Grants awarded to 13 projects for 2021

 

A total of 2,7 million SEK has been awarded in grants from the Swedish Fund for Research without Animal Experiments in 2021, divided between 13 projects. See recipients and project titles below.

Per Artursson, Uppsala University, 200 000 SEK
Project:  Filling the gaps for better prediction of endocrine disrupting chemicals

Endocrine disrupting chemicals (EDCs) are substances with the ability to interfere with the endocrine system e.g. via estrogen receptor activation. Estrogenic activity can be determined with a battery of in vitro assays, as suggested in Level 2 of the The Organisation for Economic Co-operation and Development (OECD) Conceptual Framework for Testing and Assessment for Endocrine Disrupters.

Previous publications have mostly focused on relating the in vitro activity to the in vivo uterotrophic assay in rodents (Level 3, OECD TG440). However, these studies do not address the inter-assay variability in in vitro activity, which makes in vitro-in vivo (IVIV) translation difficult. Animal experiments in rodents therefore remains the golden standard for classification of chemicals as EDCs.

In this project, we wish to investigate the reason for the in vitro variabilities by identifying appropriate EDCs using data mining of thousands of chemicals and subsequent computational modelling to fill gaps in the curated data. With the combination of curated and predicted data, the comparisons of in vitro data across assays will be feasible. The results will provide a platform for selection of a representative subset of EDCs that we then will use to investigate reasons for the poor in vitro-in vivo (IVIV) translation, using Level 2 assays combined with pathway analysis using global proteomics and determination of intracellular bioavailability.

Understanding the underlying reasons for inter-assay variability will provide a tool to normalize existing data across in vitro systems. This will increase comparability of experimental data and facilitate development of more robust predictive in silico models, which is a necessity if we want to move away from animal testing and rely primarily on in vitro and in silico methods in future risk assessment.

Gunnar Cedersund, Linköping University, 200 000 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 ground-breaking 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 there active in more general promoting and structural changes on replacement, which goes beyond my own research, e.g. creating a national replacement network. 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, 250 000 SEK

Project:  How do tendons heal? – and what happens when they don´t heal?

The general aim of this project is to elucidate molecular mechanisms in tendon healing, with emphasis on the role of loading, tendon tissue formation, and matrix contraction. Tendon ruptures are common and represent a clinical challenge, with often long-lasting muscle deficits.

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 mechano-transduction. Mechano-transduction is the way a cell converts mechanical stimulus into a biochemical response.

I´m using a model for 3D cell culture studies and load these artificial tendons with fluid flow to study the effect the mechano-transduction. I´m also using this 3D cell culture model to study the interaction between the cells and the surrounding extracellular matrix. The cells play an important role in matrix contraction but the molecular mechanisms behind this is unknown. The 3D model used in this project makes it also possible to study effects on mechanical strength of the tissue after different manipulations, e.g. drug treatment or changes in loading situations. This contrast traditional 2D cell culturing.

Studies on mechanical strength and interactions between cells and matrix are important 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.

Anna Falk, Karolinska institute, 150 000 SEK
Project:  Organoids as hosts and semi-in-vivo models of the human brain to replace animals

The main objective of this 3R program is to develop human organoid systems that will act as hosts and “in-vivo-like” brains for transplanting cells to replace the use of animals. There are two main reasons to develop brain organoid systems that mimics the human brain, one being that animal brains have been shown to be different to humans and thus sub-optimal for studies of brain disorders, second now with emerging stem cell technologies we have the chance and the responsibility to develop human “in-vivo-like” brain models for research and thereby replace animals.

In my group we are stem cell biologists with long experience in reprogramming and stem cell models of the brain. Here we use our knowledge to create organoid models of the human brain that will be used as a substitute for animal brains for transplantation of cells for cancer studies, disease modelling of neuronal maturation and for cell replacement therapy studies.

The three different types of transplantation schemes we would like to investigate and develop are the following (listed in the order of number of animals that can be replacement), organoids as hosts:

a) For transplantation of tumour cells for studies of brain cancer
b) For transplantation of patient derived neural cells to investigate differentiation, migration, integration, maturation into a healthy “brain-like” environment
c) For studies on neural cells replacement in for example organoid models of Parkinson’s disease

We have previously standardised stem cell culture and neuronal differentiation using defined, serum free and xenofree reagents, which creates robust stem cells showing close to no variability. Here we will develop the brain organoid system by replacing Matrigel with the defined and animal free 3D material BioSilk. The produced organoids will be investigated for their ability to host cell transplantation and thereby replace animal brains.

