Understanding the physiopathology of the progression of chronic kidney disease (CKD), a major socioeconomic burden on national health care systems, is a prerequisite for developing efficient preventive strategies. Over the last years we have characterized a murine animal model of CKD based on subtotal nephrectomy. Our studies have demonstrated that in this model, renal Iesions (fibrosis and cysts) are due to a degenerative epithelial proliferation linked to the activation of EGF receptor. In this model, right after nephron reduction there is a first wave of compensatory proliferation (CP) due to a properly organized lengthening and hypertrophy of the remaining tubular structures followed by a transient quiescent period (QP). Recently, we have demonstrated that after these initial steps, in specific sensitive mouse strains, a second wave of degenerative proliferation (DP) may lead to severe renal lesions. Our previous studies have demonstrated that DP propensity can be modulated by specific mutations indicating that distinct genetic programs underlie the development of lesions. One of the aims of this research project is to identify the molecular signature that characterizes the propensity to develop renal lesions. To this end we will analyze the transcriptome of sensitive and resistant strains during the crucial steps that follow the subtotal nephron reduction, CP, QP and DP. In parallel, we will analyze the cellular mechanisms that underlie the appearance of renal lesion including the maintenance of cilia and the orientation of cell division during the two proliferative phases. Finally, we will characterize the molecular and cellular mechanisms played by Lcn2, a key factor in the development of renal lesions that is involved in the EGF pathway activation. This research project is based on a multidisciplinary approach that involves the use of cell biology, genetics transcriptomics and proteomics. On the long term, our results should shed a novel light on the molecular mechanisms and possible therapeutic treatment of chronic nephropathies.

Pheochromocytomas (PHEO) and paragangliomas (PGL) are tumours that arise from chromaffin tissues of the adrenal medulla and sympathetic and parasympathetic ganglia. They are malignant in 15% of cases and hereditary in 30% of cases. Hereditary PHEO/PGL are caused by germline mutations in one of the 9 PHEO/PGL susceptibility genes: VHL, RET, NF1, SDHA, SDHB, SDHC, SDHD, SDHAF2 and TMEM127. Mutations in SDHB gene are responsible for metastatic form of the disease and are associated with poor prognosis. The aim of this MODEOMAPP project is to decipher the molecular mechanisms responsible for PHEO/PGL malignancy, with a particular interest in inherited forms associated with SDHB gene mutations. The completion of this project will require the generation of cell and animal models of SDHB-dependant malignant PHEO/PGL to progress in understanding mechanisms of tumorigenesis and carcinogenesis and validate new therapeutic strategies for these tumours for which surgery remains the only curative treatment. We develop mouse ES and chromaffin cell models in which SDHB will be inactivated by a Cre/lox strategy. Mice harbouring SDHB constitutive or conditional inactivation have also been generated and will be crossed with different lines of transgenic or knockout mice (PSA-Cre, Bloom, PTEN +/-) and/or subjected to conditions of environmental stress (hypoxia), which should accelerate the development of tumours and increase their invasiveness and metastatic potential. Non-invasive evaluation of tumour burden and vascularization will be assessed by MRI on the PARCC’s small animal imaging platform. The analysis of the transcriptome of 200 human PHEO/PGL has revealed differentially expressed genes in SDHB-related malignant PHEO/PGL. Among these genes, LOXL2 and TWIST appeared particularly interesting. They are involved in epithelial-mesenchymal transition, a process that participates to the invasiveness of different types of metastatic cancers. In order to obtain an increased statistical power, and a validation cohort, the transcriptome data will be combined with those obtained by Prof. R de Krijger in Rotterdam. The evaluation of chromosomal rearrangements (BAC array CGH and SNP- array), alterations of the DNA methylation pattern (methylome) and modification of microRNA expression profile (miRnome) will complete this meta-analysis. This ‘OMICs’ characterization of genomic and genetic events associated with SDHB-related malignancy will lead to the identification of candidate pathways that will then be functionally validated. After confirming their comparable regulation in SDHB-related cell and animal models, the effect of their silencing by siRNA approaches will be tested on proliferation, migration, survival, and the ability of these cells to form vascularised tumours in vivo. In mice that spontaneously develop tumours, pre-clinical trials assessing the role of these targets may be launched. Finally, prognostic biomarkers will be validated and transferred to clinics as tools to predict the malignant potential of a PHEO/PGL from initial pathological analysis of the primary tumour and to implement the first international clinical trial of targeted therapy in malignant PHEO/PGL and select patients for inclusion in this trial. This project will be a major asset to the neuroendocrine cancer field, with the development and analysis of in vitro and preclinical mouse models, essential to various projects of basic and applied research.

