Periodic Reporting for period 1 - MITI 2 (Gut Microbiota-Induced Tregs for Inflammatory-Bowel-Disease (IBD) Immunotherapy (MITI2))
Reporting period: 2023-05-01 to 2024-10-31
Inflammatory Bowel Disease (IBD) is a disabling condition that causes many life-altering symptoms including life-threatening complications with a great medical need. The main objective of the MITI2 project is to deliver an immunotherapy product ready to be used in a phase 1 clinical trial to treat IBD patients. The project thus aims at providing a preclinical package including: i) the proof of concept that a cellular immunotherapy using the identified Treg subset represents a clinically suitable treatment for IBD patients and ii) an original gut-explant-based organoid preclinical model, which recapitulates ex vivo the complexity of intestinal physiology. The project is organized in 8 different work packages, all of them closely related one to another.
WP1 will characterize, enhance and produce DP8α regulatory T cells (Tregs) in the GMP format to treat IBD patients. Proteomic approaches have been used to identify the antigen(s) recognized by these DP8α Tregs and derived from the bacteria F. prausnitzii. One GMP-compliant antibody panel was selected to maximize the quality of DP8α Tregs sorting. We are currently performing a deep characterization of DP8α Tregs, finalizing a method to enhance their suppressive activity and setting up conditions to produce them in the GMP format.
WP2 aims to develop a well-characterized library of patient-derived organoids mimicking dysfunctional IBD gut tissue by enriching the epithelial cells with selected immune cells and connecting them to a neural enteric network. Gut samples from non-IBD and IBD patients requiring surgery were collected to establish organoids made only from epithelial intestinal cells (hEIO). Some hEIO individual lines have already been established and stored. Their characterization is ongoing. Purification of immune and neural cells from the gut have started and co-cultures tests with hEIOs are in progress.
WP3 aims to establish the optimal experimental conditions to generate vascularized intestinal “mesoscale” organoids (PDLM) (several organoids fused together) using bioprinting. To develop the ex vivo model of functional and immunocompetent IBD (PDLM), as a proof of concept, we have developed the PDLM from commercial human colonic primary cells. Using the same condition, we establish PDLM derived from healthy and IBD patients. We have begun to develop the vascularization of these organoids, and to integrate autologous T cells.
WP4 aims to develop the first microfluidic device for perfusing a large vascularized system integrating PDLM and lymph node tissue. We successfully developed the first microfluidic chip prototype and hydrogel (PLMA, a photopolymerizable hydrogel obtained from platelets for 3D culture) designed to promote tissue vascularization. The microfluidic device demonstrated biocompatibility, good optical properties, and its ability to effectively confine hydrogel within the central chamber. Meanwhile, the significant progress made in capillary production will be integrated into the on-chip culture model in the next period.
WP5 aims to establish the objective criteria to assess the response of complex gut organoids to gold standard IBD therapy. Monitoring of the organoid growth over time has been performed by videomicroscopy. Characterization of all these organoids is currently being performed by immunofluorescence and qRT-PCR. Identification and functional test of Paneth cells was addressed.
WP6 aims to compare, using complex organoid models of IBD, the impact of DP8α Tregs treatment with the reference therapies evaluated in WP5. It has not really started yet.
WP7 aids project coordination and communication, supporting the MITI² coordinator in managing consortium. It ensures internal communication and disseminates project results to maximize visibility. A dedicated project management team supports the MITI² coordinator in overseeing consortium, ensuring that the project meets its objectives. Moreover, it oversees the dissemination of project results, enhancing visibility and impact.
WP8 objectives are to establish and implement all necessary ethics procedures to ensure that the project fully complies with ethical standards and regulations. It sets out the ethical requirements that the project, financed by the European Commission, must comply with.
WP2 aims to develop a well-characterized library of patient-derived organoids mimicking dysfunctional IBD gut tissue by enriching the epithelial cells with selected immune cells and connecting them to a neural enteric network. Gut samples from non-IBD and IBD patients requiring surgery were collected to establish organoids made only from epithelial intestinal cells (hEIO). Some hEIO individual lines have already been established and stored. Their characterization is ongoing. Purification of immune and neural cells from the gut have started and co-cultures tests with hEIOs are in progress.
WP3 aims to establish the optimal experimental conditions to generate vascularized intestinal “mesoscale” organoids (PDLM) (several organoids fused together) using bioprinting. To develop the ex vivo model of functional and immunocompetent IBD (PDLM), as a proof of concept, we have developed the PDLM from commercial human colonic primary cells. Using the same condition, we establish PDLM derived from healthy and IBD patients. We have begun to develop the vascularization of these organoids, and to integrate autologous T cells.
WP4 aims to develop the first microfluidic device for perfusing a large vascularized system integrating PDLM and lymph node tissue. We successfully developed the first microfluidic chip prototype and hydrogel (PLMA, a photopolymerizable hydrogel obtained from platelets for 3D culture) designed to promote tissue vascularization. The microfluidic device demonstrated biocompatibility, good optical properties, and its ability to effectively confine hydrogel within the central chamber. Meanwhile, the significant progress made in capillary production will be integrated into the on-chip culture model in the next period.
WP5 aims to establish the objective criteria to assess the response of complex gut organoids to gold standard IBD therapy. Monitoring of the organoid growth over time has been performed by videomicroscopy. Characterization of all these organoids is currently being performed by immunofluorescence and qRT-PCR. Identification and functional test of Paneth cells was addressed.
WP6 aims to compare, using complex organoid models of IBD, the impact of DP8α Tregs treatment with the reference therapies evaluated in WP5. It has not really started yet.
WP7 aids project coordination and communication, supporting the MITI² coordinator in managing consortium. It ensures internal communication and disseminates project results to maximize visibility. A dedicated project management team supports the MITI² coordinator in overseeing consortium, ensuring that the project meets its objectives. Moreover, it oversees the dissemination of project results, enhancing visibility and impact.
WP8 objectives are to establish and implement all necessary ethics procedures to ensure that the project fully complies with ethical standards and regulations. It sets out the ethical requirements that the project, financed by the European Commission, must comply with.
Through imaging approaches, we determined the distribution of DP8α Tregs in the human colon. Indeed, we showed a preferential dual localization of DP8α Tregs at the top of colon crypts and at the basal level of the mucosa. These cells were also detected at the periphery of intestinal lymphoid formations but not inside. Positive F. prausnitzii fractions recognized by DP8α Tregs have already been identified. For the suppressive activity of DP8α Tregs, we have identified a molecule that activates them and promotes their production of anti-inflammatory cytokines. Finally, the antibody panel to sort GMP-compliant DP8α Tregs has been identified. The IBD-derived organoid biobank will constitute a great asset for IBD translational research especially as the disease is polygenic. The integration of a neural enteric network within hEIOs will represent a ground-breaking advance in organoid technology. We have obtained PDLM from commercialized available primary human colonic epithelial cells. We would like to increase the size obtained and generate a vascular network within these organoids. The development of vascular networks in a fibrin matrix has been completed, and the connection of this network to the PDLM is in progress. The prototypes of microfluidic device developed lay the foundation for a ground-breaking culture model connecting PDLM to the lymphoid system via a vascular network. A setup to inject inflammatory compounds into the organoids has been successfully established. Using an Eppendorf microinjection setup allows to inject both inflammatory agents and bacterial components. This setup will be expanded to also measure Transepithelial Resistance as a marker for mucosal barrier function.