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2023 DIM PhD Fellowships laureates
NeuroMuscuLight
Cécile Martinat – Denis Furling
The NeuroMuscuLight project aims at establishing in vitro mature human neuromuscular junction models representative of neuromuscular diseases, and to develop new screening tools for drug discovery. Recent studies have demonstrated that human pluripotent stem cells can generate three dimensional, self-organized, in vitro tissue models of neuromuscular interactions named neuromuscular organoids. These more sophisticated and more mature models allow the studying of cellular interactions existing in such complex environment. The availability in I-Stem of various pluripotent cell lines, including those sourced from both patients and healthy individuals, enables the investigation of phenotypical alterations in a system mimicking the neuromuscular niche. This project also aims at developing an innovative light mediated contractile model of human neuromuscular circuit to provide a micro-physiological platform. Such a platform will be exploited for investigating mechanisms underlying neuromuscular pathophysiology, and assessing therapeutic strategies.
Characterizing the epigenetic response to AAV9-SMN1 gene therapy for SMA.
Piera Smeriglio – Giuseppe Ronzitti
Adeno-associated virus (AAV) based gene therapies are poised to have a large impact on rare diseases. Spinal muscular atrophy (SMA), a neuromuscular disorder characterized by motor neuron loss, is one such rare disease. Recently an AAV mediated gene therapy, Zolgensma, was approved for use in young children due to demonstrated increases in life expectancy and developmental milestones. However, the effect(s) of gene therapies like Zolgensma on the long-term stability of the epigenome is unknown. The epigenome, which consists of chemical modifications to DNA and histones, ensures that the right cells express the right genes at the right time. However, little is understood about how AAV mediated gene-therapies affect both the host epigenome and the AAV vector. The goal of the proposed work is to understand how the epigenome reacts to systemic gene therapy, specifically within the context of SMA, and to use this information to create better gene therapies.
Engineering of Signal peptide to improve Chimeric antigen receptor cell-based therapies
Franck Perez- Pierre Crozet
Cancer is a disease that affects millions of people worldwide making it the second leading cause of death. In the past years immunotherapies became one of the most promising and successful therapy. These include the therapeutic application of checkpoint inhibitors and the development of cell-based therapies like Chimeric Antigen Receptors (CAR). The latter allows specific arming and activation of cytotoxic immune cells (e.g. T and NK cells) to target tumor cells. CARs are usually composed by an antibody-derived fragment fused to a transmembrane domain and co-stimulatory motives required for immune cells activity. Despite of the high potential of CAR therapies, several limitations (e.g. side effects, limited efficacy due to T cell exhaustion, poor persistency, lack of infiltration) limit their deployement. We developped an approach, CARTune, that allows to control and potentiate CAR activity by tuning intracellular trafficking. Using a systems biology and screening approach we will improve essential module to potentiate further our technology.
Facilitating Advanced Stem Cell Techniques for Fat Engraftment
David Smadja – Jérome Larghero
Lipofilling has gained significant popularity; however, the long-lasting success of this technique has been limited due to low levels of fat survival. This project aims to develop innovative vascularized fat implantation strategies using progenitor cells to enhance fat engraftment and survival. In the preliminary studies, we demonstrated in a murine model that the addition of endothelial colony-forming cells (ECFCs) and mesenchymal stromal cells derived from Wharton’s jelly (MSC-WJs) to fat resulted in enhanced retention and increased neovascularization compared to fat alone. The specific aims of the project are as follows: (i) To investigate the involvement of human ECFCs in adipocyte formation and differentiation of MSC-WJs (ii) To develop a clinical-grade process for ECFCs large scale manufacturing (iii) To test the viability of fat engraftment with ECFCs and MSCs in a swine model. The findings from this project will pave the way for future clinical trials in lipofilling to improve patient outcomes and quality of life.
2023 Paris Région PhD Programme laureates
The 2023 call for the Paris Région PhD programme was reserved to projects in the 9 DIM’s thematics who were focusing on computational, AI or data-driven projects whose funding had to be complemented by an industrial partner. The DIM BioConvS led the scientific evaluation of the project and the follow-up of PhD students.
Computational and molecular approaches to reprogram BCL11A splicing and advance therapeutic strategies for β hemoglobinopathies.
Eric Allemand (Imagine institute) & Innovhem
β-hemoglobinopathies are a group of genetic diseases characterized by abnormalities in the production of hemoglobin, a protein contained in red blood cells and which carries oxygen in the blood. The clinical severity of patients in adulthood may be attenuated by persistent reactivation of the fetal globin gene BCL11A is the major repressor of fetal globin expression and its regulation by splicing leads to the production of distinct protein isoforms.