Anna Forsby, Stockholm University, 350 000 SEK
Project:  Validation of mRNA markers for prediction of developmental neurotoxicity

Developmental neurotoxicity testing according to regulatory test guidelines requires a vast number of laboratory 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 5-9 days of differentiation when differentiation markers are significantly expressed.

Our results show that acrylamide impair neuronal differentiation by interfering at least two signaling pathways, i.e. the retinoic acid and CREB pathways. Furthermore, acrylamide attenuates gene expression of brain derived neurotrophic factor (BDNF), choline acetyltransferase, the dopamine D2 receptor, syntaxin and syntaxin-binding protein 2, monoamine transferase A and transforming growth factor β.

Data analysis of the effects of 32 other compounds on the expression of over 3300 genes is in progress.

The focus in this project will be on genes that are involved in neuronal function and development. Functional endpoints such as viability, calcium signaling, mitochondrial potential and function, and neurite length can be associated with transcriptomic markers. The identified genes of interest as candidates biomarkers for developmental neurotoxicity will be validated by quantitative PCR (Prime-qPCR) after exposure with positive and negative model compounds.

Robert Fredriksson, Uppsala University, 200 000 SEK
Project:  Development of a novel animal free method for detecting potency of Botulinum Toxin Type A
Botulinum neurotoxin type A (BoNT/A) is produced by the bacterium Clostridum botulinum and is today produced commercially to be used in the esthetic industry to reduce wrinkles as well as in clinical practice to treat migraine, chronic pain, excessive sweating and symptomatic treatment in Parkinson’s disease. Currently the potency of each production batch is determined with the LD50 method and despite the fact the European drug product agencies in principle have banned this method it is still widely used. In the most recent annexes of the European Pharmacopeia LD50 is still noted as the standard method.

There are currently only a few non-animal assays published and despite that some producers of BoNT/A products claim to have cell-based assays in place, the number of mice used for potency testing is increasing from the most recent estimate of 600 000 animals per year (Bitz Silke, 2010, Altex 27,2/10). The reasons for this discrepancy is most likely due to the difficulty to of maintaining cell lines without drift. A huge advantage in our method where we use stem cell derived neurons rather than cell lines. Therefore, the purpose of this project is dual:

– Showing in scientific publications that stem cells provides a better alternative than cell lines for this purpose and that this overcomes the difficulties with cell-based methods. This should be a factor for regulatory agencies when they write guidelines.

– To show that this approach is viable from a commercial point of view to attract interest from commercial actors.

If more scientific publications showing that cell-based assays for BoNT/A potency determinations are actually possible, regulatory agencies and BoNT/A producers will show increased interest in cell-based assays for BoNT/A potency determination.

Our aim is to develop a proof-of-concept method that is robust enough to replace mice in all phases of the life cycle of BoNT/A products, including batch testing of drug substances, testing of actual drug products and for stability testing of drugs. If the method turn out to be valid and useful in only one or a few of these aspects it will still highly reduce the number of mice required. We believe that if more assay formats are presented, with solid data showing that these are indeed useful, both BoNT/A producers as well as regulatory agencies will show interest in replacing the LD50 method completely for BoNT/A potency testing.

Erik Hjort, Karolinska institute, 200 000 SEK
Project:  Monocyte-derived microglia from Alzheimer patients as disease model and biomarker

The presence of inflammation in the brain afflicted by Alzheimer’s disease (AD) is well known and is believed to be in a bidirectional stimulatory relationship with the molecular pathology of Ab and tau, as well as causing neurodegeneration in itself. In the AD brain, there is also a decrease in the beneficial factors associated with the resolution of inflammation and tissue healing.

Developing treatments aimed at stimulating the good aspects of inflammation while inhibiting the bad ones requires relevant and valid cellular models.

Models based on rodent prenatal microglia are predominantly used today, but such microglia exhibit differences compared to their human counterparts and are associated with animal suffering. The validity in using embryonal cells to model responses and mechanisms related to a disease of the elderly is also questionable.

