2019

Grants awarded to 12 projects in 2019

A total of 2,3 million SEK has been awarded in grants from the Swedish Fund for Research without Animal Experiments in 2019, divided between 12 projects. See recipients and project titles below.

Gunnar Cedersund, Linköping University, 250 000 SEK
Project: Knowledge-driven drug development without animal experiments
In the last few years, mathematical modelling has displayed an unprecedented impact on drug development. For instance, recent specific approvals, and even more recent general guidelines, imply that computer simulations now can be used for regulatory approvals. Since model simulations are cheaper and faster, such approvals often end the use of test animals for that application. We have modified such an approved type 1 diabetes model to also describe type 2 diabetes, a much more widespread and rapidly growing disease. Our award-winning model has been developed through numerous experimental/modelling iterations, and is already at use in several major pharmaceutical companies. However, this usage is still mostly done in traditional drug development pipelines, involving a series of cell and animal systems, which often display highly different outcomes. To allow us to move to a radically new, knowledge-driven, more efficient, and increasingly human-centered pipeline, I will use uniquely informative data to do the four most important additions still needed in the model: i) storage and release of fatty acids, ii) specific drug-targets in heart and adipose tissue, iii) multi-level and long-term translations, iv) mapping from other animal-free systems. For these groundbreaking developments, the Swedish Fund for Research without Animal Experiments awarded me the first edition of their award – “Nytänkaren”. I am also a member of the steering group for the new government-initiated National Centre for 3R, and I am the only active scientist there to represent the field of replacement. With this grant, I could demonstrate in real ongoing drug development projects, done together with AstraZeneca, how our new knowledge-driven and increasingly animal-free drug development pipeline can work in a way that is economically beneficial for them, and which also helps to save both human and animal lives.

Pernilla Eliasson, Linköping University, 200 000 SEK
Project:  How do tendons heal? – and what happens when they don´t heal?
The general aim is to elucidate molecular mechanisms in tendon healing, with emphasis on the role of loading. Tendon ruptures are common and represent a significant clinical challenge. The understanding of molecular processes during tendon repair is poor. Mechanical loading improves healing, but it is unclear how. Most likely, loading can act through two mechanisms to stimulate healing, microdamage and mechanotransduction. Mechanotransduction is the way a cell converts mechanical stimulus into a biochemical response. Microdamage is associated with influx of inflammatory cells, whereas little about mechanotransduction in healing tendons. It is difficult to separate between these two mechanisms in animals. I have introduced a model for 3D cell culture studies of human primary tendon fibroblasts, in Linköping. We will use this model together with fluid flow to study the effect of loading without micro-damage associated inflammation (which is present in animal models) to investigate the mechanotransduction component during loading. Moreover, I have previously observed that tendon fibroblasts from some patients lack the ability to form tendon tissue, in vitro. We will study the differences between cells that can form tendon tissue with those that can´t. This might help to identify factors that are vital for tendon formation. Furthermore, constructs will be cultured with serum containing high levels or normal levels of cholesterol to understand how this influences tendon tissue. This 3D model also makes it possible to study effects on mechanical strength of the tissue after different manipulations, e.g. drug treatment or changes in loading situations, in contrast to 2D cell culturing. Studies on mechanical strength is an important tool in orthopedic research and this has previously only been done in animal models. The goal with these studies is to increase the understanding on tendon repair, so treatment and rehabilitation protocols can be improved.

New project!
Anna Forsby, Stockholm University, 250 000 SEK
Project: Validation of mRNA markers for prediction of developmental neurotoxicity
animals. The objectives of this study are to map transcription of the whole genome of the human neuroblastoma SH-SY5Y cell line and to validate neuronal mRNA markers that are identified to be significantly expressed during retinoic acid-induced differentiation for prediction of developmental neurotoxicity. The SH-SY5Y cells are exposed during differentiation to chemicals that are known to induce developmental neurotoxicity and to chemicals that are considered to be non-neurotoxic. mRNA is isolated after 6-9 days of differentiation when differentiation markers are significantly expressed. Our hypothesis is that compounds that induce developmental neurotoxicity will give a specific transcriptomic “fingerprint” that differs from the mRNA expression after exposure with non-neurotoxic compounds.