BCL11A-XL is the isoform primarily responsible for fetal globin repression, while BCL11A-L/S/XS lacks this activity.
Our project is based on the hypothesis that a thorough analysis of the alternative splicing of BCL11A will allow us to reprogram its regulation in order to reactivate the expression of fetal globin. We will use third-generation sequencing to obtain an exhaustive repertoire of BCL11A transcripts in fetal versus adult erythroid cells, and develop an alternative splicing analysis algorithm to achieve this. Next, we will use antisense oligonucleotides (ASO) to reprogram the splicing of BCL11A, decreasing the production of the BCL11A-XL isoform and activating the expression of fetal hemoglobin. The best-performing ASOs will also be tested on cells from patients with hemoglobinopathies to assess the correction of the pathological phenotype.
This project will be carried out in collaboration with Innovhem, a company with valuable expertise in the measurement of fetal hemoglobin at the level of a single cell. Their technique is patented and applicable to circulating red blood cells of patients treated by gene therapy to measure the expression of fetal hemoglobin. This collaboration therefore aims to establish a new fetal globin reexpression therapy in patients with β-hemoglobinopathies.
Optimisation de la biosynthèse en bioréacteur et de la purifi cation de métabolitesvégétaux par une approche de plan d’expérience et de modélisation
Alexandre Maciuk (Université Paris Saclay) & Alkion Bioinnovations
The adjuvants for vaccines QS-21 and QS-7 have experienced significant growth since COVID-19, validating their superior efficacy without proven toxicity at equivalent doses. Their limited availability and cost make it impossible for industrial use of this adjuvant for populations in low-income countries. Optimizing the productivity of these saponins remains necessary to establish local industrial production. To achieve this, the implementation of an experimental production testing protocol on 5-7 parameters (ranges of 3 concentrations or intensities) at 2-4 light frequencies, influencing biomass production and saponin productivity, will allow the creation of a multifactorial database (culture parameters, plant images, metabolomic data on 10 metabolites). Using numerical models of biological behavior associated with image recognition through artificial intelligence using software like Matlab, R+, (…), these data will generate optimal solutions for productivity in temporary immersion bioreactors, which will be tested and analyzed before validation. Finally, the purification process will be optimized through preparative chromatography process modeling.
Microfluidic stimulation and multi-parametric analyzes by deep learning of the production of extracellular vesicles by circulating tumor cells
Catherine Villard (Université Paris Cité) & Fluigent
This thesis project integrates innovative instrumental development in the field of microfluidics, led by the socio-economic partner Fluigent, to answer a central question in oncology, that of tumor progression by dissemination through the bloodstream. This question is closely linked to mechanisms of bioproduction of extra-cellular vesicles (EVs) by a still unexplored mode of hydrodynamic stimulation, inspired by the stresses encountered by circulating tumor cells in the blood. As a result, this project of bioproduction of EVs by microfluidic technologies also have an opening towards the clinic in its dual diagnostic and mechanistic dimension. Artificial Intelligence approaches are an integral part of this project, on the one hand at the instrumental level for the real-time control of flows operated by the instrument, and on the other hand for the multi-parametric analysis (morphological, genetic, proteomic) of biological samples (cells and EVs).
From an academic point of view, this thesis will shed light on one of the possible origins of EVs participating in the metastatic cascade. From a technological point of view, it will offer a new way of bioproducing EVs, of cancerous origin or not, as well as a new instrument allowing the recirculation of fluids containing cells through microfluidic chips.
2023 Postdoc call laureates
Genome editing with RNA-based programmable synthetic organelles
David Bikard – Ariel Lindner
The ability to introduce genetic modifications at specific positions in genomes, mediated by CRISPR/Cas, accelerated research in genetics and enabled countless potential applications, from engineered microorganisms for bioproduction to gene therapies. However, off-target activity of CRISPR/Cas remains a primary concern. Inspired by recent discovery of RNA- and protein-mediated (liquid-liquid phase separation) LLPS at DNA repair sites in health and disease, we wish to set a novel strategy to localize genome modifying enzymes to specific loci while excluding undesired proteins. To this end, we will explore how the efficiency and precision of genome editing strategies could be improved by recruiting engineered RNA liquid-liquid phase separated (LLPS) organelles that harbour DNA-modifying enzymes to specific DNA positions within bacterial genomes. As all components were shown to work in Eukaryotic systems, success in this project will carry significant impact on genome editing at large.
