Team 2 : Cell and gene engineering in tolerance, fertility and regenerative medicine

Team 2 leaders: Dr. Carole Guillonneau & Dr. Jérôme Jullien

Research program

The team main objectives are the analysis of immune responses and tolerance induction in transplantation, both basic and translational research, with the development and application of genetic engineering and stem cell approaches. We integrate as much as possible these different topics and technologies and reinforced our valorisation strategy with the creation of 2 start-up with wich we collaborate. Our research projects are organized along 3 main axes.

1) Immune tolerance and immunotherapies (PIs: Carole Guillonneau, Ignacio Anegon, Matthieu Giraud)

- We work since several years on CD8⁺ T regs both on basic (phenotype, function, TCR-peptide interactions) and clinical aspects (in pathological situations and as cellular therapy product like CAR-Tregs) and are part of a European consortium RESHAPE to perform the world first trial using polyclonal CD8⁺ Tregs in kidney transplant patients in 2022.
- We develop immunotherapies to induce tolerance using anti-CD45RC mAbs and IL-34 cytokine.
- We investigate the control of expression of self-antigen genes in the thymus through the identification of activating factors that operate either synergistically with or without AIRE, or drive the development of thymic cells (TECs).

2) Transgenesis, genome and epigenome editing (PIs: Ignacio Anegon, Matthieu Giraud, Jérôme Jullien)

- Through our IBiSA-labeled platforms (TRIP and GenoCell Edit) gene editing of immune genes using CRISPRs and different viral vectors are applied to a) human CD8⁺ Tregs to KO or KI genes (TCRs or CARs directed against alloantigens); b) human CD34⁺ cells derived from ES/iPS KO or KI for different immune genes used to humanize immunodeficient mice or rats to explore the function of these genes; c) The generation of KO and KI rats (KO for IgM, Rag1, IL2Rγ, Aire or KI for Foxp3-GFP) using gene-specific nucleases (ZFNs, TALENs, CRISPRs).
- We also apply epigenome editing tools to probe the function of epigenetic marks in gene regulation, notably in self-antigen expressing thymic cells.

3) Stem cell fate, developmental biology and fertility (PIs: Jérôme Jullien, Laurent David, Thomas Fréour)

- Cell fate during human preimplantation development: We focus on the analysis of the epigenetic contribution of sperm to cell fate establishment and on the signalling pathways regulating embryonic cell commitment. We have conducted a very active clinical research on fertility.
- We investigate somatic cell reprogramming to early embryonic fate to reveal the epigenetic barriers to cell fate change.
- We generate human GMP hepatocytes derived from ES/iPS cells to transplant in liver diseases, we correct iPSC from patients with liver genetic diseases using CRISPRs and to confer them immune stealth properties.
- We differentiate human ES/iPS towards CD34⁺ cells (to humanize immunodeficient rats) and the to CD8⁺ Tregs or toward mTECs a source of therapeutic cells.

PIs from the team are managing platforms:

- The Rat Transgenesis TRIP platform is a platform led by Ignacio Anegon that generates genetically modified rats (2015-2020: 41 projects, most outside of our INSERM unit, that resulted in 32 publications). TRIP has a working force of 7 persons (ETP 3). TRIP services also include the distribution of rat models created for our own research but that also have widespread interest. Importantly, TRIP performs research and development on genome editing and transgenesis in the rat and other organisms as well as immunophenomic techniques.

- The GenoCellEdit platform is a platform led by Ignacio Anegon opened in June 2014, employ 2 persons and provides CRISPR-Cas services to academic and privated researchers. GenoCellEdit also has R&D projects to improve the technology. The platform generated over 200 different CRISPRs/sgRNAs for projects from international, national and local partners, especially for CR2TI teams. Transgenesis projects include applications in rats, mice and sheep.

- The iPSC Induced Pluripotent Stem Cell core facility led by Laurent David (4 technical staff, platform part of our local cluster of research (SFR)) mission is to accelerate research in the stem cell field by facilitating the derivation and distribution of induced pluripotent stem (iPS) cell lines, training and assisting partners in the setup of differentiation protocols. The platform is opened to local (45%), national (45%) or international (10%) users. R&D: the iPSC core facility has optimized new reprogramming protocols and is currently developing genome editing protocols, in collaboration with the TRIP core facility.

- The Rodent facility and microsurgery led by Carole Guillonneau (3 technical staff). This facility covers 400 m² and hosts mice and rats used for CR2TI research projects. Rodent facility technicians also perform microsurgery (heart, kidney and skin grafts), as well as basic animal care, other surgical procedures (blood sampling, organ harvesting) and immunomonitoring.