Scientific background Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the first cellular immunotherapy developed to cure hematologic malignancies. It is based on the anti-tumor allo-immune response (graft versus tumor effect) induced by the donor immune system also transferred during the transplant process. Despite its efficiency, hematologic malignancies relapse accounts for half of deceases and to date, no biomarker allow to predict whose patient will relapse after allogeneic HSCT and to identify these patients early before relapse. Traditional statistical methods used for biomarker identifications are limited, mostly by their parametric nature, and could benefit from advanced machine learning and optimization techniques to select relevant variables and link them to the relapsing process. This unmet medical need is of critical importance to improve prognosis of patients who are currently treated for a hematologic cancer with allo-HSCT and to adapt their treatment before relapse. Hypothesis Here, we assume that integration of clinical data with immune and metabolic variables could provide metadata for a mathematical model to predict relapse occurrence. Aims To characterize circulating immune subsets and metabolome in the donor and to compare them at 3 months and one year after transplantation in patients with or without relapse To build a calibrated stochastic simulator for the relapsing process, accounting for post-transplant events and integrating clinical data with immune and metabolic variables. Methodology This project will rely on a multicentric cohort of 369 patients who received an alloHSCT. We will use mass cytometry and mass spectrometry to decipher circulating immune subsets and metabolites associated with relapse and other post-transplantation events. We will then create a simulator that model the dynamics of post-transplant events to identify relevant biomarkers using advanced optimization techniques and to generate a tool to predict relapse after alloHSCT. Validation in animal model will finally help to identify relevant new therapeutic targets. Expected results and impact This project will use data from an already constituted large cohort of patients to develop a machine learning tool for clinicians to estimate the probability of relapse based on various clinical and immune-metabolic data.

Histone variants act through the replacement of conventional histones by dedicated chaperones. They confer novel structural properties to nucleosomes and change the chromatin landscape. The functional and physiological requirement of the replacement of conventional histones by histone variants during organ formation and post-natal life remains poorly described. The incorporation of the histone variant H3.3 into chromatin is DNA-synthesis independent and relies on two different chaperone complexes, HIRA and DAXX/ATRX, which have different genomic deposition domains. While most epigenetic studies are performed in vitro, we intend to study them in an in vivo context where cell behavior can be properly addressed and where consequences for tissue formation, growth, homeostasis and repair can be fully investigated. Skeletal muscle provides the possibility to address yet poorly explored biochemical, cell biology, and developmental aspects of chromatin biology during development and postnatal life. Based on published and preliminary data from the three partners involved in this project, we hypothesize that: (i) HIRA and DAXX play a key role in muscle stem cells identity and muscle fibers organization (ii) H3.3 contributes to genome stability and prevents premature aging in adult muscle fibers (iii) a third H3.3 chaperone exists, which allows H3.3 incorporation into chromatin in the absence of HIRA and DAXX. Therefore, the main objectives of this proposal are defined in three work packages as follows: WP1: Conserved and divergent functions of H3.3 and DAXX-ATRX/HIRA pathways in muscle progenitors: we have recently shown that in the absence of HIRA, the muscle stem cell pool is lost during muscle regeneration. In addition, conditional HIRA inactivation in muscle progenitors during development have reduced myoblast numbers and smaller muscle size. In this context, our investigations will be extended to DAXX and H3.3. Our preliminary results indicate that DAXX is regulates myogenic gene expression via its histone chaperone activity. WP2: Role of H3.3 and DAXX-ATRX/HIRA pathways in adult myofibers structure and function: H2A.Z inactivation in adult muscle causes accelerated aging due to accumulation of DNA damage consecutive defective DNA repair by non-homologous end joining (NHEJ). H3.3 is also required for NHEJ. We therefore predict that H3.3 inactivation in muscle fibers will cause DNA damage and premature aging. Many evidences indicate that H3.3 regulates gene expression. We will determine if similarly to H2A.Z, H3.3 function in muscle fibers is restricted to DNA repair or if it also regulates gene expression. Finally, the roles of H3.3 chaperones have not yet been investigated in post-mitotic muscle fibers. To address these points H3.3, HIRA and DAXX will be inactivated in muscle fibers. We have recently shown that muscle fibers contain several myonuclear domains with specific identity and function defined by nuclei-specific expression profiles. The epigenetic landscape and myonuclei identity will be evaluated by single nuclei RNA seq and ATAC seq in the KO muscles. WP3: characterization of a new H3.3 deposition pathway that can bypass DAXX-ATRX/HIRA: H3.3 Chip-seq in Hira KO and Daxx KO myoblasts show HIRA and DAXX independent H3.3 deposition at specific loci, suggesting the presence of a third chaperone. Like other chaperones, this new chaperone should be part of a large multiprotein complex. We will isolate this complex from myoblasts and identify its composition. The complex will then be reconstituted with recombinant proteins to analyze its deposition properties. We will also invalidate the expression of some of the important components of the new deposition complex in vivo and we will determine the presumably perturbed H3.3 distribution pattern and the resulting cell phenotype at molecular level. Taken collectively, the expected data should shed in depth light on the intimate mechanism of H3.3 deposition and H3.3 function.

