Exploiting genetic biomarkers of neurodegenerative diseases for developing early diagnostic tests and possible treatments

Dementia is a general term for decline in mental ability and cognitive dysfunctions severe enough to interfere with everyday life. Dementia encompasses different but often overlapping pathologies including Alzheimer diseases (60-80% of the diagnosed cases) along with Parkinson’s, Pick’s (also known as frontotemporal degeneration, FTD) and Charcot’s diseases (also known as amyotrophic lateral sclerosis, ALS). These neurological disorders occur because nerve cells involved in cognitive functions undergo degeneration. There are currently no convenient, reliable clinical assays and therapeutic approaches to diagnose and treat dementia, which make them a societal issue of utmost importance. A breakthrough came in 2011 when scientists discovered a common genetic basis for two types of dementia, FTD (the second most common cause of early-onset dementia) and ALS. These two diseases, which display considerable pathological overlap, are so intertwined that they are now commonly referred to as FTD/ALS diseases. It was thus discovered that a defect in chromosome 9 (more precisely, in the open reading frame 72 of chromosome 9, so called C9orf72) is responsible for FTD/ALS diseases. This genetic aberration is characterized by the expansion of the hexanucleotide repeat GGGGCC, or d[G4C2], with an average of 2 repeats for healthy people while FTD/ALS patients carry up to thousand repeats. The G4C2-repeats thus represent a reliable genetic biomarker that could help clinicians diagnose FTD/ALS earlier. In this project, named DEMENTIA, we first describe the current state of knowledge on FTD/ALS diseases, from the mechanistic bases of the G4C2-mediated or -promoted neurotoxicity to the therapeutic strategies that are currently investigated to tackle this critical societal issue. We next exploit the wealth of data recently acquired on C9orf72 repeats, with a particular focus on the RNA r[G4C2] repeats that are more readily accessible in nerve cells, with a two-fold purpose: 1- to study precisely the secondary structures they might fold into (i.e., hairpin and/or quadruplexes) in order to decipher the conditions that govern their folding dynamics; we will then exploit these data to develop an in vitro fluorescent assay capable of detecting these structures from cell and RNA extracts for diagnosis purposes; and 2- to exploit this in vitro assay to identify new chemicals that could disrupt these higher-order structures (known to be responsible for neural injuries) to identify new drugs capable of curbing or reversing neurodegeneration, therefore addressing an unmet need for effective therapies to treat dementia. To this end, DEMENTIA relies on the complementarity of the people and the institutes involved (ICMUB at Dijon, Institut Curie at Orsay and IECB at Bordeaux) and is built on the firmly established expertise of the project’s partners, according to a two-way organization that makes it a low-risk, yet ambitious and resolutely interdisciplinary research program.

InsidePores
InsidePores proposes an original and low cost approach to elaborate nano-structured inorganic materials (NPs) by a solution process. The novelty relies on the use of dedicated porous supramolecular materials acting as matrixes for template-assisted growth of the NPs. We aim at using the confined space within the supramolecular template to grow metal and metal-oxide materials, which size, shape, and spatial organization will be controlled by the host's porosity.

An essential step of the overall process is the insertion of the molecular precursors of the NPs into the porosity of the matrix. To achieve efficient and fast loading, it is desirable that the incoming guests have weak interactions with the matrix. For this reason, the selected matrixes have the walls of their channels functionalized by chemical groups able to form such weak links with the guests.

InsidePores is an exploratory project dedicated to the optimization of the chemical procedures allowing the preparation of shape and size controlled nano-objects, either as composites (NP@matrixe) or free materials. It focuses primarily on ZnO and targets a material that will exhibit only the excitonic emission in the UV energy range (desirable for UV laser and blue-LED applications). An extension concern metallic NPs of noble metals (Ag, Au...) for the elaboration of plasmonic nanocomposite metamaterials.