Robert Fredriksson, Uppsala University, 200 000 SEK
Project: Development of a novel method for an animal free method for Botulinum toxin testing
Botulinum toxin type A (BoNT/A) is one of the most potent neurotoxins known and its mechanism of action is to cleave SNAP-25 in neurons to make them unable to signal. BoNT/A is used in the beauty industry to reduce facial wrinkles, marketed under the trade name BoTox, as well as in medicine to treat various conditions such as chronic pain and excessive sweating. Accurate potency determination is vital for its safe and effective use as a commercial substance and potency measurements must be assessed for each batch produced. Today potency measurements are done by all BoNT/A producers using LD50 assays in mice. There are a few patented protocols for animal free BoNT/A potency measurements, but these seem to be used to a limited extent in practice. The challenges has been to identify a cell-line with high enough sensitivity towards the BoNT/A and a good enough readout of the BoNT/A effect, which could be done by measuring amount of cleaved SNAP-25. We have several candidate immortal cell-lines as well as a stem cell derived line that has very high sensitivity towards BoNT/A as well as high expression of SNAP-25. We have also identified and tried out monoclonal antibodies that are specific enough for detection of cleaved and uncleaved forms of SNAP-25 in these cells. Within this project we will refine the methods for detecting cleaved SNAP-25 as well as the cell-culturing methods. The main outcome we are aiming for is to have a Proof of Concept protocol including cell-culture, BoNT/A treatment and detection of cleaved SNAP-25 including preliminary determination of internal assay parameters such as sensitivity, variability and reproducibility. This can be used to show that there is a possibility to establish a cell based assay for detection of BoNT/A potency determination.

Maria Karlgren, Uppsala University, 250 000 SEK
Project: Human cell based models for accurate assessments of brain drug uptake
Poor predictions of CNS drug exposure is a major problem in CNS drug development and is primarily the result of relying on animal-based models although major species differences are well-known for the blood-brain barrier (BBB) and the expression of drug transporters. Here, we will solve this problem by developing predictive models taking the drug transporters expressed in human BBB into account and thereby accurately quantify drug delivery to human brain. This will be done by combining in vitro kinetics from mechanistic transporter models and human BBB in vitro models with quantitative proteomics and physiological parameters to predict the human in vivo situation. The project will be performed at Uppsala University in close collaboration with UDOPP (part of DDDp/SciLife Lab) giving a unique opportunity for validation in real-life drug development projects. With our model drug candidates with predictable and favorable human BBB properties will be identified and selected early in drug discovery/development. As a result an initial reduction of animal PK experiments of 50-60%, with further reduction potential, is estimated. Thereby providing the refined tools needed to substantially reduce and, in a longer perspective, replace animal BBB studies throughout the drug discovery/development process.

Hanna Karlsson, Karolinska Institute, 150 000 SEK
Project: New cell models for improved assessment of genotoxicity and cancer risk of nanoparticles
The production and use of small nanoparticles is increasing in society and this leads t o a requirement to test the toxicity of a substantial number of nanoparticles in the future. Despite research during recent years it is still unclear how risks associated with nanoparticles should be assessed, particularly considering chronic effects. To assess cancer risks, genotoxicity assays can be used both in cell-based assays and in animal studies. For particles, it is believed that a so-called secondary, inflammatory driven, mechanism is important for genotoxicity. This mechanism is believed to be related to release of reactive oxygen species (ROS) by immune cells (macrophages, neutrophils) that infiltrate the lung upon particle exposure (called oxidative burst) leading to DNA damage. Such a mechanism is, however, presently considered to be hard to detect in vitro and this has been used as an argument for continuing to use animal studies. The aim of this study is therefore to develop an in vitro assay that can detect an inflammatory-driven genotoxicity. The assay should also be functioning under serum-free conditions in order to avoid animal serum. We will culture both epithelial cells and macrophages and test two approaches: 1) Exposure of epithelial cells to “conditioned medium” from macrophages and 2) Co-cultures of epithelial cells and macrophages. We will test both particles considered to be carcinogenic to humans (e.g. quartz) as well as non-carcinogenic particles (iron oxide particles) as benchmark particles leading to a form of “validation” of our assay. We will then test a range of nanoparticles. The goal is to develop a standard assay that can take the interplay between different cell-types into account in order to completely avoid the use of animals.