Quantitative interactions between synthetic therapeutic bacteria and the tumor microbiota
Pascal Hersen – Maria Carla Parrini
Synthetic biology has produced engineered therapeutic bacteria that successfully deliver cancer-killing payloads to solid tumors in murine models. However, these therapeutic bacteria don’t seem to replicate their functionality in humans. On the other hand, very recently, the tumor microbiota has emerged as a key player influencing tumor development with evidence for both inhibitory and stimulatory effects. Because both therapeutic and native intra-tumoral bacteria grow isolated from the immune system, such microbial interactions appear as unexplored territory and a timely convergence of subjects at the interface of microbial ecology, synthetic biology, and cancer biology. Here we will develop a platform to culture tumor-derived bacteria with spheroids and quantify the action of key bacterial therapeutics, allowing a better understanding of the dynamic processes governing intra-tumoral bacterial growth and competition, and guide the design of new effective bacterial therapeutics.
Bacteria delivered anticancer therapy in heterogeneous tumor microenvironment
Philippe Nghe – Olivier Tenaillon
In this project, we propose to develop a technology to recreate tumor microenvironments in co-cultures of bacteria and human cells, measure growth by time-lapse microscopy and obtain endpoint transcriptomics of both bacteria and human cells. Until a few years ago it was not accepted that the tumor microenvironment may contain microorganisms. In contrast, it is now considered as key property of tumor physiology. This also supports the concept of engineering microorganisms to cure cancer, a major goal of synthetic biology. Here, we propose to combine the research on the tumor microenvironment and engineering of microorganisms with the end goal of better fundamental understanding of their interactions, enhanced anti-cancer therapy, and application in precision and personalized medicine by developing a device for ex vivo diagnostics.
2023 Small to Medium Equipment Call laureates
Gene therapy approaches for rare neuromuscular disorders
Project leader: Valérie Allamand
Equipment: electroporation system Neon NxTTM (InVitrogen)
Neuromuscular disorders regroup numerous phenotypes that are clinically and genetically heterogeneous. Work in the Center of Research in Myology focuses on several subtypes for which there is no curative treatment to date. We strive to understand the pathomechanisms at play in order to develop and test innovative therapies. Our projects rely on patient-derived cell models (dermal, myogenic, neuronal, iPSCs), to assess therapeutic efficacy in vitro, prior to pre-clinical testing in animal models. The proposed project focuses on gene therapy strategies aiming to re-express or modulate the expression of mutated proteins of interest, using tRNAs, overexpression systems and/or RNA interference. These approaches are hampered in vitro by the difficulty to efficiently transfect human and mouse primary/immortalized cells. The acquisition of the Neon™ NxT electroporation system will greatly facilitate our project by bridging a critical technical gap.
Future biotherapies for severely burned patients
Project leader: Sébastien Banzet
Equipment: Nanoparticle characterization device VIDEODROP (Myriade)
Cell therapy has proved its worth in a number of tissue repair indications, notably wound healing. Severe burns are of thermal or radiological origin. They cannot heal on their own, and so require skin grafting, which involves collecting healthy skin from the patient and grafting it onto the burned area. The graft needs to expand considerably to cover large areas, which poses problems of graft take. Our group has developed an adjuvant treatment to surgery, consisting in taking cells from the patient, multiplying them and injecting them at the time of grafting. This improves engraftment and reduces inflammation. We’re working on a simpler approach involving treatment not with the cells themselves, but with substances they secrete, including extracellular vesicles (EVs). These products would be easier to preserve and immediately available. The acquisition of the requested equipment would enable us to quantify these EVs at all stages of production.
Identification and isolation of beneficial mucosal commensals
Project leader: Benoit Chassaing
Equipment: SH Cell Sorter (SONY)
The human intestinal tract is colonized by a myriad of microorganisms, collectively named the
intestinal microbiota, which contains both beneficial and harmful members. For example, in the normally
nearly sterile mucus layer that protects human intestinal epithelium, the balance between beneficial and
harmful microbiota members is highly associated with health status. Our aim here is to isolate, biobank
and characterize new mucus-resident bacteria. We propose to biobank live commensals from the mucus
lining using a cutting-edge equipment able to sort stained single bacteria isolated from mucosal biopsies
and placed in an oxygen-free environment to sustain bacterial viability. Such live mucus-associated
bacteria cannot be isolated with currently available equipment, and will highly contribute to the
understanding of the role played by mucus colonizers in health and disease. This project will significantly
expand the range of microbiota-derived candidates with beneficial properties (next-generation
probiotics).