Chernobyl accident fallout and major radioactive water leaks from the damaged Fukushima Daiichi nuclear power plant arouse concerns regarding the potential radioactive contamination of aquatic systems nearby and set a necessity for a reliable risk assessment tool in case of accidental radioactive discharge in the environment. Determination of trace levels of environmental plutonium, typically at femtomolar concentration levels, and other tetravalent metals requires a thorough radiochemical separation and very sensitive analytical methods. DGT technique (Diffusive Gradient in Thin-films) is widely used for sampling trace metals in various media (water, pore-water of soils and sediments). It has been successfully applied for determining radioisotopes and radioelements (134Cs, 137Cs, U, etc.). For the moment, there is no specific DGT able to discriminate different tetravalent species (An4+) from di or trivalent transition metals. We propose to develop specific An4+ resins usable in various physico-chemical environments (water, pore-water of soils and sediments). For this purpose, it is mandatory to design and select the optimal chelators, which should exhibit very high binding affinity and selectivity towards the tetravalent elements at the pH values of natural waters, over all other metals. The chelator metal binding properties in solution will be characterized from a structural and thermodynamic point of view in order to assess their efficiency in terms of sensitivity and specificity. The next step will be to immobilize these ligands onto organic polymers to afford original chelating resins (i) for analytical separation and (ii) for hitherto unavailable DGT-type devices. The pre-concentration capacities of the analytical DGT tool will be validated with respect to Pu and other tetravalent metals in laboratory (lab tests) and real environmental conditions (field tests). Last but not least, the industrial partner is committed to produce and commercialize the various resins and DGTs. 

Principal Investigator: Dr Michel Meyer

Bioconjugates have become essential tools for biomedical research, diagnosis and therapy. A second generation of bioconjugates begins to emerge. It consists in doubly-modified proteins, which have optimized properties and/or open new fields of applications such as bimodal medical imaging or theranostics. For instance, these last years have seen the emergence of bimodal imaging agents based on a biovector labeled with both a fluorescent dye and a chelator for a radiometal (for PET or SPECT), and some of them are used nowadays in clinic for surgery assistance. Some tremendous advances have also been made in the field of Antibody Drug Conjugates (ADC), which became a major class of highly potent drugs for targeted therapy. Attaching two different cytotoxic payloads, or both a drug and an imaging reporter, to an antibody (theranostic), will contribute to the development of improved ADC. 

The development or these new bioconjugates is currently hampered by the technical difficulties associated with their construction. Increasing attention has been paid in the last years to the use of site-specific labeling of antibodies, allowing access to chemically defined constructs, more reproducible procedures, a better control of the degree of labeling, which finally may facilitate approval process. However, in many cases, advanced level of protein engineering is skill required to obtain such bioconjugates. Thus, the development of technologies or procedures able to facilitate the production of such biomolecules should enable the explosion of this next generation of bioconjugates and their translation into the market. These innovative technologies must fulfill several criteria: ideally they should be i) easy to perform, ii) site-specific, iii) tolerant/biocompatible, iv) modular. 

The ZINELABEL project aims at developing a robust, modular, small, ternary platform, so-called dichloro-s-tetrazine, enabling the direct or indirect, site-specific, double-labeling of proteins in fully biocompatible conditions. The reactivity of dichloro-s-tetrazine will be exploited to prepare a library of mono- and di-substituted derivatives, by successive introductions of different moieties (NIR dye, macrocyclic chelator, PEG chain, targeting agent). The capacity of these modified tetrazines to undergo iEDDA click reaction will be studied and these platforms will be used for i) site-specific labeling of an Affibody, ii) dual modification of a protein aptamer targeting HSP70. These proofs of concept will serve at demonstrating the high potential and versatility of the ZINELABEL technology for the easy and efficient dual-labeling of proteins.

Organophosphorus nerve agents (OP) are highly toxic for human beings and are unfortunately used as chemical warfare agents and pesticides. Considering the serious threat that they represent to the worldwide security and health, much efforts have been devoted to the development of sensitive and selective devices for their detection. Nowadays, none of the developed strategies have led to a detection system that is at the same time sensitive, fast, wearable, robust, reliable, universal and visual. The DetectOP-BChE project aims at developing such a system by designing a warning detection system towards OP and an advanced tool to identify the nature of OP responsible for the alert. The two systems are complementary and will be devised as a single one device. The strategy is based on the highly sensitive, visual and simply operational fluorescent/colorimetric detection approach associated with the characteristics of cholinesterases as excellent, fast and selective biomarkers of OP exposure. This novel OP-detection approach will dramatically decrease the false positive alerts, thus enhancing the reliability of the system, and should allow to reach low detection limits. The DetectOP-BChE devices will be suitable to the French and International Armed Forces for the protection of their military troops as well as for the protection of first rescuers and surrounding civilians in the context of the perverted use of OP in armed conflicts and terrorist attacks. OP being also used as pesticides, the DetectOP-BChE devices will be applicable to the protection of researchers and staff of the chemical industry as well as farmers.

