Bile secretion is an essential function of the liver necessary for fat digestion and xenobiotic elimination. This function mainly rely on transporters localized at the canalicular membrane of hepatocytes, such as the ATP-binding cassette (ABC) transporters including ABCB4, ABCB11, responsible for the secretion of phosphatidylcholine (PC) lipids and bile salts into bile, respectively. Genetic variations of the loci encoding these two ABC transporters are correlated with rare cholestatic liver diseases, the most severe forms being progressive familial intrahepatic cholestasis (PFICs). Unfortunately, current pharmacological treatments remain inefficient or disappointing, the ultimate option for these patients remaining liver transplantation. Substantial efforts are thus required to develop alternative pharmacological therapeutic strategies. Our CHOLLID project focuses on clinically relevant ABCB4 and ABCB11 genetic variants that are associated with defects in their expression, traffic or function. The therapeutic challenge of CHOLLID, a bedside-to-bench-to-bedside project, is to characterize these variants at the molecular and cellular levels (structure, function, localization, molecular partners…), and to identify and develop new efficient targeted pharmacotherapies as an alternative for patients with severe forms of ABCB4- and ABCB11-related diseases. Such strategy is inspired from pharmacotherapies proposed for patients with cystic fibrosis, due to mutations in ABCC7/CFTR (cystic fibrosis transmembrane conductance regulator), but also from previous works of the coordinator to which the CHOLLID consortium gathers a broad range of expertise from the atomic scale to preclinical approaches. CHOLLID aims to develop a doctoral network that meets criteria of excellence in terms of training and science in the frame of the HORIZON-MSCA-2024-DN-01 call. For that, we are bringing together beneficiaries/partners with outstanding backgrounds in the various aspects of our project, from very basic in silico (molecular modeling, molecular dynamics…) and in vitro (cell models, organ-on-chips…) approaches to in vivo preclinical studies (mouse models mimicking the human pathologies), including innovative strategies (cryogenic electron microscopy, solid-state NMR, acellular in vitro functional assays…). So far, the enrolled beneficiaries have an excellent track record in their respective fields, ensuring a robust and valuable training for the recruited PhD candidates. These PhD candidates will be recruited in the frame of a fair, transparent and gender-equality process. During their PhD training, each candidate will have to opportunity to visit other labs of our consortium (“secondments” of up to six months on the overall PhD period) in order to benefit from different working environments, develop new skills and learn new techniques; these aspects are crucial for the excellence of our program and make it eligible under this MSCA-DN call. Moreover, non-academic beneficiaries/partners will be integrated in the consortium, allowing students to discover as many postdoctoral opportunities as possible. The basis of our consortium has already been settled and additional beneficiaries/partners still have to be confirmed/integrated. For that, we will have online meetings but we believe that in-person meetings are mandatory to discuss in details about our project and to finalize the building of a solid and coherent consortium. This will be done in a European capital/big city in a member state involved in this project. Organization and holding of such meeting(s) requires funding, which is the principal object of the present application to this ANR MRSEI call. In addition, part of this funding may be used to hire a private firm competent to improve and correct our application file.

Bile secretion is an essential function of the liver necessary for fat digestion as well as elimination of xenobiotics and endogenous metabolites. This function mainly depends on ATP-Binding Cassette (ABC) transporters located at the canalicular membrane of hepatocytes and responsible for the secretion of hydrophobic components into bile: ABCB4 for phospholipids and ABCB11 for bile acids. Genetic variations of these transporters are associated with rare cholestatic liver diseases, the most severe being Progressive Familial Intrahepatic Cholestasis (PFIC) type 2 (defects in ABCB11) and 3 (defects in ABCB4) in which children are affected during the first months of life. While ursodeoxycholic acid has exhibited satisfying relief in most patients with mild forms of ABCB4- and ABCB11-related diseases, most of PFIC2/3 patients do not or only poorly respond to this treatment, for whom liver transplantation remains the only alternative. In order to avoid or at least delay transplantation, our research project aims at identifying new small molecules of therapeutic interest able to correct the molecular defects of ABCB4 and ABCB11 due to mutations of the genes encoding these two transporters. Based on previous collaborative projects involving the three partners of this project, we will use structural kinase-inactive analogues of roscovitine, a trisubstituted purine, to rescue the intracellular traffic and the function of defective variants of ABCB4 and ABC11 identified in patients with rare cholestatic diseases. The ideal drug candidates must rescue the intracellular traffic without impairing transporter function. The candidate molecules will be selected using the following research approach: 1) Kinase-inactive roscovitine analogues will be synthesized by varying the chemical scaffold and substituents of the parent molecule, giving rise to a potential chemical library of more than 70,000 compounds. 2) The capacity of the newly synthesized molecules to rescue the intracellular traffic and the function of defective ABCB4 and ABCB11 genetic variants will be investigated in relevant cell models. The molecules displaying the best rescuing capacities and the less cytotoxic effects will undergo further validation in mouse models. 3) The best molecules will be chemically optimized in order to improve their benefit/toxicity ratio, by e.g., increasing efficacy at low doses. Such optimization will benefit from in silico experiments providing a realistic atomic-scaled pictures of structural defects of ABCB4 and ABCB11 variants. Particular attention will be paid to the lowering of inhibitory interactions between drug candidates and canalicular ABC transporters by means of molecular dynamics simulations. 4) Finally, the selected drug candidates will undergo a series of preclinical tests in mouse models in order to: i) determine their ADMET profile (Absorption, Distribution, Metabolism, Elimination and Toxicity); ii) validate their efficacy to rescue bile secretion in mouse models mimicking the human diseases (AAV8-mediated expression of the mutated human transporters in a KO background). This project gathers the expertise of three partners (two academics and a private biotech company) in the fields of medicine, pharmacology, biology, biochemistry, medicinal chemistry and molecular modelling. Our main goal is to provide a solid proof-of-concept that selected roscovitine analogues could be considered as relevant pharmacological alternative to liver transplantation for patients with rare cholestatic diseases related with molecular defects of ABCB4/ABCB11. This preclinical proof-of-concept is a prerequisite for the industrial development and the marketing of new drug candidates. Moreover, new targeted pharmacotherapies identified in the frame of this project might also benefit to more diseases caused by other impaired intracellular traffic processes related with genetic variations in patients.

