Periodic Reporting for period 1 - SPHERO-NASH (A novel NASH model for target and drug candidate identification)
Periodo di rendicontazione: 2023-09-01 al 2025-02-28
Metabolic dysfunction-associated steatotic liver disease is an important global health burden that affects an estimated 1 billion individuals, or 25% of all adults worldwide. While initially benign, the condition can progress to metabolic dysfunction-associated steatohepatitis (MASH), which has an annual prevalence rate of 2.8% in the EU that is expected to further increase by more than 40% by 2030. Clinically, MASH and its progression to fibrosis remain an unmet medical liability with only a single approved pharmacological treatment, resmetirom, that only results in measurable benefits in <30% of patients. One of the main reasons for the paucity of treatment options is that non-human models of MASH do not accurately mimic the characteristics of human liver pathophysiology. Consequently, there is an urgent need to develop human-based in vitro models that encompass the essential cell lineages of the human liver that underlie the etiology and progression of MASH.
In this ERC-PoC project, we focused on the translational and commercial potential of a 3D spheroid model (SPHERO-NASH) we have developed to meet this need. A particularly important aspect of this model is that it allows the study of (i) inducers and mechanisms of MASH formation, (ii) drug-induced inhibition of MASH by different mechanisms, and (iii) degradation of the extracellular matrix (ECM), including collagens, and its role in the development and treatment of MASH. The MASH phenotype depends on both an increase in profibrotic stressors, such as those present during metabolic syndrome, and on molecules that inhibit proteinases involved in the degradation of the ECM. The versatility of the spheroid model enables the identification of putative drug targets involved in the regulation of liver fibrosis as well as the identification of new chemical entities that can reverse the NASH phenotype suitable for further commercialization. We performed high-throughput screening (HTS) using chemogenomic libraries to discover new targets for the development of anti-steatotic, anti-inflammatory and anti-fibrotic therapies. In addition, we have successfully commercialized the SPHERO-NASH model and have made it available to pharmaceutical partners in the EU and beyond. The fully completed licensing of the platform to a Chinese CRO partner can serve as a testament for the utility of the developed approach. The SPHERO-NASH model thus has a great fundamental and commercial potential in MASH drug discovery and development.
In this ERC-PoC project, we focused on the translational and commercial potential of a 3D spheroid model (SPHERO-NASH) we have developed to meet this need. A particularly important aspect of this model is that it allows the study of (i) inducers and mechanisms of MASH formation, (ii) drug-induced inhibition of MASH by different mechanisms, and (iii) degradation of the extracellular matrix (ECM), including collagens, and its role in the development and treatment of MASH. The MASH phenotype depends on both an increase in profibrotic stressors, such as those present during metabolic syndrome, and on molecules that inhibit proteinases involved in the degradation of the ECM. The versatility of the spheroid model enables the identification of putative drug targets involved in the regulation of liver fibrosis as well as the identification of new chemical entities that can reverse the NASH phenotype suitable for further commercialization. We performed high-throughput screening (HTS) using chemogenomic libraries to discover new targets for the development of anti-steatotic, anti-inflammatory and anti-fibrotic therapies. In addition, we have successfully commercialized the SPHERO-NASH model and have made it available to pharmaceutical partners in the EU and beyond. The fully completed licensing of the platform to a Chinese CRO partner can serve as a testament for the utility of the developed approach. The SPHERO-NASH model thus has a great fundamental and commercial potential in MASH drug discovery and development.
We developed a novel, patient-relevant three-dimensional (3D) liver spheroid model composed of primary human hepatocytes and non-parenchymal cells (hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells). This model replicates key histopathological features of MASH, including steatosis, inflammation, and fibrosis, closely matching patient biopsy data. The spheroids spontaneously develop fibrosis, which worsens with free fatty acid treatment. Liver endothelial cells (LSECs) were found to drive fibrosis by releasing tissue inhibitor of metalloproteinases 1 (TIMP1), which is upregulated under lipotoxic stress and enriched in fibrotic liver zones. Knockdown of TIMP1 reduced collagen accumulation, confirming its role in fibrosis regulation.
To advance therapeutic discovery, the model was combined with high-content imaging, multi-omics profiling, and chemogenomic screening. This revealed promising therapeutic targets such as muscarinic M1 receptor (CHRM1) activation and TRPM8 inhibition. Mechanistic studies showed that CHRM1 inhibits TRPM8 via phospholipase C activation, leading to strong anti-fibrotic effects.
Additionally, we developed a long-term 3D spheroid model for screening conjugated siRNAs for specificity, stability, off target effects and toxicity. These spheroids maintained viability and asialoglycoprotein receptor expression for five weeks, enabling sustained, non-toxic siRNA uptake and durable gene silencing. This human-relevant platform provides a crucial bridge between traditional in vitro systems and animal models for siRNA therapeutic development.
We built a strong network of over 20 partners, including pharmaceutical companies and academic researchers focused on MASH. The 3D MASH model has been successfully integrated into its service portfolio, with multiple industry requests for testing candidate molecules’ anti-fibrotic and anti-steatotic effects. These results validate the model’s commercialization potential for early preclinical testing. Chemogenomic screens have also identified novel drug targets, with lead-like molecules in development and evaluation through genetic tools like KO mice. Intellectual property from these efforts will be carefully managed. Notably, HepaPredict entered a licensing agreement with Shanghai Hepo Biotechnology Ltd, granting exclusive rights to deploy the 3D MASH technology in China, demonstrating the model’s maturity and commercial attractiveness for future investment.
To advance therapeutic discovery, the model was combined with high-content imaging, multi-omics profiling, and chemogenomic screening. This revealed promising therapeutic targets such as muscarinic M1 receptor (CHRM1) activation and TRPM8 inhibition. Mechanistic studies showed that CHRM1 inhibits TRPM8 via phospholipase C activation, leading to strong anti-fibrotic effects.
Additionally, we developed a long-term 3D spheroid model for screening conjugated siRNAs for specificity, stability, off target effects and toxicity. These spheroids maintained viability and asialoglycoprotein receptor expression for five weeks, enabling sustained, non-toxic siRNA uptake and durable gene silencing. This human-relevant platform provides a crucial bridge between traditional in vitro systems and animal models for siRNA therapeutic development.
We built a strong network of over 20 partners, including pharmaceutical companies and academic researchers focused on MASH. The 3D MASH model has been successfully integrated into its service portfolio, with multiple industry requests for testing candidate molecules’ anti-fibrotic and anti-steatotic effects. These results validate the model’s commercialization potential for early preclinical testing. Chemogenomic screens have also identified novel drug targets, with lead-like molecules in development and evaluation through genetic tools like KO mice. Intellectual property from these efforts will be carefully managed. Notably, HepaPredict entered a licensing agreement with Shanghai Hepo Biotechnology Ltd, granting exclusive rights to deploy the 3D MASH technology in China, demonstrating the model’s maturity and commercial attractiveness for future investment.
We have advanced the liver spheroid model into a 3D MASH platform that offers a physiologically relevant and scalable system for investigating the molecular mechanisms underlying liver fibrosis. This model serves as a powerful tool for phenotype-driven screening of potential anti-MASH therapeutics. In addition, we have developed applications within this platform to study siRNA pharmacokinetics, focusing on how structural modifications to the siRNA backbone influence cellular uptake, stability, toxicity, and target specificity during long-term cultivation.