Bipolar disorder is a severe chronic psychiatric disorder affecting 1% of the population. Lithium is its gold standard treatment. Human MRI and preclinical studies suggest that it may increase neurogenesis, neuroprotection, myelination and modulate synaptic plasticity and neuroinflammation. However, many aspects of its mode of action remain unknown: which cellular effects are associated with the “MRI effects” of lithium in patients? Are its therapeutic cellular effects region specific or brain wide? We will conduct 2 parallel studies (in rats and humans) using similar (longitudinal) designs and methods ([11C]-UCB-J PET and MRI) plus histology and immunochemistry in the rats. We will assess synaptic plasticity, myelination, oligodendrocytes, neurogenesis and neuroinflammatory aspects associated with lithium in the same study. We will thus be able to draw inferences and inter species correspondences between PET/MR findings and immunohistological findings

Résumé: SARS-CoV-2 is a betacoronavirus that has recently emerged as a human pathogen in the city of Wuhan in China’s Hubei province. The disease caused by this newly identified virus has been named COVID-19 and symptoms include fever, severe respiratory illness and pneumonia. As of March 2020, the World Health Organization (WHO) has declared that SARS-CoV-2 is pandemic, and the number of confirmed cases is exponentially increasing. SARS-CoV-2 virus is closely related to SARS-CoV, which was responsible for the Severe Acute Respiratory Syndrome (SARS) in 2002. Similarly, to SARS-CoV, SARS-CoV-2 is of zoonotic origin and was demonstrated to cause life-threatening diseases in humans. So far, there is no vaccine or treatment for COVID-19. It is therefore critical to generate vaccines and drugs that will either prevent or treat COVID-19. At present, there are 8 candidate vaccines in clinical evaluation and more than 100 candidate vaccines in preclinical evaluation. Vaccine in clinical trials are represented by inactivated SARS-CoV-2 virus, non-replicating viral vectors, DNA and RNA vaccines (https://www.who.int). Surprisingly, no protein subunit vaccine are reported to be tested currently in clinical trial, as proposed in this project, which are generally safer and easier to produce. Moreover, all Human coronaviruses enter their host cells using the trimeric transmembrane spike (S) glycoprotein. The coronavirus’ S protein represents a major target for the human humoral immune response following SARS-CoV-2 infection. However, a large set of data from previous SARS-CoV-1 or CoV-2 infected individuals, or generated in preclinical models, pointed out the potential protective effect of cellular immunity. In this study, we propose to test the immunogenicity and the preventative effect of a combination of two vaccine platforms already in phase 1 to 3 clinical development; i.e the DNA-derived DREP platform and the anti-Dendritic cell (DC) targeting epitope-based vaccine. A series of DREP and DC-targeting constructs against SARS-CoV-2 are already available. A large set of data showed that these vaccines, either administered alone, or in a prime boost combination, elicited strong and durable T and B-cell immune responses against infectious agents. We develop here an original strategy aimed to induce a polyepitopic T and B cell responses. These vaccines are ready to be tested in two preclinical models, in humanized mice (mice reconstituted with a human immune system), allowing to study in depth human immune responses of different vaccine combinations, and in transgenic knock-in mice expressing the human CD40 and human ACE2, the receptor of SARS-CoV-2 to demonstrate the protective effect of these vaccines. The overreaching goal of this study is to identify within the 12-months time line of this project, vaccine (s) that will be moved forward to the clinical development.

Coronary microcirculation (i.e coronary flow in vessels smaller than 300µm) plays a key role in the control of cardiac perfusion. The importance of coronary microcirculation for relevant pathological conditions including angina in patients with normal or near-normal coronary angiograms is increasingly recognized. Indeed, a large number of patients with anginal symptoms and ischemia on stress testing have a normal coronary angiogram and current evidences suggest that about two third of these patients have coronary microvascular dysfunction (CMD), also known as microvascular angina (MVA). Patients with CMD have poor prognostic with significantly higher rates of cardiovascular events, including hospitalization for heart failure, sudden cardiac death, and myocardial infarction (MI). Another important and frequent alteration of the microcirculation is associated to sustained myocardial hypoperfusion during acute myocardial infarction despite coronary revascularization. This so called no-reflow phenomenon remains largely underdiagnosed, and is associated with adverse outcome. Finally, there are also major evidences for CMD during heart failure with preserved ejection france (HFpEF). Despite the urgent clinical need, there are simply no techniques available routinely in clinic, to directly visualize the coronary microvasculature and assess the local coronary microvascular system. Up to date, only global indirect measurements through functional testing (PET, CMR and contrast echocardiography) or invasive measurements can provide hemodynamic information such as Myocardial Blood Flow (MBF) and Coronary Flow Reserve (CFR) in response to vasodilator effects. In CorUS, a novel ultrasound technology will be developed to image the anatomy and the function of coronary vessels at the microscopic scale using a non-invasive and non-ionizing technology. This approach relies on ultrafast ultrasound imaging of the heart at 5,000 images/s, a breakthrough technology pioneered about twenty years ago by researchers of the laboratory Physics for Medicine Paris and more recently on the new technology of Ultrafast Ultrasound Localization Microscopy (ULM) which was introduced to resolve blood vessels at a micrometer scale in deep organs by tracking ultrasound contrast agents (microbubbles) circulating in the blood flow. Cutting-edge technology will be developed in CorUS for local and direct imaging of the coronary blood flows at the microscopic scale to provide new anatomical and functional markers of the coronary microcirculation. Preliminary proof of concept experiments in perfused porcine hearts and in perfused beating rat hearts have demonstrated the feasibility of 2D and 3D coronary microcirculation imaging. This technology will be translated to large hearts application and the approach will be validated in vivo on preclinical large animal models with alteration of the coronary perfusion. Finally, a clinical proof of concept study will be performed on patients with coronary microcirculation alteration. The main objectives of CORUS are: 1. To develop a new ultrasound technology for coronary microcirculation imaging 2. To validate the technology on large animal preclinical models of coronary microcirculation alteration 3. To perform a proof of concept clinical study on patients with coronary microcirculation disease CorUS will have major impacts in the understanding, the management and the treatment of coronary artery diseases and the non-ionizing, non-invasive imaging technology developed in this project could become a major tool for the clinical investigation of microvascular coronary circulation at the patient’s bedside.