Aims. Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic ß-cells. The HLA DQB1*0302 class II HLA gene carry the highest risk for T1D, but class I MHC alleles modulate the risk, in particular HLA-A*0201. Among ß-cell autoantigens, insulin has been ascribed a key role in T1D. While this knowledge has boosted efforts to prevent T1D using insulin-specific immunotherapy, these efforts have been unsuccessful when translated into clinical trials, pointing to the need for new preclinical T1D models. The aims are to: 1) characterize the immune and metabolic phenotypes of humanized mice (YES) expressing human insulin (hPPI), HLA-A*0201, DQ8 transgenes and lacking their mouse counterparts, 2) evaluate YES mice as a new preclinical model for insulitis and diabetes, 3) characterize hPPI epitopes targeted by CD4+ and CD8+ T cells and develop T-cell assays in these mice, 4) evaluate strategies to induce immune tolerance to hPPI as a therapy in T1D. Methodology. We have obtained YES mice that lack the expression of murine class I, class II and insulin genes, express HLA-A*0201, DQ8 transgenes and human insulin (hINS) transgenes. Analysis of pancreases from YES mice shows normal islet morphology, but we have preliminary evidence that YES mice develop insulitis following immunization against a human ß cell line. To characterize the immune and metabolic phenotypes of YES mice, three YES lines will be studied. As the replacement of murine class I and class II MHC molecules may interfere with the selection of class I- and class II-restricted T cells and with antigen presentation, the main immune subsets, in particular T lymphocytes, will be quantified by FACS in thymus, spleen, lymph nodes and pancreas. Cytotoxic and proliferative responses to conventional antigens will be evaluated. Insulin gene expression will be quantified in liver, pancreas, thymus, spleen, kidney, inguinal lymph node and the metabolic phenotype of YES mice further characterized and compared to that of the parental mouse lines used. To evaluate YES mice as a new preclinical model for insulitis and diabetes, attempts will be made to characterize further and optimize the induction of insulitis and immune-mediated ß cell destruction using different immunization procedures and introduction of B7.1 expression on ß-cells. To characterize hPPI epitopes targeted by autoimmune CD4+ and CD8+ T cells and apply T-cell assays to detect ß-cell autoimmunity in this model, DQ8-restricted CD4+ and A*0201-restricted CD8+ T cell responses to human insulin will be evaluated and the repertoire of epitopes recognized defined. Immunogenicity of human insulin peptides and recognition by CD4+ T cells will be studied using proliferation assays and evaluating production of cytokines. Insulin-specific CD8+ T cells will be studied – immunization against a library of insulin peptides, cytotoxicity assays, single cell PCR of tetramer-sorted cells. T cell clones will be generated and tested for recognition of P815 cells cotransfected with HLA-A*0201 and hINS. The same T cell assays will be used to study insulin-specific CD8+ and CD4+ T cells along induction of insulitis/diabetes. To evaluate the feasibility of inducing immune tolerance to insulin, and more generally ß-cells, in this model, as preventive or therapeutic strategies in T1D, we will assess whether delivery of a human insulin-Fc fusion protein can induce immune tolerance in YES mice, and identify immune correlates and mechanisms of diabetes protection. Insulin-Fc fusion proteins will be delivered to YES offsprings upon treatment of mothers during pregnancy or lactation, or to YES mice at 3 week of age or following induction of insulitis/diabetes. Mice will be evaluated for induction of T cell tolerance to human insulin and for prevention of insulitis/diabetes. Setting up a new preclinical T1D model may help the development of T-cell assays and pave the way to “vaccination” strategies in T1D.

Rationale: 45 million individuals worldwide are bilaterally blind, among them 4.9 million are suffering from corneal defects. Corneal disorders are consequences of congenital diseases, or environmental insults such as physical injury, viral infection, chemical and thermal burns. The general result is corneal neo-vascularization that leads to loss of corneal transparency and blindness. Corneal transplantation is widely used with high success at the short term but is limited by the shortage in post mortem cornea and immunorejection prevalence is significantly increasing with time within the first 5 years. Therefore, there is a major need for alternative allo- or autologous sources for corneal cell therapy. Background: Oral epithelial cells, mesenchymal stem cells, hair-follicle stem cells and embryonic stem cells were investigated for their therapeutic potential for corneal diseases. However, only partial success has been achieved by the use of these cells since they were not able to uniformly commit into corneal epithelial cells and/or no experimental data have been provided to demonstrate their ability to stratify and reconstitute in vitro a corneal tissue or displayed abnormal thickness and barrier function. Artificial cornea is an alternative acellular solution that is suggested only for patients with complex ocular diseases who are at high risk for donor graft failure. These complicated surgeries unfortunately allow limited vision improvement and are expensive. Innovative product: we propose here to design a standardized and reproducible technology to produce in large amount "ready to use" cornea from human pluripotent stem (induced or embryonic) cell lines. We will take advantage of the unlimited growth capacity of these cells and their pluripotent ability to differentiate into any cell type. As a proof of concept, we have recently developed an efficient technique to generate corneal cells expressing putative corneal-specific markers from either human embryonic stem cells or human induced pluripotent stem cells. We have shown that these corneal cells are able to stratify in vitro for the production of a corneal tissue. Potential applications of the innovative product: the finalized product will be used in two main applications: a. corneal regenerative medicine: here, we aim to produce unlimited cornea from pluripotent stem cells. Such tissue will serve as “ready-to-use” source to be transplanted immediately into injured eye as allo-graft. This product will be easily accessible for surgeons around the world, standing by for grafting into patients. In addition, for patients presenting immuno-rejection problem, autologous cornea will be produced directly from their own cells. b. cellular model for cosmetical toxicity and drug testing: the corneal tissue obtained from pluripotent stem cells should become a valuable tool to perform, in a reproducible and physiological manner, toxicity assays of cosmetic products and pharmacological tests of drugs. The EU Cosmetic Directive lead to the ban of animal testing for cosmetic ingredients (acute tests). Major efforts are still made to find reliable and relevant alternative methods to eye irritation testing. So far, quite unsuccessfully as no suitable model is yet established. Our reproducible in vitro tissue model will allow predicting the safety and efficacy profiles of actives and formulations. Specifically, it will be used in ocular irritation assays, as tests for corneal permeability and metabolism and mucin production. To be done within 2 years: our current goals are (1) to standardize the quality of iPSC/huESC-derived corneal tissues, (2) to evaluate their therapeutic potential in vivo, (3) to demonstrate that this model may serve as a useful in vitro tool for toxicity assays, thereby allowing the reduction in animal testing.