In this project microglia will be produced from monocytes isolated from the blood of AD-patients, and their responses when exposed to Ab compared those of healthy controls. Outcome responses will cover the harmful as well as beneficial aspects of inflammation, including phagocytosis of Ab42, expression of activation markers, cytokine, chemokine and neurotrophin production, and pathological Ab-associated activation of the NLRP3 inflammasome. Our hypothesis is that AD-derived MdMs express exaggerated harmful responses while beneficial ones are inhibited. Furthermore, the responses from AD-derived MdMs will be correlated to data on cognitive impairment and cerebrospinal fluid biomarkers of Ab and tau pathology. By surveying the available antibodies etc. needed, we have found animal-free alternatives in all cases.

By performing this project we may promote the replacement of animal microglial models with a human model with validity and relevance for the study of AD, and increase awareness of animal-free alternatives for antibodies and sera, thereby reducing animal suffering while improving the quality of research.

Pekka Korhonen, Karolinska institute, 200 000 SEK
Project:  Toxicogenomics for chemical’s toxicity prediction

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. This continuation project develops a programmatic PTGS-based Adverse Outcome Pathway (AOP) assessment workflow that can encompass all the AOPs in the international AOPWiki database. The aim is to enable direct and effective coupling of in vitro model-derived omics data to key events (KEs) in AOPs, generating thereby a useful novel way to link cytotoxic influences to the rapidly developing AOP principle for replacing animal experiments.

The PTGS project has thus far processed and generated more than 800 million data points which will be leveraged to validate the tool for toxicity prediction (> 500 million liver-related data points). The TG-GATEs toxicogenomics database will be applied to take an Artificial Intelligence (AI) and “Big Data”-driven approach to address the subject. Focusing initially on liver toxicity prediction, the project is set to generate needed proof-of-concept for a widely applicable AOP-driven solution to toxicity prediction. This goal covers creating standard procedures for a real product-based applicable testing tool.

Agreeing fully with the currently Forska Utan Djurförsök-supported work, the 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 further expand the applicability of the PTGS concept.

Per Malmberg, Chalmers university of technology, 200 000 SEK
Project:  Chemical imaging for skin permeation studies – an analytical approach to replace animal experiments

Every day, our skin is exposed to many different metals and chemicals when it encounters everything from tools, utensils and electronics to cosmetic products. Exposure to metals and chemicals can cause contact allergy and can also long-term health effects such as increased cancer risk. In occupational health, healthcare and legislation, it is therefore of great interest to be able to follow where metals and chemicals end up in the skin, how large the absorption is and how cells in the skin are affected.

Today there is no direct method for measuring and visualizing skin uptake of metals and chemicals together, and therefore no hygienic limit values that regulate this. Furthermore, it has not been investigated in detail to what extent metals and chemical substances penetrate the skin and how combination exposure affects cells and tissue.

In this interdisciplinary project, we want to validate and further develop an animal-free method for studying skin uptake using chemical imaging, where we examine skin absorption of exogenous substances in an in-vitro method based on donated human skin. During the project time we will validate and establish this approach so that it can replace current in-vivo and in-vitro approaches. The method will also provide a refinement, since it provides a unique way of looking at the skin and is a cost-effective animal-free alternative that can be used for occupational studies, basic research and product testing.

Ivan Nalvarte, Karolinska institute, 200 000 SEK
Project:  A new model to study Alzheimer’s disease in human mini-brains

Experimental research of Alzheimer’s disease (AD) is extensively based on animal models due to the lack of relevant and reliable in vitro methods that recapitulate the disease progression. However, human disease relevance of animal models has been questioned, warranting the development of alternative models for human AD.

The hallmarks of AD include cortical and hippocampal deposition of amyloid plaques, neurofibrillary tangles and activation of local immune response that all contribute to extensive neuronal death and cognitive decline.

The proposed project aims at developing an immune competent in vitro model that recapitulates this pathology and that can be used in semi high-throughput. We will establish cerebral brain organoids from induced human pluripotent stem cells harboring driving AD mutations and controls. These will be grown in a tailored simple semihigh throughput microgravity bioreactors to allow long term culturing. To address the impact of immune response on AD pathogenesis, we will establish co-cultures of cerebral organoids with isogenic microglia. This model will be validated against human AD brain samples available in the KI Brain Bank. In this way, we aim at replacing molecular studies using animal AD models by offering a human relevant in vitro model, thereby greatly reducing the number of mice included in AD research today.

