EU legislation¹ makes it a legal obligation to replace, reduce and refine the use of animals in research (the ‘3Rs’ principle), including a specific focus on restricting the use of the non-human primates (NHPs) unless scientifically justified. The development of in vitro models for human safety assessment is still challenging due to complex biological responses in various organ systems following drug treatment. Therefore, laboratory animals will still be requested in the safety testing of new therapeutics and innovative medical technologies until non-animal approaches have reached the necessary level of maturity and validation to ensure that only safe treatments reach patients, and that patients get timely access to the most innovative therapeutics.

A substantial amount of work has already been conducted to increase the scientific knowledge and understanding of the role of minipigs in toxicity testing² and the pig is often used e.g., in the toxicological evaluation of small molecules. Replacing NHPs with minipigs in the safety testing of new therapeutic modalities has been, however, more difficult, due to lack of translational knowledge, but will be an important ethical step towards minimising the use of NHPs. New drug modalities are often designed to engage human targets with high specificity, which is the rationale for selecting NHPs in the safety testing of this kind of drug candidates. By expanding the translational knowledge in minipigs versus NHPs and humans, the scientific justification for selecting pigs as an alternative to NHPs can be improved.

The project funded under this topic adheres to the principles of the 3Rs by: i) closing the current translational knowledge gaps regarding minipigs versus NHPs and humans, offering the opportunity to replace NHPs with pigs, improve the reproducibility of pig studies, and advance the underlying knowledge of biological processes to facilitate the development of non-animal alternatives (reduce, refine and replace); ii) creating scientific and technological opportunities in animal housing facilities to collect, digitalise and generate more reproducible data in freely moving, undisturbed animals with the potential to reduce the total number of animals, and improve animal welfare and data quality (reduce, refine).

Closing the translational knowledge gap regarding minipigs versus NHPs and humans will enable the development of new, refined, and digital research tools, which will contribute to:

• reducing and replacing the overall number of NHPs in research without compromising human safety.
• improving disease understanding that will open up new research pathways, and enhanced use of non-invasive digital technologies that can improve animal welfare (refinement), and furthermore, are potentially applicable to humans.
• improving the sustainability and quality of biomedical research and development (R&D) in areas of unmet medical need by ensuring access to well-characterised minipig models in R&D of new therapeutics and innovative medical technologies.
• optimising knowledge sharing between academia, regulators, and the health care industry to accelerate the generation of knowledge and medical innovation.
• fostering the development and validation of non-animal models and approaches by implementing translational data obtained in the future project, which could pave the way to such models. Data generation will be based on early discussions with regulatory authorities and academic partners, thereby ensuring the contribution to the development and validation of non-animal approaches.

¹ _{DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes; https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex%3A32010L0063}

² _{The RETHINK project on minipigs in the toxicity testing of new medicines and chemicals: Conclusions and recommendations. https://doi.org/10.1016/j.vascn.2010.05.008}

• Obtain and share biological knowledge of minipigs, thereby facilitating the development of innovative solutions by improving the translational understanding between minipigs versus NHPs and humans, including further understanding of the minipig immune system, with the overall aim to replace, reduce and refine the use of animals in non-clinical safety assessment.
• A regulatory pathway for nonclinical safety assessment of biologicals and other new therapeutic modalities in minipigs with the potential to impact regulatory strategies.
• Publicly available databases and software for physiological, genomic, transcriptomic, metabolomic, proteomic and epigenetic minipig data to understand underlying mechanisms of disease/toxicities and find new mode of actions for pharmaceutical interventions.
• Characterised and validated genetically modified minipig models:
  • genetically modified minipig models based on the CRISPR/Cas9 gene-editing technology.
  • minipigs with ‘humanised’ immune system components and effectors for testing biologicals.
  • small–sized micropig for efficacy/safety assessment to facilitate compound availability in pharmaceutical R&D.
• Assessment of the utility of the minipig as a relevant toxicology species for immuno-safety testing using therapeutics which have been tested preclinically and clinically. Assisting and synergising the already existing translational and regulatory efforts related to immunological safety evaluation. Developing validated antibodies and in vitro immunoassays to characterise the immune system and assess the immuno-safety of therapeutics in minipigs.
• Minipig-specific technology for automated study data: validated medical devices, biosensors, algorithms, software, and digital animal housing. Machine learning and artificial intelligence (AI)-based tools to monitor abnormalities in behaviour and physiological systems in undisturbed animals.