The development of new biomedical technologies creates an increasing demand for improved types of adhesives able to be interfaced in a highly controlled and tunable manner with biological tissues. Current fixation methods are poorly satisfactory in this respect: mechanical fasteners, sutures and staples, are usually too damaging and induce unwanted inflammatory reactions; surgical polymer glues still suffer from insufficient strength, excessive swelling and/or toxicity. There is today a major unmet need for a biocompatible and easily-applicable technology to secure devices and implants onto the soft and highly hydrated surfaces of internal organs. The NANOBIOTAPE project integrates ex and in vivo evaluation into materials design to create a new type of bioadhesive surgical membranes. The NANOBIOTAPE project starts from a paradigm-shifting approach to bioadhesion recently invented at ESPCI Paris. This method relies on the use of nanoparticle solutions or powders in place of polymer glues: adhesion is produced by the adsorption of the macromolecules composing biological tissues at the surface of particles. By tailoring these adsorption processes, the project team proposes to design novel adhesive membranes, which can bind to tissues during a surgical operation and be detached harmlessly on demand. These novel functional surgical tapes will be specifically devised for the development of improved surgical practices in the treatment of liver cancer, which is the 5th most frequent localization of cancer worldwide and has been increasing by almost 3% per year since 2000. This multidisciplinary project is based on the close collaboration of four complementary teams uniting materials scientists to clinical researchers and practitioners in veterinary surgery and human hepatic surgery. Chemists and physicists from the Soft Matter and Chemistry laboratory at ESPCI Paris and the Centre des Matériaux at Mines ParisTech will design, fabricate and characterize the performances of the bioadhesive membrane. Veterinary surgeons from the Ecole Nationale Vétérinaire d’Alfort and hepatic surgeons from the Centre Hépato-Biliaire at Paul Brousse Hospital will use these adhesives in vivo in animal models and assess their clinical relevance. They will be assisted by histologists to determine the biocompatility of the implants. New surgical bioadhesives will be delivered that offer a new set of functions of interest to medical practitioners. These functions will be tailored to liver tissues and to procedures in hepatic surgery and could be rapidly transposed to other internal organs of interest. This research will provide the demonstrations of safety and effectiveness required prior to the first clinical trials. From a “One Health” perspective, the results obtained in NANOBIOTAPE will not only benefit to Human but also to companion animal patients in veterinary medicine. Discoveries and breakthroughs are also anticipated in the control of interfacial phenomena occurring between hydrogel-based devices and biological tissues.

