The pathophysiological mechanisms for most primary antibody deficiencies (PADs) are not well understood in contrast to those underlying other primary immunodeficiencies. Lymphoproliferation, chronic diarrhea, autoimmune cytopenia and increased incidence of lymphomas are the main clinical complications associated to PADs. Our project is based on the hypothesis that most PADs have underlying genetic defects. For pediatric cases of PADs we assume that they are more frequently related to monogenic causes. PADs diagnosed in adulthood have also likely underlying genetic defects, however, the late disease onset and diagnosis in these patients could be explained due to further genetic modifying, epigenetic and environmental factors. The genetic delineation of PADs is essential to enable precise diagnosis, better treatment and follow-up und thus improve the quality of life of chronically affected patients and their families. By combining clinical, immunological, histopathological, functional and genetic data analysis, we aim to establish new diagnosis biomarkers, identify new therapeutic targets and decipher novel pathophysiological mechanisms for PADs. Exploration of PAD pathogenesis and validation of genetic defects will be performed by in-depth characterization of patients' cells and generation of in vitro cellular models. This project will contribute to a better understanding of the fundamental mechanisms underlying human antibody mediated immune responses and will improve the diagnosis and care of PAD patients.

Rare diseases are a major public health topic because of the difficulty of their management, both in terms of diagnosis and treatment. Composed of a large and heterogeneous group of pathologies, they are individually rare but collectively frequent, with more than 350 million people affected worldwide, including 3 million in France. It is estimated that 80% of them are of genetic origin, 65% are severe and disabling and 50% develop in childhood. But above all, 95% of them have no curative treatment. There is a real need for diagnosis: 25% of patients suffering from these conditions are forced to wait 5 years or more for a diagnosis. This diagnostic wandering delays the implementation of an adapted medical follow-up, the prevention of complications, the development of personalized therapeutic strategies and genetic counseling. Created in 2007 as a scientific cooperation foundation with AP-HP, Inserm, the University of Paris, the AFM, the Fondation Hôpitaux de Paris-Hôpitaux de France and the City of Paris as founding members, the Imagine Institute for Genetic Diseases is a research and care institute, an international leader in the field of rare genetic diseases. It was awarded the IHU label in 2011 in recognition of its translational research activities, and the Carnot Institute label in 2020 in recognition of its commitment to strongly develop its partnership research activities with industry. Located on the campus of the Hôpital Necker (Paris), Imagine is a unique place, bringing together a range of multidisciplinary and multi-professional skills, experts in care, teaching and research, patients and patient associations, all committed to the fight against rare genetic diseases. As part of its societal mission, the Imagine Institute wishes to continue its openness and explore concrete actions of mediation and communication with the general public, and more particularly with young people, on genetic diseases and the advances and hopes brought about by research. It has thus set itself 4 ambitious societal missions: - Disseminate knowledge about rare genetic diseases - To share with as many people as possible the challenges, methods and results of research. - To stimulate vocations as doctors and researchers, particularly among young people. - To work on the social inclusion of patients suffering from rare genetic diseases. In this context, the institute is carrying out various communication and mediation actions for the general public (conferences, visits, etc.), patient associations (annual FAIR forum), as well as school group (visits, mediation workshops, development of new playful formats). These formats take shape both in person and remote, throughout the year, regularly punctuated by regional and national highlights (Fête de la Science, Rare Disease Days, etc.). The researchers are literally the actors of these events, together with the communication service and the person in charge of Science and Society programs. One example is the Bourse de Diffusion Scientifique program, dedicated to doctoral students at the Institut Imagine. Through this program, students have the opportunity to contribute to societal issues related to rare genetic diseases, with an educational and inclusive purpose. SAPS-CSTI-AAPG18/19 call for proposals concerns a maximum of 5 innovative projects of the institute for which we propose 3 main approaches of communication and scientific mediation: inclusion of dedicated contents within the next Forum of patient associations, development of a ludic mediation format under the format of an Escape Game, construction of transdisciplinary "Regards Croisés" conferences around the innovations and approaches of these projects.