Pekka Kohonen, Karolinska Institute, 150 000 SEK
Project: Deepened mechanistic validation of toxicity pathway functionality in a patented analysis tool
This application combines computational analyses with cell culture data in order to de velop alternatives to animal experiments within the research area of “toxicogenomics”. The Predictive Toxicogenomics Space (PTGS) tool has been developed for application to dose-response analysis with benchmark dosing technology. Importantly, the work from last year shows that the PTGS-based analysis provides 10-100 times higher sensitivity that common assays for diverse genomic and cellular toxicity endpoints. The proposed continuation incorporates independent validation and completion of the project into a novel, sensitive and mechanism-driven benchmark dosing tools. Specifically, project funding is now applied for validating the workflow outlined in the application with a TempO-Seq highthroughput transcriptomics study utilizing Trichostatin A (TSA), analyzing thereby a promising new class of compounds (histone deacetylase inhibitors). Furthermore, increasingly automated report generation software tools are to be developed for streamlining the analysis work flow. Overall, the project is set to generate needed proof-of-concept for a widely applicable solution towards toxicity prediction of environmental agents. This goal covers creating standard operating procedures for a real product-based applicable testing tool. Agreeing fully with the currently Forska Utan Djurförsök-supported work, the ultimate goal is to act in a translational manner relative societal needs of improving environmental health and safety with precise and cost-effective in vitro and in silico assays that fully replaces animal testing. The proposed analyses in this application build fully on the previously funded proposal, and are aimed to complete the tool development for publication in a high impact journal.

New project!
Tobias Lammel, Göteborg University, 200 000 SEK
Project: Development of a 3D in vitro model (spheroids) from the continuous rainbow trout liver lline RTL-W1
At present primary hepatocytes are the preferred model for mimicking liver-typic functions in vitro, in particular for studies investigating xenobiotic biotransformation and clearance. However, they have certain shortcomings:
1. Rapid loss of metabolic competencies and limited longevity make them unsuitable for long-term toxicity studies.
2. Isolation of cells from the animal and subsequent functional characterization/quality control is time- and labor intensive.
3. Use of cells originating from fish with different genetic and biochemical background increases interstudy variability.
4. Killing of fish rises ethical concerns.
Continuous liver cell lines are superior to primary hepatocytes regarding most of the above aspects, but their capability to reproduce complex toxic responses as they would occur in vivo is limited due to partial or complete loss of tissue-specific functionalities (e.g. metabolic competencies) at time of their establishment. Culturing cells in a more natural, three-dimensional (3D) environment allowing for cell-cell interaction and communication has been demonstrated to assist restoring tissuespecific cell shape, polarity, and functionalities. Our objective is to develop and functionally characterize 3D spheroidal aggregate cultures from the rainbow trout liver cell line RTL-W1 and investigate their potential for toxicity testing

Stina Oredsson, Lund University, 150 000 SEK
Project: Novel 3D Cell Culturing Methods in Cancer Research
Today, millions of animals are used in cancer research. However, before testing new d rugs in animals transplanted with human tumours, initial experiments usually are performed with cancer cell lines. The correlation between data obtained in cell culture and those obtained in animals is not satisfactory and it results in a long time before new medicines can reach the patient. It has come to the attention of many cancer researchers that traditional twodimensional cell culturing does not give a good reflection of cancer growth in vivo or of sensitivity to drugs. Among differences between in vitro and in vivo is the difference in dimensions of growth. Therefore, there is a considerable interest in three-dimensional (3D) cell culturing among cancer researchers as this better reflects tumour growth in the body. Most of these systems use animal-derived products such as matrigel and collagen as extracellular matrix support and fetal bovine serum in the medium. We have developed a 3D tumour outside the body using biocompatible and biodegradable polycaprolactone fibres mimicking the collagen network of a tumour. Human breast cancer cells or pancreatic cancer cells are seeded into the network together with human normal cells found in a tumour such as fibroblasts. Together they form a tumour resembling breast and pancreatic tumours, respectively, in patients as evaluated by different microscopic techniques. We also add immune cells as they are important from different aspects such as being targets for drug treatment that increases their activity and drugs that decreases the immune inhibiting activity of cancer cells. We show that NK cells kill the cancer cells and that this effect can be modulated by drug treatment. Our 3D in vitro tumour will contribute to faster development of anti-cancer drugs to the benefit of humans while also reducing animal experiments.