The project DILI-on-chip addresses the development of a microfluidic device to assess in vitro adverse drug reactions, on a human-like liver lobule. We will focus on the particular case of DILI (drug induced liver injury). From a unique donor, hiPSCs (human induced pluripotent stem cells) will be differentiated to obtain the different types of cells, hepatocytes as well as non-parenchymal cells, that compose the liver lobule. Hepatocytes forming the hepatic cord, bordered by endothelial cells forming the sinusoid, and cholangiocytes forming the bile ducts will be organized in 3D on the chip, in order to get a functional unit mimicking the Hering channel. Kupffer cells will also be introduced in the vascular channels, as they are involved in the immune aspects of DILI. Multiple hepatic cords will be contained in microfluidic chambers, organized in circular lobule-like structures, and vascularized in order to induce the hepatocyte polarization, and bile canaliculi formation. Bile ducts will be formed in our chip, for the first time, by cholangiocytes maturation at the continuation of the hepatic chamber. Such biliary ways reconstructed on a chip is of prime importance as bile extraction is mandatory for the long term viability required for DILI assays. The microfluidic device will be composed of two parts ; i) a supporting substrate where will be organized the fluidic connections level, with the vascular and biliary flows. This substrate will be made out of Polydimethylsiloxane (PDMS) using the conventional soft lithography techniques or replicated in plastic (PolyMethylMetAcrylate) by hot embossing ii) on the top of this substrate will be assembled cell culture units, made out of polysaccharides gel, that have the suitable stiffness properties near to the one of the liver parenchymatic tissue, and the appropriate porosity for the nutrient and oxygen exchanges. The geometry of the microfluidic circuitry molded in this gel will be optimized in order to induce the tubular self-organization of cholangiocytes and endothelial cells, with the help of specific functionalization of the gel. An important feature of the device is the possible biodegradability of the gel part, that will render possible the collection of cells or cell content, for proteomics or transcriptomic analysis after drug exposure of the organ on chip. The polysaccharide gel structured units will be arrayed on the microfluidic subpart, in order to prove the feasibility of parallelization of the concept, with possible drug screening by the spotting of several drugs at different concentrations on the array, and collection of bile and vascular flows, for toxicology analysis in the context of DILI. Hepatotoxic compounds effects on the organ on chip will be analyzed, characterizing firstly the drug metabolism activity of the cells, then monitoring the damages induced to liver cells. Cumulative mechanisms of DILI will be finally addressed, by the drug exposure to cells presenting pre-existing pathologies. The project is organized in five tasks, including the management task. The tasks 1 will focus of the obtention of the different cells types that will compose the liver on chip, that will be differentiated from Induced pluripotent stem cells (iPSCs) from the same donor. The consortium will benefit from the huge experience of partner 2 in iPSCs cells differentiation. The second task concerns the fabrication of the microfluidic device, combining the expertises of the partners in microfluidics systems fabrication, and in the preparation and patterning of biodegradable gels. Task 3 will be devoted to the cell loading and culture within the device, including the fluidic instrumentation of the experiment. The formation of a functional hepato-cholangiocyte transition will be characterized on the chip in real-time. Finally, the task 4, led by the industrial partner, will focus on the drug assay on the functional liver on chip, in the DILI application context.

Despite significant progress in medical imaging technologies, there currently exist no tools capable of objectively helping healthcare professionals during liver transplantation surgeries. Instead, surgeons still rely on their own senses (vision and touch, primarily) to determine whether a transplant is healthy either before, during or after the procedure. In turn, surgery remains subjective and dependent on the experience of the surgeon, resulting in unacceptable failure, recurrence and morbidity rates, as well as in significant quality of care disparities across hospitals. As of January 1st, 2018, 1437 patients are on the wait list for liver transplantation, a number that is continuously increasing, with only 574 patients a decade ago. Every year, 10 to 12% of these patients will not survive the wait-time for getting a transplant. By large, these numbers are the result of the lack of objective decision criteria to determine whether a donor liver is healthy enough to be transplanted. In addition, following liver surgery, 7 to 10% of the patients will suffer liver function deficiencies. While the reasons behind these deficiencies are well known (combination of defects in micro-circulation, venous congestion, arterial thrombosis, asynchronies in hepatocyte regeneration leading to physiological disorganization), no tool currently exists to detect these deficiencies early during surgery and intervene in a timely manner. In turn, these deficiencies lead to costly emergency re-operations, and a survival rate decrease as important as 15%. Because these failures and complications can be mainly related to the lack of information regarding the liver, they could be avoided if tools were available to assist surgeons in visualizing the viability of the liver tissue both before and after transplantation allowing to select viable donors, as well as proper & timely intervention. Instead, surgeons subjectively rely on their own visual senses to assess the quality of the procedure, leading to an unacceptable morbidity and mortality rates. The hypothesis underlying our study is that Near-Infrared (NIR) light travels relatively deeply into tissues and is capable of providing critical information during surgery. In particular, oxy- and deoxy-hemoglobin, water and lipids can provide functional information, while scattering can provide micro-structural information. We recently developed a novel method called Single Snapshot of Optical Properties (SSOP) that relies on the analysis of the tissue response in the Spatial Frequency Domain to extract its optical properties (absorption and scattering). Because SSOP works entirely in the frequency domain, it is today the first and only method amenable to provide video-rate quantitative images during surgery. In this project we propose to develop innovative solutions to allow quantitative multispectral optical imaging in real-time during liver transplant surgery. To solve this challenging problem we have assembled a multidisciplinary team of scientists, engineers and surgeons. Pr Vibert from the Paul Brousse Hospital (HPB) in Paris is an expert in liver transplantation procedures. Dr. Diana from the University Hospital Institute (IHU) in Strasbourg is an expert experimental surgeon specialized in innovative surgical guidance techniques. Finally, the coordinator has invented SSOP, and our group and others at the ICube laboratory have a proven expertise in advanced photonics methods and the design of surgical guidance systems.