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Final Report Summary - INTERDRUG (Epigenetic mechanisms underlying inter-individual differences in drug response and hepatic disease.)

Final summary report – InterDrug

1. Background.
The liver is a vital organ serving a variety of important physiological functions including blood sugar homeostasis and blood turnover. In addition, the liver is central for the metabolism of drugs and xenobiotic substances. Importantly, though the response to drugs differs considerably between individuals due to a combination of genetic and environmental factors, which can result in lack of efficacy or drug-induced liver injury (DILI).
Upon damage, the liver has a unique regenerative capacity that is tightly linked to the capability of hepatocytes to rapidly respond to various signaling molecules or changes in environment. Whereas hepatocytes are functionally highly specialized and do not divide under homeostatic conditions, upon liver damage, they transiently lose expression of key hepatic genes and dedifferentiate into fetal-like progenitor cells which enter the cell cycle to regenerate the damaged tissue.

2. Project scope.
In the project we analyzed the genetic basis of these inter-individual differences and developed tools to further our understanding of epigenetic contributors. Furthermore, we assessed the molecular frameworks that convey hepatocytes their unique regenerative capacity.

3. Results.
3.1 Genetic variability in pharmacogenes and their impact on drug response
In light of recent extensive population-scale sequencing efforts, we analyzed and quantified the genetic variability in genes with importance for drug absorption, distribution, metabolism and excretion (ADME). By integrating sequencing data from more than 6500 individuals, we found that important phase I and phase II enzymes as well as drug transporters were genetically highly variable1, 2. Importantly, the vast majority of these genetic variants (>90%) were rare with and we estimated that these rare variants account for 30-40% of the phenotypic variability in drug response. This data is of importance as it indicates the shortcomings of current pharmacogenetic tests and highlights the importance to include these rare variants in order to be able to understand clinically relevant genetic variation. Yet, multiple challenges on technical, interpretative and ethical levels need to be overcome to enable the reasonable dissemination of comprehensive pharmacogenetic genotyping into clinical practice and we provide a roadmap for its clinical implementation3, 4.

3.2 Novel tools for epigenetic analysis of hepatic gene expression
To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing5. Using this approach, we describe three types of variability of hepatic hmC profiles: (i) sample-specific variability, where the local hmC values correlate to the global hmC content of livers, (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene- and site-specific components of hmC variability might con- tribute to the epigenetic control of hepatic genes.

3.3 Comparison of available in vitro models for pharmaceutical and toxicological applications
To study drug response and its inter-individual variability in vitro, we compared multiple cell system in a series of multi-center studies in order to define a gold standard model that reflects in vivo phenotypes as closely as possible. Using a panel of 13 hepatotoxins, it was found that primary human hepatocytes (PHH) were the most sensitive cell model6. In addition, by employing a toxicogenomic approach, we found that their response to chemical insults closely recapitulated molecular changes observed patients experiencing adverse drug reactions7.

3.4 Molecular mechanisms of dedifferentiation and liver regeneration
However, when maintained in conventional 2D monolayer cultures, PHH dedifferentiate and lose hepatocyte-specific functions after few hours in culture, which significantly limits their utility for long-term functional studies. To understand the molecular basis of this dedifferentiation process, we analyzed the early events in proteomic and transcriptomic signatures in time course experiments with high temporal resolution8. Using an integrative bioinformatics approach, we found that massive changes in non-coding RNAs, including miRNAs occurred already after few hours in culture, which preceded the downregulation of hepatic genes. Importantly, inhibition of the miRNA machinery using small molecule inhibitors drastically delayed dedifferentiation and the loss of hepatic markers. Interestingly, as changes in miRNA patterns during dedifferentiation in culture resembled alterations reported during liver regeneration in vivo, this approach might provide a compelling experimental paradigm to study molecular events during liver regeneration in vitro.

3.5 Novel physiological 3D culture systems
To circumvent dedifferentiation and thus to develop a culture system in which cells exhibit physiological phenotypes, we developed an integrated 3D hepatic model system. Strikingly, in this system hepatocytes remained viable and functional for >5 weeks in chemically-defined, serum-free conditions9. Furthermore, the setup supports co-culture of hepatocytes with other hepatic cell types, such as Kupffer, stellate and biliary cells, thus capturing the full cellular repertoire of the intact liver. Furthermore, spheroid cultures derived from different donors retained their inter-individual variability. This experimental platform provides a versatile tool for a variety of pharmaceutical applications, such as determination of acute and chronic hepatotoxicity, pharmacokinetic analyses and hepatic target validations. Furthermore, we currently expand the technology to serve as a pathophysiologically relevant model system for a variety of liver diseases, including steatosis, cholestasis, fibrosis and hepatitis.

4. Socio-economic impact.
The InterDrug project delivered significant positive outcomes for society:
i) The comprehension that rare variants considerably contribute to inter-individual differences in drug response provides valuable new insights for the adoption of personalized medicine in clinical practice. Furthermore, in the frame of this project, this project contributed to the development of standardized guidelines for pharmacogenetic result reporting to facilitate acceptance by clinicians and to increase result transparency for prescribers10.
ii) A deeper understanding of the mechanisms regulating hepatic gene expression will contribute to the discovery of potential biomarkers for drug response.
iii) To make the novel 3D cell culture technology that we developed more accessible, we founded a company (HepaPredict AB) that already successfully deployed this platform to the pharmaceutical industry.

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