To ensure long-term sustainability, all the interdisciplinary science-based knowledge obtained and generated in the project arising from this topic will be shared, integrated, digitalised, and published in peer-reviewed journals, encouraging industry and academia to develop innovative medical science solutions and technologies, such as scientifically and ethically sound animal models, assays, biomarkers, monitoring devices, biosensors for normal physiological behaviour, and algorithms. Based on the close collaboration with regulatory bodies, the knowledge generated in the project is further expected to impact regulatory guideline strategies. All outputs will require long-term sustainability and maintenance to fulfil the scope of the project.

Challenges

• Increasing need to find alternatives to testing in NHPs in line with EU legislation.
• Almost no precedence in minipig use for safety testing of biologicals and new therapeutic modalities [e.g., oligonucleotides, small interfering RNAs (SiRNAs), crystallisable fragments (Fcs), antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), monoclonal antibodies (mAbs), vaccines, gene-editing and cell-based therapies].
• Lack of scientific knowledge to scientifically justify a de-selection of NHPs in the non-clinical safety assessment of new therapeutics. Lack of public minipig reference ’omics’ with good quality annotation: Full genome sequencing, in parallel with baseline transcriptomics, proteomics, metabolomics and epigenetic information.
• Lack of ‘humanised” and genetically modified models available for efficacy/safety testing, including genetically modified smaller micropigs to address cases of limited substance supply.
• Significant knowledge gap on the minipig immune system and reduced number of laboratory tools and reagents when compared to other toxicology species (rodent and non-rodent).
• Lack of widespread use of biosensors, medical devices, ‘intelligent’ animal housing for automated data collection and analysis in minipig studies.

Objectives

The overall objective of this topic is to characterise the minipig for use in R&D of new therapeutics and innovative medical technologies. The knowledge generated in this proposal may facilitate innovative health solutions and improve disease understanding and human predictions. The goal is to advance biomedical R&D by generating background scientific data to evaluate if the minipigs could be a viable and feasible alternative to NHPs in key therapeutic areas, with a special focus on translatability from minipigs to humans.

Key activities

1. Compile and publish existing historical safety data in minipig biomedical R&D and discuss data with regulators.
2. Evaluate the translatability of minipigs in human risk assessment following treatment with biologicals and new therapeutic modalities, and discuss future perspectives of the minipigs with regulatory agencies, e.g., by requesting regulatory interactions with European Medicines Agency (EMA) such as scientific advice and/or novel methodology qualification advice to understand possible regulatory hurdles in using minipigs for safety assessment.
3. Minipigs multi-omics and imaging: Generate omics reference data (genomics, transcriptomics, proteomics, metabolomics, and epigenetic information) to enable translational research in minipigs. To further characterise the minipig, imaging technologies such as magnetic resonance imaging (MRI), computed tomography (CT) scans and positron emission tomography (PET) scans are also of interest.
4. Genetically modified pig models including the micro-pig: Characterise and validate humanised and genetically modified minipig models, including the micropig to generate translatable animal models in non-clinical safety assessment.
5. iPig: Digital technologies, clinical data collection and AI: Create, validate, qualify, and benchmark digital solutions that can objectively measure clinically relevant and functional biomarkers in minipigs for use in preclinical toxicity studies in line with the regulatory agencies’ requirements.
6. Minipig immune system: validate reagents, assays, and biomarkers for immunological investigations: Conduct investigative studies in minipigs to support their translational significance in immuno-safety assessments and validate reagents/assays.
7. Project management: Compile, digitalise, and publish existing and newly-produced data.