Trans-splicing gene therapy for Stargardt disease: construction of molecular and cellular tools to target ABCA4 gene mutations
This project aims to develop an innovative therapeutic approach for Stargardt disease, a macular degeneration caused by mutations in the ABCA4 gene. The strategy is based on SMaRT (Spliceosome-Mediated RNA Trans-splicing) technology, which enables mutation correction at the transcriptome level by replacing mutated exons of endogenous mRNA by trans-splicing with an exogenous RNA (PTM). Since the PTM contains only a part of the mRNA to be corrected, this approach can overcome the obstacle of the large size of the ABCA4 cDNA, which exceeds the carrying capacity of AAV vectors. The project will consist of several phases using molecular and cell biology techniques: construction of viral vectors for the expression of PTMs, production of cell lines to test the efficacy of binding domains (BD) to induce trans-splicing, and screening of BDs to optimize PTMs. Selected PTMs will then be tested in retinal organoids and animal models to demonstrate their therapeutic potential for the treatment of this genetic disease. As AAV is currently the most effective vector for retinal transduction, this project could open new therapeutic perspectives for Stargardt disease.
DEFENSOMES, COUNTER-DEFENSOMES, AND THE REMODELING OF MICROBIAL COMMUNITIES
Horizontal gene transfer (HGT) enables bacteria to swiftly adapt to new ecological niches and challenges. This process is primarily facilitated by mobile genetic elements (MGEs), such as bacteriophages (phages), plasmids, and transposable elements, which are prevalent in most genomes, often in multiple copies. The potential for conflicts arising from the interactions between MGEs and bacteria has driven the evolution of sophisticated defense mechanisms to filter, tame, or inactivate these elements. Well studied examples of anti-MGE immunity include restriction-modification (R-M), abortive infection, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Altogether, they revolutionized the field of genome engineering as precise cleavage / stabilization / editing tools, and further propelled the quest for additional defense mechanisms as well as MGE counter-defense strategies capable of curbing their action. The last decade has witnessed the identification and, in some cases, the mechanistic characterization of an extensive arsenal of previously unknown anti-MGE defense systems. These systems can be deployed at various stages of the MGE infection process, either by degrading invading nucleic acids, inhibiting their replication, or inducing dormancy or death of infected cells to stop the mobile element’s spread through the microbial population. With the growing number of anti-MGE families identified, so has the discovery of MGE-encoded counter-defense systems. Such counter-defensome deploys multiple mechanisms to inactivate host immune systems (beyond bacteriophage gene mutations), that include direct binding to immune proteins, post-translational modification of immune proteins, targeting of secondary messengers, and counteracting metabolite-depleting defense systems.
Many of the defense and counter-defense systems known to date have been uncovered through bioinformatic exploration of reference genome databases (e.g., NCBI RefSeq). Yet, the latter overrepresent organisms that can largely be cultivated in laboratory, and therefore provide a limited snapshot of the uncharted fraction of environmental microbial diversity that remains uncultured. To characterize this hidden diversity, we recently performed a large-scale screening of high-quality bacterial population genomes reconstructed from environmental metagenomes, highlighting the diversity of defensomes and the potential for functional cooperation and generation of novel functions between different defensive modules [1]. Findings stemming from this study raised further questions related to the nature of conflicts and alliances between defense system families, breadth of counter-defense strategies in the environmental phageome, as well as the tantalizing prospect of prioritizing core-defense genes for the development of antimicrobials capable of targeting an entire bacterial species. We propose to address such questions in the current proposal as follows:
1) The analysis of defense system co-occurrence / co-localization and synergistic immunity across bacterial species and biomes;
2) A first-of-its kind large-scale mapping of the counter-defensome of phageomes across multiple environments;
3) Analysis of the core layers of the defensome across bacterial species, with additional proof of concept that such genes (many of them now known to be essential), can be used as targets to develop antimicrobials aiming at eliminating an entire bacterial species.