Beta-hemoglobinopathies are caused by mutations affecting the production of the adult hemoglobin beta-chain. Persistence of fetal gamma-globin chain synthesis in adult life substantially ameliorates the clinical phenotype of beta-hemoglobinopathy patients. Currently, allogenic hematopoietic stem cell transplantation is the only definitive therapy for patients affected by severe beta-hemoglobinopathies. Transplantation of autologous, genetically modified hematopoietic stem cells represents a therapeutic option for patients lacking a suitable donor. Gene therapy strategies currently in clinics include the transplantation of hematopoietic stem cells transduced with an integrating lentiviral vector expressing a functional beta-globin gene. Genome editing approaches based on the use of site-specific nucleases have been explored by many groups, including ours. Clinical trials have been recently initiated exploiting genome editing to down-regulate BCL11A, a master repressor of fetal hemoglobin, via the inactivation of its erythroid-specific enhancer. However, a non-trivial genotoxicity risk associated with the use of these approaches raises concerns when applied in the clinics. In this project, we will use cutting-edge epigenome editing technologies to dissect the molecular mechanisms underlying gamma- and beta-globin gene regulation, and to develop novel potential therapeutics for beta-hemoglobinopathies. We will identify critical regions in two regulatory elements that control gamma-to-beta globin switch during development, such as the BCL11A erythroid-specific enhancers and the gamma-globin gene promoters. We will identify epigenetic marks that are differentially deposited in fetal or adult cellular models at these regions to secure silencing of gamma-globin and activation of beta-globin. We will then both use previously established designer epigenome modifiers (DEM) and develop novel effectors to alter these epigenetic marks in order to restore gamma-globin expression in a cellular model of adult erythropoiesis. To this end, we will use the DEM technology to erase activating marks and deposit repressive epigenetic marks at the BCL11A enhancers in order to inactivate these elements. We will then pursue a similar approach to deposit activating epigenetic marks at the gamma-globin promoters. We anticipate an increase in gamma-globin expression as a result of both the inactivation of the BCL11A enhancers and the direct activation of its own promoter. The best-performing reagents selected from the in vitro model will be tested in primary human cells from healthy donors to identify the most efficient to induce fetal globin expression in the context of clinically-relevant cells. Ultimately, the most efficient reagents will be used to reactivate fetal globin in patient-derived cells and their safety profile will be thoroughly evaluated to reveal the potential of this approach for clinical translation. Overall, the knowledge acquired in this project will be instrumental to develop novel therapeutic approaches aimed at restoring gamma-globin expression in patients affected by beta-hemoglobinopathies.

Life-threatening influenza pneumonia is a major public health problem, by its number, and a longstanding scientific enigma, because most cases of influenza are benign. Clinical manifestations and outcome of Influenza A virus (IAV) infections may possibly be determined by both viral and host factors. While life-threatening influenza pneumonia can be favored by co-morbidities, most cases in otherwise healthy individuals remain unexplained. The French team previously identified single-gene inborn errors of type I and III interferon (IFN) immunity as genetic etiologies of lifethreatening influenza pneumonia, including autosomal recessive (AR) IRF7, AR IRF9, and autosomal dominant (AD) TLR3 deficiencies. The German team discovered that rare, deleterious variants of the IFN-stimulated gene (ISG) MX1 increase susceptibility to zoonotic infections with the avian IAV subtype H7N9. On this evidently complementary basis, the two teams now hypothesize that these and other genetic determinants of innate immunity may underlie critical influenza in many more patients than hitherto suspected. In a highly synergistic effort, we will analyze whole exomes of a large, diverse cohort of more than 300 patients with severe influenza, searching for inborn errors of immunity (IEI), with a particular - but not exclusive - interest in type I and III IFN IEI recently found to underlie critical COVID-19 pneumonia. We will also test the candidate rare variants at the molecular, cellular, and immunological levels. Finally, we will search for autoantibodies (auto-Abs) neutralizing type I and III IFNs that were recently shown to account for about 20% of COVID-19 deaths and to reach 4% of individuals older than 70 years old in the general population. Our preliminary data are exciting. We have recruited pediatric and adult patients with life-threatening IAV infections, in collaboration with multiple international partners. We have also found three new candidate genetic disorders, including X-linked recessive (XR) TLR8, AR NLRC3, and AR MX2 deficiencies. Finally, we have identified auto-Abs neutralizing IFN-alpha and/or -omega in 10-20% of a small cohort of patients with severe influenza. The biological and clinical implications of our study are multiple and important. Biologically, we will provide additional evidence that life-threatening influenza pneumonia can be driven by human genetic and immunological determinants that undermine type I and III IFN immunity to IAV in the respiratory tract. Clinically, we will provide a rationale not only for annual influenza vaccination of individuals at risk, but also for specific therapeutic options in patients with deficiencies in type I and III IFN immunity due to genetic disorders or anti-IFN auto-Abs.

Mastocytosis is a heterogeneous disease characterized by an accumulation of mast cells (MCs) in the skin or various organs. The systemic form includes indolent and aggressive forms such as mast cell leukemia. Somatic mutations activating the KIT receptor tyrosine kinase (TK) are found in 90% of cases, but several arguments indicate that other genetic events are necessary to trigger the disease. We have recently demonstrated the synergistic cooperation between germline GLI3 hedgehog (Hh) mutations and somatic KIT mutations in mastocytosis onset, and have proposed a new therapeutic strategy combining anti-Hh and anti-TKs for patients overexpressing Hh genes. Seeking other genetic events, we recently identified mastocytosis patients carrying germline mutations (stop codon FASR250X and missense FASLGH122N) in the FAS-FASLG apoptotic pathway. The deficiency of this pathway is known to be the main driver of autoimmune lymphoproliferative syndrome (ALPS) but has never been studied in mastocytosis. We also found that MCs from patients with aggressive mastocytosis express very low levels of FAS at the membrane, indicating that deregulation of FAS is involved in the pathophysiology of the disease via resistance to apoptosis. Interestingly, 75% of ALPS patients carry germline mutations in FAS gene with variable expressivity. Mutations induce partial or total inhibition of FAS, or production of a dominant negative form that blocks apoptosis. Among the patients, 15% develop urticaria and/or severe allergies indicating that MCs are activated in this pathophysiological context. Here, we propose to study post-transcriptional defects of FAS expression resulting from pathogenic mutations or splicing dysregulation in mastocytosis and ALPS diseases. Moreover, our collaborative work will shed light on the contribution of mast cells in ALPS.