tRNA-mediated therapy for collagen VI-related disorders due to nonsense mutations

Valérie Allamand (Sorbonne Université, Inserm) and Yves Fromes (Myology Institute)

At least 10% of human genetic diseases are caused by nonsense mutations that introduce premature termination codons (PTCs), often leading to loss of function. Collagen VI (COLVI) deficiency causes rare neuromuscular disorders covering a large phenotypic spectrum, for which there is no curative treatment. Patients carrying homozygous nonsense mutations in the COLVI-encoding genes display the most severe clinical presentations The project aims at correcting PTCs using anticodon-engineered tRNAs delivered by gene therapy vectors, in vitro (human and mouse cells) and in vivo (mouse model). This approach should allow re-expression of full-length COLVI and restoration of muscle homeostasis, which will be monitored by state-of-the-art techniques.The strength of this strategy is to enable faithful cognate amino-acid integration in place of the PTC, independly on target gene or PTC location, while sparing native stop codons. Thus, it has the potential to address an unmet clinical need for numerous untreatable genetic diseases, benefiting numerous patients.

Design of regulatory RNA with integrated machine-learning models and evolution in the lab

Simona Cocco (Ecole Normale Supérieure (ENS), CNRS) and Bruno Sargueil (Université de Paris ,CNRS)

Designing new functional RNAs, with controlled specificity, affinity, thermal stability,allostery … is a fundamental challenge in bioengineering, with great promises for therapeutic diagnosis and treatments. In this PhD project, we will focus on two classes of functional RNAs: bistable molecules, changing structure upon the binding of cognate metabolites, and aptamers binding specific targets. We will develop new Machine Learning (ML) methods to learn model from sequence data collected from public databases and from high-troughput directed selection (SELEX) experiments. We will then use these ML approaches to design functional RNAs, whose structural properties will be validated using chemical probing (SHAPE) methods and binding assays and to guide the SELEX experiments. The thesis will be carried out in the Laboratoire de Physique de l’ENS from PSL and in the Therapeutic Targets and Drug Design lab from Paris Cité, in collaboration with the Laboratory of Neurodegenerative Diseases at CEA-Paris Saclay.

Evaluation of calcium transients in muscle cells

Sonia Albini (Genethon)

Calcium mishandling in Duchenne muscular dystrophy (DMD) suggested that dystrophin may regulate calcium-signalling cascades such as calcium entries. Calcium overload in human DMD myotubes is associated with muscle fiber injury indicating that calcium homoestasis is critical for muscle integrity. With this equipment, we aim at monitoring calcium transients to detect abnormal calcium regulation in DMD to be able to use it as readout for gene therapy testing. Thus, calcium flux restoration and consequently, membrane stability can be assessed using the proposed system

Organoids and Organ-on-CHIps technologies to resolve paradigms in basic biology and REgenerative Medicine

Thierry Jaffredo (IBPS)

Organoids and organs-on-a-chip technologies are becoming increasingly important in biology and regenerative medicine. We are resolutely embracing this trend, which makes it possible to take the complexity out of biological issues while maintaining a 3D approach close to living organisms. We are in the process of pooling and rationalising these technologies by creating optimised conditions for observing and preserving the samples needed to resolve the biological and medical questions we are asking ourselves. These conditions include the acquisition of an EVOS M500 type culture microscope specially dedicated to the fine observation and recording of 2D and 3D cell cultures in visible light and fluorescence, as well as a 231-litre freezer at -150°C that will ensure the stable and safe conservation of pluripotent cells, their derivatives and the biological and medical samples received by the teams.

Automated platform to engineer pathogenic strain libraries via optimized conjugation

Sara Napolitano and Angelica Frusteri (Institut Pasteur)

Many CRISPR-based strategies have been recently developed to probe the functioning of bacterial cells. In clinical microbiology, these tests need to be performed on collections of clinical isolates to obtain robust results. However, pathogenic strains are often more difficult to engineer than lab strains. The construction of collections of engineered pathogenic bacteria is a challenge. The goal of our project is to develop an automated platform that enables robust and high-throughput construction of such collections through optimized bacterial conjugation. The platform will be based on a cheap liquid handler robot, a compact plate reader, a large class 2 biosafety cabinet, and control software. Several applications will be performed, notably for dissecting the mechanisms of unexpected resistance to antimicrobial treatments of clinical isolates. No such platform seems to be available in the Paris area. It will be shared within the BioConvS community upon reasonable request.

Microvesicles, Cell and Gene Therapies Alternative For Blinding Eye Surface Lesions

Benoit Souquet (Hôpital Fondation Adolphe de Rothschild)

Stem cells derived, for example, from umbilical cords, produce nano-sized vesicles with great therapeutic properties. We want to bring new properties to these stem cells to give specific therapeutic action of the vesicles they produce, to treat diseases of the cornea. The cornea is the watchglass of the eye, whose function is to protect the interior of the eye and to be transparent. Some corneal diseases have no cure yet, and will progressively opacify the cornea, leaving patients blind. Our aim is to produce an eye drop containing these modified vesicles to treat these diseases. But to manipulate and observe these vesicles, invisible to the naked eye and difficult to observe with conventional microscopes, specific instruments are necessary.