Mutagenesis and selection of enzymatic catalysts for biotechnological applications: development of an integrated in vivo method
Due to their properties as catalysts producing highly enantio- and regioselective compounds from target substrates under mild reaction conditions, the use of enzymes in biotechnological processes is rising. However, their often insufficient activity on non-natural compounds and narrow substrate ranges still limit their use in industrial setups. To obtain enzymes with enhanced activities, methods of directed evolution are available, involving mutant gene library generation and high throughput testing of individual variants in a cellular context. Linking of the targeted enzymatic activity to cell growth by constructing strains conditionally auxotrophic for essential metabolites or for energy carriers have significantly enlarged the application range of directed evolution (Chen et al., 2022). To achieve spatial and temporal connection between mutagenesis and variant screening, in vivo mutagenesis approaches have recently been developed. Among them are inducible systems employing different deaminase base editors tethered to T7 RNA polymerase (T7 RNAP), provoking base substitutions concomitant to transcription depending on the deaminase used (Cravens et al., 2021; (https://2021.igem.org/ Team:Evry_Paris-Saclay). However, these techniques have not yet been applied for the amelioration of industrial biocatalysts.
The components of the systems, i.e. target genes, T7 RNAP-deaminase fusion proteins and regulatory modules, are plasmid borne. The PhD student will further develop this method by inserting the T7 RNAP editor and the target gene into the E. coli chromosome, thus stabilizing the system and opening the possibility of multiple rounds of mutagenesis and selection steps in GM3 automated continuous culture devices available in the laboratory. He/she will establish a mutagenesis and selection protocol, using a native gene enabling conditional metabolic selection as reporter. The validated protocol will subsequently be applied to heterologous NADPH-dependent dehydrogenases using a generic NADPH sensor selection strain constructed and used in the lab (Lindner et al., 2018). These will include the screening for alcohol and amine dehydrogenases, activities already studied by our group (Ducrot et al., 2020), to obtain variants with broadened substrate specificity. Their potential for synthetic applications will be assessed in laboratory scale, using targets chosen in collaboration with national and international partners. In vitro characterization of the enzymatic activity of enhanced variants will also be undertaken. The PhD student will benefit from multiple expertise and equipment of the UMR Génomique Métabolique, covering molecular genetics, synthetic biology, directed evolution, chemical analytics and enzymology.
Elucidation of the homarine degradation pathway in the oceans
Context:
Primary biological production in the oceans exerts significant control over atmospheric CO2. Every day, phytoplankton transform 100 million tonnes of CO2 into thousands of different organic compounds (1). Most of these molecules (as metabolites) are biologically labile and converted back into CO2 within a few hours or days. The climate-carbon feedback loops mediated by this reservoir of labile dissolved organic carbon (DOC) depend on this network of microbes and metabolites. In other words, the resilience of the ocean to global changes(such as temperature rise and acidification) will depend on how this network responds to these perturbations.
Because of its short lifespan, this pool of labile DOC is difficult to observe. Yet these microbial metabolites are the most important carbon transport pathways in the ocean and are assimilated by marine bacteria as sources of carbon and energy. Knowledge of the main metabolic pathways (from genes to metabolites) is therefore essential for modelling carbon flows in the oceans. However, the diversity of these molecules remains largely unexplored and many of them have no annotated biosynthetic and/or catabolic pathways. This is the case for homarin (N-methylpicolinate), an abundant compound in the oceans. Homarine content can reach 400 mM in the marine cyanobacterium Synechococchus (2) and this ubiquitous organism contributes between 10 and 20% of global net primary production (3).Because of its abundance, homarine is probably an important metabolite in the carbon cycle.
Project:
In this thesis project, we aim to elucidate the homarine degradation pathway in the oceans.
Ruegeria pomeroyi DSS-3 is a Gram-negative aerobic bacterium and a member of the marine Roseobacter clade. Its close relatives account for around 10-20% of the bacterial plankton in the mixed coastal and oceanic layer (4). In the laboratory, DSS-3 can use homarine as its sole carbon source but to date, there is no information on the genes and catabolites involved in this process.