Principal Investigator: Prof. Anthony Romieu

In the last few years, s-tetrazines have been the object of considerable interest in various fields, which spread out from energetic materials to biomedicine. Despite this significant extent of applications, the synthetic preparation modes of functionalized s-tetrazine remains very limited. They mainly rely on the initial Pinner synthesis of prefunctionalized tetrazine core with serious steric and electronic limitations. The present project aims now at providing new selectivities in C–H activation of s-aryltetrazine core by extending the methods and scope of utilizable transition metals to meta- and para- remote C–H functionalization with electrophilic and nucleophilic reagents: the applied targets span a large domain from molecular materials to bioactive drugs. In-depth mechanistic study will be achieved by analytical electrochemistry and DFT studies, in order to solve the issue of rate limiting step (OA, RE, TM) and reagents compatibility in the reaction process.

Principal investigator: Dr Julien ROGER

Among the different disciplines in medical imaging, molecular imaging is an emerging powerful tool, which enables the detection of pathophysiological changes in living subject at the cellular and molecular levels. More particularly, it can be used to perform a fast and accurate diagnosis, or to monitor the success of a treatment. Even if it plays a central role in oncology, the use of molecular imaging opens up an incredible number of possibilities for other medical applications as for cardiovascular diseases, infections, bone disorders, thyroid disorders, brain disorders, gastrointestinal diseases… Among the different molecular imaging methods, optical imaging (OI) is the method of choice for in vitro and ex vivo studies, and is used more and more for small animal imaging studies. It is a non-ionizing technique, which presents the high resolution and the high sensitivity needed for molecular imaging investigations. It is more restricted for clinical applications, due to its tissue limited penetration, but it is of major interest for endoscopy investigations, as well as for fluorescence imaging guided surgery. The increasing interest in OI is also due to two emerging fields: bimodal imaging and trackable therapeutics. Indeed, OI is a modality of choice for bimodal imaging: its high sensitivity is suitable for a coupling to a PET/SPECT probe, which will take over the fluorophore for in vivo imaging. Concerning trackable therapeutics, grafting a fluorescent probe to a therapeutic moiety enables to track in real time the resulting therapeutic in vitro and in vivo, giving crucial information on its biodistribution and its cellular targets.

Aiming at performing in vivo studies, the use of near infrared (NIR) light offers several advantages, such as low absorption coefficient of most of the biomolecules, lower scattering, minimization of autofluorescence signals and risks to disturb the normal metabolism of the biological system to study. It therefore requires the use of called NIR fluorescent probes, which absorb and emit in the “diagnostic window” (between 650 and 900 nm). However, the choice of the fluorescent dye is almost always limited to commonly used cyanines. Despite some advantages, these fluorophores suffer from poor fluorescence efficiency, but above all from a chemical instability, rapid photobleaching, and today, there is no ideal NIR-fluorescent probe suitable enough for biological applications (water-soluble, stable, low toxic…), enabling a large scope of functionalizations, and able to be synthesized in large scale.

We are offering to solve this problematic and to develop a NIR-fluorescent platform displaying the properties above-mentioned, and we are targeting an original and promising family of fluorophores, the aza-BODIPYs, because their synthesis is straightforward, they absorb and emit in the NIR region, they present outstanding chemical and photochemical stability, and can be produced easily in the gram scale. Therefore, the aza-BODIPYs compounds presents all the advantages for biomedical applications, except their high lipophilic character and their very poor solubility. That is why we will develop a simple strategy to solubilize them, and we aim at elaborating a highly funtionalizable water-soluble aza-BODIPY platform, and to design, starting from this platform, new Monomolecular Mutimodal Imaging Probes (MOMIP) and optical metal-based trackable therapeutics. In the scope of the project, we will suggest simple synthetic routes to get access to the water soluble aza-BODIPY platforms, the resulting optical imaging probes, MOMIP and theranostic constructs. The most promising systems will be bioconjugated to different biovectors, and, in the last part, we will demonstrate, through an imaging pilot study, the potential of the new systems.

Principal Investigator: Dr Christine Goze