even with this level of animal use, preclinical assays are cited as only 71% predictive of whether a drug will be toxic in humans. Poor predictability is due to species differences and the inability to develop test platforms that mirror diverse human genotypes, particularly those associated with the high drug susceptibility seen in cardio- and hepato-toxicity. Adverse drug reactions account for 100,000 deaths per year in the US alone. To facilitate reduction and replacement of animal use, and to increase predictability to human toxicity, this proposal brings together a new consortium with skills in genome engineering (Skarnes, Rosen; Sanger Centre), human pluripotent stem cell biology & robotic automation (hPSC; Denning, Young; Nottingham) and toxicity testing (Goldring, Park; Liverpool). The aim is to engineer different patient-relevant mutations associated with drug susceptibility or resistance into the protein coding regions of otherwise genetically healthy hPSCs. Differentiating these cells will produce cardiomyocytes and hepatocytes that carry specific mutations within a common genetic background, allowing unbiased evaluation of how genotype-drug interaction affects cell structure, function and viability. Co-culturing cardiomyocytes with hepatocytes of different genotypes will allow the impact of altered hepatocyte function on cardiomyocytes to be assessed. Comparing these results with data from the literature and industrial partners will allow the predictive value of this humanised in vitro model to be determined. A 0.1% reduction in animal-based in vitro, ex vivo and in vivo tests has the potential to save up to 54,000 animals in Europe alone.

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