Comparative analysis of RNAseq experiments conducted on DSS-3 cultures grown with homarine or glucose (control) as a carbon source will enable us to identify the candidate genes involved in the degradation pathway. This pathway will also be studied using a metabolomic approach based on liquid chromatography coupled with very high resolution mass spectrometry. The difference in profile between DSS-3 metabolomes from cells grown on glucose as a carbon source and those from cells grown on homarine will help to detect catabolites in the pathway. Finally, the candidate genes will be cloned for recombinant expression in E. coli, the corresponding proteins purified and their activity characterized in order to reconstruct the entire homarine degradation pathway in vitro.
Analysis of the expression of these genes in data from the Tara Oceans project (5) will be the first step towards a better understanding of the role of homarine in the carbon cycle.
References :
(1) doi.org/10.1038/358741a0
(2) doi.org/10.1128/mSystems.01334-20
(3) doi.org/10.1073/pnas.1307701110
(4) doi.10.1038/nature03170
(5) https://fondationtaraocean.org/expedition/tara-oceans/
Towards a cellular factory producing biohydrocarbons: biology and biotechnology of an emerging streptophyte microalgal model
In the evolutionary history of living organisms, the gradual adaptation of certain aquatic microalgae to an aero-terrestrial way of life was a crucial period, as it gave rise to all present-day terrestrial plants. Recent sequencing of the genomes of streptophytic algae, a group little studied until now, has begun to shed light on this evolutionary process. The appearance in ancestral streptophytic algae of the ability to synthesize and excrete hydrophobic compounds such as hydrocarbons, capable of forming a water-impermeable protective layer on the cell surface, was necessarily an important step in survival and adaptation to the aerial environment. Today, the inability of industrial algae to excrete hydrocarbons is a major biotechnological barrier to the biosourced production of hydrocarbons for green chemistry and fuels. The aim of this thesis project is therefore twofold: firstly, to characterize the synthesis and excretion pathways of hydrophobic compounds in an algae that is an emerging model of streptophyte algae and the only one in which hydrocarbon synthesis enzymatic equipment similar to that found in plants is present; secondly, for applied purposes, to use genetic engineering approaches to determine a set of proteins that will maximize hydrocarbon synthesis and excretion in this model alga.
Unraveling the mechanism of enzymatic carbon fixation
The Synchrotron Group at the Institut de Biologie Structurale in Grenoble is currently developing an innovative method called TR-FOX (Time-Resolved Functional Oscillation Crystallography). This technique aims to elucidate, firstly, the global dynamics of biological macromolecules in action and, secondly, their fine catalytic mechanism. It relies on the use of an injector capable of depositing onto the crystal, during the course of the X-ray diffraction data collection, a nanoliter droplet containing the substrate and cofactor of the studied reaction. This triggers the enzymatic reaction within the crystal. The approach can be combined with UV-Visible absorption spectroscopy to characterize the reaction kinetics more precisely. The goal is to obtain a series of structures during the catalytic cycle in order to make a molecular movie depicting the functioning of the enzyme. This thesis has two objectives: (i) improve and validate the TR-FOX method and, (ii) study the catalytic mechanism of two enzymes involved in carbon fixation either by capture or conversion of CO2.
Towards a better understanding of membrane proteins through AI
Despite the remarkable advances in artificial intelligence (AI), particularly with tools like AlphaFold, the prediction of membrane protein structures remains a major challenge in structural biology. These proteins, which represent 30% of the proteome and 60% of therapeutic targets, are still significantly underrepresented in the Protein Data Bank (PDB), with only 3% of their structures resolved. This rarity is due to the difficulty in maintaining their native state in an amphiphilic environment, which complicates their study, especially with classical structural techniques.
This PhD project aims to overcome these challenges by combining the predictive capabilities of AlphaFold with experimental small-angle scattering (SAXS/SANS) data obtained under physiological conditions. The study will focus on the translocator protein TSPO, a key marker in neuroimaging of several serious pathologies (cancers, neurodegenerative diseases) due to its strong affinity for various pharmacological ligands.