BLOOd-Derived Products’ ACtIve Fraction Identification for Corneal healing
Project leader: Eric Gabison
Equipment: KR2I TFF System- KF COMM 2- with scale (REPLIGEN)
The cornea is the protective lens of the eye, perfectly transparent. The most superficial layer of the cornea is constantly being renewed, allowing it to maintained its transparency and to cope with everyday aggression. In the case of pathologies, infections or trauma, wound healing can lead to corneal opacification, resulting in reduced visual acuity or even blindness. There are medicines in the form of eye drops, prepared from the patient’s own blood, which promote healing while preserving transparency. Our project is to identify the active molecules in order to develop more effective eye drops for corneal pathologies for which therapies are either unsatisfactory or very expensive. We want to isolate a specific fraction of blood to validate our hypotheses and we need a tangential flow filtration system that can be used routinely in our laboratory.
DACS
Project leader: Jacques Fattaccioli
Equipment: MOTIC Inverted Epifluorescence Microscope
Cell therapies have shown remarkable therapeutic outcomes for blood cancer diseases and hold great promise for solid cancers. However, their manufacturing process remains a major bottleneck, leading to its high costs. Therefore, it is crucial to develop tools that automate, standardize, and ensure flexibility and scalability. Among the manufacturing steps, cell sorting and activation are often overlooked despite their detrimental impact on the final product. Lack of control in these initial steps can increase production time, reduce treatment efficiency, and even result in production failure, worsening patient outcomes. At Ecole Normale Superieure, we aim to develop a closed-automated system based on our proprietary technology (DACS) to address these challenges. DACS allows gentle cell sorting and activation at any volume or cell concentration. This innovative solution aims to streamline manufacturing, reduce costs, and improve accessibility, ultimately democratizing cell therapies.
High throughput imaging and analysis to assess Vascular endothelial architecture upon bacterial metabolite stimulation
Project leader: Diego Garcia-Weber
Equipment: filters and lenses for Cytation5 (Agilent)
The healthy intestinal microbiota produces several families of small anti-inflammatory metabolites that reach the systemic circulation and end up in direct contact with the vascular endothelium. Although some of them have been shown to exert some protective effects on endothelial cells, the effect of most of them has not been studied in endothelial models yet, mainly due to the difficulty in accessing high throughput imaging technologies. Thus, this project aims to implement a high throughput imaging and analysis technology to screen the effect of bacterial metabolites on endothelial cell architecture. Thanks to the present call, the Cytation5 automated microscope from Agilent already present in the laboratory will be upgraded with more resolutive objectives (20x, 40x), more excitation wavelengths (GFP, RFP, Cy5) and a complete version of the analysis software (Gen5iPrime). It will be made available to other members of the laboratory, the CRSA institute and the network of present and future collaborators.
Glucose Delivering Scaffolds for promoting survival and functionality of MSCs
Project leader: Hervé Petite
Equipment: SPARK Multi-Mode Microplate Reader (TECAN)
Mesenchymal stromal cells (MSCs) are promising candidates for regenerative medicine, but the initial ‘proof of concept’ of most MSC-based therapies has not yet been translated into routine clinical practice due to their massive death after implantation. At B3OA, we demonstrated for the first time that hMSCs exposed to near-anoxia, but not glucose deprivation, remained viable and functional. From a tissue engineering perspective, these data demonstrate that MSCs loaded into glucose-supplemented scaffolds can survive an ischaemic episode and remain functional, prompting the development of glucose-releasing scaffolds. The proof of concept of such scaffolds containing starch (a polymer of glucose) and amyloglucosidase (an enzyme that releases glucose from starch) has been established at B3OA. The proposed project aims to further optimise these scaffolds and then evaluate their functionality in the context of bone repair. Ultimately, such materials will have numerous applications in cell-mediated regeneration.