Producing recombinant monoclonal antibodies without animal-origin products

David Tareste (Institut de Psychiatrie et Neurosciences de Paris (IPNP))

Monoclonal antibodies play a crucial role in various scientific and medical applications, from detecting molecules in research to developing treatments for conditions such as cancer and infectious diseases. Current methods for antibody production involve injecting antigens into animals – causing stress and pain – and ultimately require their sacrifice for antibody purification. Our project addresses this ethical concern by eliminating the need for animal-derived products in antibody production. Our approach leverages available antibody sequences, cloning them into cell lines that enable high-level protein expression without relying on animal-derived components. These recombinant monoclonal antibodies will be purified using the requested AKTA system and qualitatively analyzed using systems already available in our facility. This approach not only provides a more ethical means of production but also enhances the quality and specificity of monoclonal antibodies by avoiding animal-origin products that can affect reproducibility.

Multidisciplinary approach to stimulate the bioproduction and engineering of Gram+ bacteria’s Extracellular Vesicles (EVs) for therapeutic applications to treat inflammation.

Jean-Marc Chatel (MICALIS)

We propose a biotherapy based on therapeutic extracellular vesicles (EVs) as an alternative to live probiotics. EVs will be produced in a scalable bioreactor at high yield from Gram+ bacteria abundant for intestinal microbiota and characterized at biophysical and molecular levels (proteins, lipids, sugars, nucleic acids, metabolites). We will also engineer the content of EVs to enhance their intrinsic immunomodulatory activity. The anti-inflammatory and neuromodulatory properties of EVs from an isolated species and a pool of EVs from a combination of species will be tested in vitro and in vivo in preclinical models of acute inflammation (Inflammatory Bowel Diseases-IBD), low-noise inflammation (Irritable Bowel Syndrome-IBS) associated with cognitive-emotional and behavioral disorders, and neurodegenerative pathology (Alzheimer’s).

Rational Shuffling: Optimizing AAV for Targeted Liver Gene Therapy

Tiziana La Bella (Genethon)

Adeno-associated virus (AAV) are the vectors of choice for gene therapy in vivo due to their excellent safety profile and the proven efficacy in pre-clinical and clinical studies which led to achieve marketing authorization for some of the AAV-based products. Despite these enormous successes, limitations in their use still exist, including the specificity of the tissue targeting and the immune response against the AAV capsid in human. This project aims to develop a new generation of AAV vectors for liver gene therapy by using an innovative engineering method. Our approaches involves modifications of the AAV capsid surface which plays a central role in both tissue tropism and antibody recognition. The project seeks to identify capsid regions crucial for liver tropism and gather valuable insights for designing improved AAV variants.

Phenotyping Impact of Optimized Gene Therapies in Rett Syndrome Mice

Nicolas Panayotis (SPPIN – Saints-Pères Paris Insitute for the Neurosciences)

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations of the transcriptional regulator MeCP2. While RTT is incurable, mouse studies could rescue the pathology upon MeCP2 reexpression. Gene therapy (GT) still faces challenges in controlling MeCP2 levels. MeCP2 is nuclear but the mechanisms involved in its nuclear shuttling, including the importin machinery have never been targeted in RTT. We aim to develop a new therapy combining AAV-based MeCP2 replacement with manipulation of the importins. The T158M mutation disrupts MeCP2’s DNA binding, leading to severe RTT. Acquiring Ethovision XT, a versatile system to track and analyze mouse behavior, will help us to study cohorts of T158M mice treated with various vector designs and define how fine-tuning MeCP2 nuclear levels potentiate GT. Parallel research includes morphological analyses and transcriptomics. This work will provide insights on how to ameliorate pathologies caused by alteration of other transcription factors’ subcellular levels.

High content screening for myelinated neurons On a CHIp

Hugo Salmon (Université Paris Cité – Faculté de Sciences)

Peripheral neuropathies affect about 5% of the general population after the age of 50 and are caused by a wide set of factors among whom diabetes, genetic causes, toxic (such as chemotherapies, pollutants) and life habits. Extracellular vesicles (EV) seem to play an important role in regulating myelin physiology. They constitute a potential avenue for EV based biotherapeutics, especially for the demyelinating peripheral neuropathies. In HCS-MOCHI, we will develop the high content screening (HCS) to explore a neurofluidic myelinating model of the nervous system. Taking advantage of compartmentalized architectures and soft styrenic block copolymer for microfluidics, the HCS will allow a better control on neuron and SCs differentiation starting at precursor state until myelination, compatible with high content screening for neuropharmacology.

New methods of bioproduction of Extracellular Vesicles for the treatment of trauma patients

Marina Trouillas (Hopital Paul-Brousse – Insitut André Lwoff)

Severe trauma is the principal cause of death among young people worldwide. Trauma, from all causes, induces deregulation of major physiological crossroads which contribute in the short term to cellular and tissue destruction and lead to repair abnormalities. Our unit has already demonstrated that mesenchymal stromal cells and their secretion factors are capable of modulating these pathways, and thus limiting the worsening of lesions or promoting tissue repair. In a translational research approach, we are confronted with regulatory constraints for clinical quality production. The acquisition of a bioreactor will help develop our means of producing acellular products optimized for tissue repair, prepared under conditions compatible with regulatory requirements.

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.