The work will involve predicting the structure of TSPO, both in the presence and absence of ligands, acquiring SAXS/SANS data of the TSPO/amphiphile complex, and refining the models using advanced modeling tools (MolPlay, Chai-1) and molecular dynamics simulations. By deepening the understanding of TSPO’s structure and function, this project could contribute to the design of new ligands for diagnostic and therapeutic purposes.
Effects of the combination of ionizing radiation and radio-enhancing molecules in breast cancer models
The proposed program aims to evaluate the efficacy of molecules enhancing the effects of radiotherapy, in in vitro and in vivo models of breast cancer. Two types of molecules, namely an inhibitor of mitochondrial genome maintenance and an inhibitor of the Base Excision Repair pathway, will be tested for radiopotentiation efficacy in the models.
The proposed inhibitors, whether targeting mitochondrial genome maintenance or the BER pathway, are already being investigated in vitro, both in the laboratory and by collaborators. We have shown that inhibition of the mechanisms targeted leads to an impairment in DNA damage repair following genotoxic stress. During this project, we will evaluate the effects of inhibitors on DNA damage repair induced by irradiation of different types (conventional, ultra-high dose rate, even extreme dose rate) and the associated mechanisms.
Variability in response to therapeutic combinations is frequently observed when moving from in vitro to in vivo models. We will therefore evaluate the inhibitors on cell line models well characterized in the laboratory, and corresponding to different breast cancer subtypes. On the other hand, the studies will be completed by a validation of the effects observed in vitro on a murine model of breast cancer. This xenograft model, developed in immunocompetent animals, will enable us to monitor the clinical, histological and immune response of the animals and their tumors, in order to confirm the interest of the molecules for therapeutic application in support of radiotherapy.
The proposed program will benefit from the laboratory's collaborations with physicists and chemists, and IRCM's experimental facilities and platforms (irradiation, animal experimentation, microscopy, cytometry, etc.).
Scaling of cytoskeletal organization in relation to cell size and function
Each cell type, defined by its function and state, is characterized by a specific size range. Indeed, cell size within a specific cell type displays a narrow distribution that can vary from as much as several orders of magnitude between smaller cells, such as red blood cells, and large muscle cells. Interestingly, this size characteristic is essentially maintained during the life cycle of an individual and highly conserved among mammals. Altogether, these features suggest that maintaining “the right size” for a given cell could play an important role in performing its function.
The actin cytoskeleton, that can form different stable while dynamic intracellular architectures, plays a major role in the structural plasticity of cells in response to changes in shape and size. Our recent work suggests that actin networks developed within a cell scale with the actual size and volume of the cell. However, how cells adapt the turnover and organization of their numerous structures assembled from a limiting pool of actin monomers remains to be understood.
In this project, we thus propose to study the organization and dynamics of actin networks in selected cell types displaying distinct sizes. In particular, our study will focus on characterizing the impact of such networks organization/dynamics on different cellular functions such as cell migration or adaptability to environmental cues. The feedback between cytoskeletal architecture dynamics, cell size and function will also be addressed by perturbing the organization and dynamics of the actin cytoskeleton in these cells.
Development of a dosimetry system to track alpha particles in in vitro assays for Targeted Alpha Therapy
Targeted Alpha Therapy (TAT) is a promising new method of treating cancer. It uses radioactive substances called alpha-emitting radioisotopes that are injected into the patient's body. These substances specifically target cancer cells, allowing the radiation to be concentrated where it is needed most, close to the tumors. Alpha particles are particularly effective because of their short range and ability to target and destroy cancer cells.
As with any new treatment, TAT must undergo preclinical studies to test its effectiveness and compare it to other existing treatments. Much of this research is done in laboratory, where cancer cells are exposed to these radioactive substances to observe their effects, such as cell survival. However, assessing the effects of alpha particles requires special methods because they behave differently than other types of radiation.
Recently, a method for measuring the radiation dose received by cells in laboratory experiments has been successfully tested. This method uses detecto