New project!
Lena Palmberg, IMM, Karolinska Institute, 200 000 SEK
Project: Development of lung models with multiple cell types for treatment
strategies of chronic lung diseases
Air pollutions caused 9 million premature deaths in 2015. Chronic obstructive pulmon ary disease (COPD) can be caused by air pollution and is the third leading cause of death worldwide. By using multi-cellular lung mucosa models with human primary bronchial epithelial cells, combined with immune effector cells (macrophages), air pollution related health hazards will be assessed. In the research team pre-clinical and clinical experts as well as scientist from the pharma industry warrant a comprehensive assessment of scientific methodology and data interpretation. The goal including evaluation of different treatment strategies.The studies explore the interaction of exposure, therapeutic effects, innate immunity, protease/antiprotease balance and oxidative stress as well as the interactions of various cell types. This knowledge can be utilized to develop preventive measures and treatment strategies for patients with COPD and chronic bronchitis. Validation of the models enables us to develop a systematic in vitro-testing strategy during drug development in order to reduce the requirement for animal inhalation studies.

Peter Sartipy, University of Skövde, 150 000 SEK
Project: Stem Cell-Derived Human Cardiomyocytes as test model, an alternative to animal studies
Anthracyclines, such as doxorubicin, are highly efficient chemotherapeutic agents against a variety of cancers. However, anthracyclines are also among the most cardiotoxic therapeutic drugs presently on the market. Chemotherapeutic-induced cardiomyopathy is one of the leading causes of disease and mortality in cancer survivors. The exact mechanisms responsible for doxorubicin-induced cardiomyopathy are not completely known, but the fact that the cardiotoxicity is dose-dependent and that there is a variation in time-to-onset of toxicity, as well as gender- and age differences suggests that several mechanisms may be involved. In the present project, we have investigated doxorubicin-induced cardiotoxicity in human pluripotent stem cell-derived cardiomyocytes using proteomics. In addition, different sources of omics data (protein, mRNA, and microRNA) from the same experimental setup were further combined and analyzed using newly developed methods to identify differential expression in data of various origin and types. Subsequently, the results were integrated in order to generate a combined visualization of the findings. In our experimental model system, we exposed cardiomyocytes derived from human pluripotent stem cells to doxorubicin for up to two days, followed by a wash-out period of additionally 12 days. Besides an effect on the cell morphology and cardiomyocyte functionality, the data show a strong effect of doxorubicin on all molecular levels investigated (mRNA, microRNA, and protein). Differential expression patterns that show a linkage between the proteome, transcriptome, and the regulatory microRNA network, were identified. These findings help to increase the understanding of the mechanisms behind anthracycline-induced cardiotoxicity and suggest putative biomarkers for this adverse reaction.

Lena Svensson, Lund University, 150 000 SEK
Project: Cancer-on-the chip: using microfluidic in vitro models to study cancer
Transendothelial migration of cancer cells is a key process behind metastasis, allowing cancer cells to spread from the primary tumor via the blood and lymphatic vessels to other sites in the body and form new tumors. Despite the fact that metastases cause the majority of deaths in cancer patients, the mechanisms involved in the metastatic process are poorly understood. This is mostly due to difficulties in studying the process, and today’s research is heavily based on implanting and studying tumors in animals. Such experiments are very invasive and often not very relevant to human disease. To overcome these issues, more advanced in vitro models are needed in order to study cancer cell metastasis in an efficient and accurate fashion. In our lab we have developed a microfluidic vascular model and currently we want to establish this model for investigations of metastases in different microenvironments as well as immune surveillance. The microfluidic model we will use in this project will enable us to study the above mentioned processes in real-time and with high resolution in three dimensions under physiologically relevant conditions. By utilizing this model, we are refining such studies due to the technique allowing for greater experimental control and by using human cells we are removing the species barrier, thus increasing human relevance. Microfluidic models have great potential in substituting and reducing the number of animal experiments in cancer research.

 

Senast uppdaterad: 8 januari 2019