Penny Nymark, Karolinska institute, 150 000 SEK
Project:  Toward risk assessment without animals: Development of Adverse Outcome Pathways for lung cancer.

The risk for chemicals and other types of substances, including nanomaterials, to cause cancer is complex to assess and currently based mainly on animal experimentation, even though non-animal alternative methods exist. Some of these cell-based methods and assays are accepted by regulatory instances for risk assessment, however, are mainly applied as weight of evidence information in addition to currently required animal data. In addition, novel high-throughput systems, capable of handling increasing numbers of samples in short time frames, require validation towards regulatory acceptance.

The new Adverse Outcome Pathway (AOP) concept is expected to simplify and improve the integration of alternative methods towards becoming gold standard regulatory accepted methods for risk assessment. AOPs describe toxic mechanisms causally connected to human organ injury and disease. They provide a basis for development of mechanism-focused alternative methods predictive of the risk for toxicity. This project will develop AOPs for lung cancer, one of the most common and deadly forms of cancer associated with exposure to a wide range of inhalable substances. The project will apply toxicological big data derived from in vitro high-throughput testing and analyses of around 40 diverse nanomaterials. A novel bioinformatics pipeline will be utilized to integrate data and guide the development and refinement of AOPs for lung cancer.

The overall aim will be to map assays, information and data in line with the AOP concept and thereby support further development and integration of AOP-aligned animal-free methods for cancer risk assessment of inhalable nanomaterials.

Stina Oredsson, Lund University, 200 000 SEK
Project:  Novel 3D Cell Culturing Methods in Cancer Research to Reduce Animal Experiments

The main final goal of the present project is to create a human 3D tumour outside of the body that is totally animal product free and that can be used at a reasonable cost. The purpose is to use this model in pre-clinical testing of novel potential chemotherapeutic drugs that not only target the cancer cells but also target normal cells as well as secreted molecules in the tumour environment that provide protection to toxic insult to the cancer cells.

To enable this goal, there are two major issues related to animal-derived products to be solved: the extracellular matrix and the medium. As an artificial 3D collagen network, we use electrospun polycaprolactone (3D) fibres. We have shown that this network is biocompatible, and that normal cells and cancer cells thrive in the 3D fiber mesh. The growth pattern of co-cultures of cancer cells and fibroblasts are treated with experimental compounds and chemotherapeutic drugs. The preliminary data show that fibroblasts protect the cancer cells from toxic insult which is in corroboration with present knowledge and thus contributes to validation of our model. Preliminary data of adding NK cells and dendritic cells demonstrate a role for immune cells in the tumour environment.

We will further validate the model e.g. treating with immune checkpoint inhibitors. So far, we have used donor heard horse serum supplemented medium as a means to eliminate fetal bovine serum but we are now taking this to the next level by developing a totally defined medium. Thus, a number of cell lines are being adapted to totally defined medium and in preliminary experiments we have found that these cells also thrive in the 3D PCL fiber mesh. Thus, we are not far from our goal of human 3D tumour outside of the body that is totally animal product free but much validation-work is needed.

Lena Öhman, Göteborg University, 200 000 SEK
Project:  Intestinal organoids for studying intestinal diseases and drug mechanisms

The intestine is the site of diseases of various severity, ranging from functional disorders to chronic inflammation and cancer. During recent years, the importance of the species-specific interaction between the host and the local intestinal microenvironment, thus the microbiota and metabolites they produce, for disease generation has become evident. The overall aim of this project proposal is to develop a tissue-based model system making use of our in house developed intestinal organoids for determining epithelial events of intestinal diseases, alongside assessing cellular mechanisms of therapeutic drugs.

In contrast to other organoid systems, our unique model is based on organoids from healthy donors, which are stimulated with fecal supernatants containing luminal factors, from patients and healthy subjects, giving rise to this gives rise to organoids with different characteristics, potentially disease-like and healthy-like “mini-guts in a dish”.

Thus, our hypothesis, supported by our preliminary data, is that organoids cultured in the presence of fecal supernatants reproduce the intestinal conditions of the donors who provided the fecal material. The model system will a useful tool for studying and comparing epithelial events in different intestinal diseases and explore cellular mechanisms of therapeutic drugs. Our model system will in part replace experimental animal models exploring epithelial events of human intestinal diseases such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and colon cancer.

Senast uppdaterad: 31 januari 2022