Speeding up precision Medicine in Cystic Fibrosis
Project leader: Isabelle Sermet-Gaudelus
Equipment: MTECC setup
Cystic fibrosis (CF) is a life-limiting genetic disease affecting approximately 70,000 people worldwide. CFTR is the main Chloride channel of the airway epithelium. Absence or dysfunctional of the CF transmembrane regulator (CFTR) protein leads to the accumulation of thick airway mucus in the lungs, chronic infection and inflammation, and finally respiratory insufficiency. CFTR modulators which treat the underlying cause of the disease by directly correcting the CFTR protein, lead to considerable improvement of respiratory function and quality of life for 85% of the patients. For the 15% remainings, we have demonstrated that evaluation of CFTR activity rescue in their nasal primary cells, mimicking a reconstituted airway mucosa, can be used at the patient level (personalized medicine) to predict response to current CFTR modulators and at the mutation level (precision medicine) to search for novel therapeutic strategies. This provides the path to use CFTR activity rescue as a biomarker predictive of the clinical respiratory response in patients treated by strategies aiming to restore CFTR. This funding will support buying a set-up which will considerably speed up our research program, by enabling perform many simultaneous experiments in an automated way, to assess CFTR activity in different conditions.
Deep Inside Muscle Engineered Tissues
Project leader: Myriam Reffay
Equipment: 920 nm Pulsed Fiber Laser
Within the 2-years DIMET project, we have the ambition to outmatch the challenge of artificial muscle micro-tissues to replace animal models and open up new avenues of investigation into biotherapies to treat muscle pathologies. Using magnetic nanoparticles, we are transforming the living cell itself into an actuator to enable these challenges to be met to form stimulable aggregates for the study of muscle tissues formation, organization and properties. While our work to date has focused on labelled cell models, how can we generalise this original all-in-one approach to real muscle tissue? The challenge of imaging living tissue is therefore at the heart of the technological challenge we propose to overcome. We therefore request a femtosecond pulsed laser that will enable us to study the endogenous fluorescence of tissues and their organization to evaluate the action of biotherapies in myopathies.
Culturing intestinal microbiota, a tool for mechanistic and therapeutic discoveries
Project leader: Nathalie Rolhion
Equipment: Anaerobic culture chamber (Labo and Co)
The intestinal microbiota is a large and diverse microbial community that inhabits our intestine. Modifications of the intestinal microbiota are associated with numerous human diseases. The link between intestinal microbiota and human health is mainly based on correlatives studies and functional studies are now needed to decipher the mechanisms and offer new microbiota-based therapeutic approaches such as probiotics. In vitro culture of intestinal microbiota or intestinal micro-organism(s) is extremely challenging and relies on an anaerobic atmosphere, as most of the intestinal micro-organisms are sensitive to oxygen. In this context, we aim to acquire in our laboratory an anaerobic chamber to cultivate intestinal microbiota or intestinal micro-organism(s) to tackle mechanistic and therapeutics questions on the role of microbiota in human health.
A cell therapy approach to improve radiotherapy efficiency
Project leader: Germain Rousselet
Equipment: Nucleofector 4D core unit (LONZA)
Radiotherapy exerts its effects through direct killing of tumor cells, but also through stimulation of an anti-tumor immune response. However, methods for modulating this radio-induced immune response are still lacking. Interferon beta (IFNb) is a central player in the response. We have shown that a protein called TRIM33 restrains IFNb expression in myeloid cells, a sub-population of immune cells. Indeed, radiotherapy is more efficient in mice carrying a deletion of the Trim33 gene in myeloid cells, qualifying TRIM33 as a potential target for radiotherapy improvement. In order to treat patients, we chose to develop a strategy to delete the Trim33 gene in myeloid cells ex-vivo, and then inject these cells back into the patient. We want to develop a proof of principal for this method in mice. One key step is to delete Trim33 ex vivo in primary myeloid cells by CrispR/cas9, in a method that requires a nucleofection device for which we apply.
Miniaturized asymmetrical flow field flow fractionation on a soft thermoplastic chip: a high performance purification and analysis system for a large range of size of biotherapeutics
Project leader: Hugo Salmon
Equipment: Zeiss SteREO Discovery.V20 Stereo Microscope
To accelerate the bench to bed translation of emerging nanomedicines and
biotherapeutics, a growing need in flow-based separative methods (asymmetrical flow field flow fractionation (AF4) and tangential flow filtration (TFF)) is required for their purification and characterization. There have been attempts to miniaturize them to increase their throughput and general performances but the membrane encapsulation remains a bottleneck. With MISOSOUP, we acquire a stereomicroscope and a thermostator and implement a patented method to perform miniaturized AF4 and TFF on a soft thermoplastic chip in a thermostated manner. We then demonstrate the performances on a set of nanoparticle standards and extend it to therapeutics.
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