Periodic Reporting for period 1 - ORGESTRA (Organoid technologies for disease modeling, drug discovery and development for rare diseases)
Période du rapport: 2024-01-01 au 2025-12-31
ORGESTRA is a Joint Doctoral Network training thirteen PhD researchers in organoid technologies for disease modelling, drug discovery and clinical development for rare diseases including cystic fibrosis and cystinosis. An innovative training programme has been developed to provide unique international, intersectoral and interdisciplinary training experience for our researchers.
We are developing both healthy and diseased organoid models that phenotypically correspond to affected organs. The developed advanced disease models will be used to identify druggable targets, through screening of existing drugs for the ability to correct diseased phenotypes (candidates for drug repurposing) and using gene-based strategies. In addition, we will develop scalable bioprocesses for improved production and cryopreservation of tubuloids and/or kidney organoids in cGMP compatible conditions using 3D cell culture approaches and stirred-tank bioreactor technology.
Our work on cystic fibrosis (CF) and primary ciliary dyskinesia models aims to develop large scale, cheap and animal free protocols for airway cell expansion and modulation, and image-based functional and molecular readouts (subcellular) through integration of automation and AI image analysis and high-content imaging.
The efficiency of different therapeutic strategies, including mRNA delivery, viral gene integration, site-specific gene editing and DNA repair will be compared in the different models developed by the project.
To maximise impact our work also includes proposing innovative clinical trial designs using novel outcomes under clinical and trial success relevance and linked to patient’s perspectives.
We are developing both healthy and diseased organoid models that phenotypically correspond to affected organs. The developed advanced disease models will be used to identify druggable targets, through screening of existing drugs for the ability to correct diseased phenotypes (candidates for drug repurposing) and using gene-based strategies. In addition, we will develop scalable bioprocesses for improved production and cryopreservation of tubuloids and/or kidney organoids in cGMP compatible conditions using 3D cell culture approaches and stirred-tank bioreactor technology.
Our work on cystic fibrosis (CF) and primary ciliary dyskinesia models aims to develop large scale, cheap and animal free protocols for airway cell expansion and modulation, and image-based functional and molecular readouts (subcellular) through integration of automation and AI image analysis and high-content imaging.
The efficiency of different therapeutic strategies, including mRNA delivery, viral gene integration, site-specific gene editing and DNA repair will be compared in the different models developed by the project.
To maximise impact our work also includes proposing innovative clinical trial designs using novel outcomes under clinical and trial success relevance and linked to patient’s perspectives.
We have successfully cultured and biobanked kidney tubuloids from urine of cystinosis patients and we are developing tubuloid lines from patients with other hereditary tubulopathies. Work is progressing on establishing a bank of cell lines isolated from urine of tubulopathy patients and colon-derived cystinosis organoids. The tubuloids, organoids and cell lines are being phenotypically and functionally characterized. A systematic review on the current state of gene therapy/small molecules/mRNA-based approaches for paediatric genetic kidney disease is being undertaken to aid in developing our drug development pipeline.
Engineered lentiviral vectors encoding ion-sensitive fluorescent reporters have been developed and lentiviral production and transduction protocols have been optimized, resulting in stable reporter expression in both wild-type and patient-derived intestinal organoids. Homogeneous reporter-expressing organoid populations were established through manual selection and FACS sorting to ensure assay robustness and reproducibility. Functional proof-of-concept was demonstrated, with HS-YFP PDIOs showing the expected iodide quenching response following pharmacological CFTR activation. In parallel, automated fluorescence imaging pipelines and quantitative analysis workflows are being developed to support future high-content, AI-enabled screening applications. Patient-derived airway epithelial culture workflows have been established and scaled. 2D and 3D primary nasal airway models were implemented alongside functional assays assessing CFTR activity and ciliary function in a robotics platform. Fully automated ciliary beat frequency (CBF) measurements with integrated analysis were established, together with Python-based workflows for high-throughput organoid dispensing using automated liquid handlers.
Engineered lentiviral vectors encoding ion-sensitive fluorescent reporters have been developed and lentiviral production and transduction protocols have been optimized, resulting in stable reporter expression in both wild-type and patient-derived intestinal organoids. Homogeneous reporter-expressing organoid populations were established through manual selection and FACS sorting to ensure assay robustness and reproducibility. Functional proof-of-concept was demonstrated, with HS-YFP PDIOs showing the expected iodide quenching response following pharmacological CFTR activation. In parallel, automated fluorescence imaging pipelines and quantitative analysis workflows are being developed to support future high-content, AI-enabled screening applications. Patient-derived airway epithelial culture workflows have been established and scaled. 2D and 3D primary nasal airway models were implemented alongside functional assays assessing CFTR activity and ciliary function in a robotics platform. Fully automated ciliary beat frequency (CBF) measurements with integrated analysis were established, together with Python-based workflows for high-throughput organoid dispensing using automated liquid handlers.
The ORGESTRA project is in the early research phase, but results are already being developed leading to advances in knowledge and publications contributing to the Euroepan Research Area. This is advancing multiple pathways to impact including:
The development of cutting-edge disease models will facilitate analysing drug efficacy and mode of action in the most relevant in vitro systems available. New knowledge of organoids, cell lines, biomarkers, disease phenotyping, stratification of patients and disease genes will be available to aid the design of new therapies, screening and prioritising candidate drugs with high translational value for further development.
The new knowledge created on targeted viral (rAAV) and non-viral delivery (via LNPs) of mRNA will deliver significant scientific and technological advances that may be used for other oligonucleotide strategies, such as siRNA or gene editing (e.g. CRISPR) therapies. Furthermore, demonstrating kidney targeting of mRNA-LNPs and gene repair may be useful for therapy development for other rare kidney and lung diseases. Finally, mRNA-based supplementation of gene defects can be used to de-risk future gene therapy approaches and will open avenues for new treatments options to target other tissues as well.
The development of components to predict drug-patient interactions, will improve future clinical trial designs for stratified medicine models and the incorporation of insights on patient’s preferences regarding disease or treatment characteristics aims to improve understanding disease pathways for treatment development towards patient needs.
The development of cutting-edge disease models will facilitate analysing drug efficacy and mode of action in the most relevant in vitro systems available. New knowledge of organoids, cell lines, biomarkers, disease phenotyping, stratification of patients and disease genes will be available to aid the design of new therapies, screening and prioritising candidate drugs with high translational value for further development.
The new knowledge created on targeted viral (rAAV) and non-viral delivery (via LNPs) of mRNA will deliver significant scientific and technological advances that may be used for other oligonucleotide strategies, such as siRNA or gene editing (e.g. CRISPR) therapies. Furthermore, demonstrating kidney targeting of mRNA-LNPs and gene repair may be useful for therapy development for other rare kidney and lung diseases. Finally, mRNA-based supplementation of gene defects can be used to de-risk future gene therapy approaches and will open avenues for new treatments options to target other tissues as well.
The development of components to predict drug-patient interactions, will improve future clinical trial designs for stratified medicine models and the incorporation of insights on patient’s preferences regarding disease or treatment characteristics aims to improve understanding disease pathways for treatment development towards patient needs.