Mutations in genes belonging to the RHO GTPase pathway are responsible for intellectual disability (ID), psychiatric disorders and brain development anomalies. The great heterogeneity of phenotypes associated with these gene mutations renders the development of therapeutic strategies strenuous. Studying PAK3, a central gene of the RHO GTPase pathway, will help us establish a genotype/phenotype correlation, which is essential to 1- define the rules behind mutation pathogenicity, 2- understand the underlying mechanisms and 3- propose adapted therapeutic approaches. 1- Our project is to define the genotype/phenotype correlation using about 20 different PAK3 mutations in order to understand the origin of PAK3-linked ID degree of severity, as well as why ID may sometimes be associated with other neurodevelopmental defects. Thus, we will establish and characterise the broadest cohort of patients bearing PAK3 mutations ever built. In parallel, we will assess the functional defects of mutated PAK3 variants and their effects on cell biology (shape, adhesion, migration) as well as neuron differentiation (neurite growth, dendritic spine formation). Our hypothesis states that mutation pathogenicity is not simply a loss or gain of function but may involve more complex mechanisms of signalling interference. Indeed, the presence of a mutated protein is often more deleterious than the lack of a protein. 2- To go further in analysing the severe forms of PAK3-linked ID, we created a new knock-in model bearing a mutation clinically responsible for a severe ID associated with secondary microcephaly. This mouse model presents strong behavioural and cognitive anomalies, as well as secondary microcephaly, reminiscent of the clinical case. Our project consists in a more thorough analysis of the mouse model behavioural and cognitive defects, in order to compare our results with the patient’s clinical traits. Our ex-vivo and in-vitro preliminary analyses allowed us to propose a new molecular mechanism of mutation pathogenicity, which we will investigate thoroughly. 3- We will test two phenotypical rescue strategies with the aim of further developing therapeutic solutions. The first strategy concerns severe forms of the disease. The degradation of stable pathogenic PAK3 proteins should, at least partially, restore phenotypic anomalies usually associated with severe ID. This strategy of specifically degrading stable pathogenic variants was never explored in the context of neurodevelopmental disorders, even while it is being developed as potential cancer treatment. It would also be applicable to over-activating mutations in genes belonging to the RHO GTPase pathway. The second rescue approach targets Cofilin, a convergence point of the RHO GTPase pathway. Several strategies targeting this actin polymerisation regulator were already explored to rescue behavioural anomalies and synaptic plasticity defects. We aim to demonstrate that this approach would also correct neuronal differentiation anomalies appearing during post-natal development. Thus, the efficiency of a cofilin-blocking peptide to restore neuritic arborisation and dendritic spine formation in mutated mice will be evaluated. This project is based on strong preliminary results and an already operational consortium composed of 2 clinician teams and 3 research teams (1 team being knowledgeable in the two fields). This project will allow us to understand the genotype/phenotype relations regarding PAK3 gene mutations as well as mutations on other genes belonging to the RHO-GTPase pathway. Our results will greatly help advance genetic counselling and patient monitoring. The post genomic and preclinical aspects of this project will also enable us to pave the way for new therapeutic approaches in the optic of personalised medicine.

Vitamin D plays key roles for calcium homeostasis. Biallelic loss-of-function variants of CYP24A1, the enzyme that converts the active 1,25D3 into inactive metabolite, are responsible for autosomal recessive Idiopathic Infantile Hypercalcemia (IIH, ORPHA 300547, 1/80000), inducing hypercalcemia, nephrocalcinosis, nephrolithiasis and ultimately kidney failure. At the opposite of this rare disease, nephrolithiasis will affect during lifetime 20 to 30% of the general population, with a significant financial impact on health systems and an important burden of disease. The frequency of CYP24A1 heterozygosity is estimated to be 1/130 using the most frequent pathogenic variants. Thus, whether CYP24A1 heterozygosity is associated with haplo-insufficiency inducing a renal phenotype by itself or whether it is “only” a risk factor of nephrolithiasis remains debatable. Recent reports indicate that patients with heterozygous variants of CYP24A1 present increased 1,25D3 levels, and might present a higher risk of nephrolithiasis, but human data are scarce and somehow heterogeneous. In contrast, the murine model of CYP24A1 heterozygosity spontaneously displays a IIH-like phenotype, therefore positioning this pre-clinical model as a relevant tool to unravel the pathogenesis in heterozygous subjects. The objective of the translational HeteroCYP project is to deepen the understanding of heterozygous CYP24A1 variant-associated phenotypes in humans, using mouse models, clinical data and human samples. Such a demonstration will be instrumental to improve the genetic counselling and clinical care of these patients, so as to provide a personalized medicine. Last, novative therapeutic options will also be evaluated in our preclinical model, and might provide rationale for clinical trials in heterozygous patients.

Nucleic acid-based therapeutics have emerged and represent today, key targets in a wide range of diseases. Since natural oligonucleotides (ONs) are rapidly metabolized in biological media by nucleases and suffer from a poor cellular uptake, several chemical modifications were envisaged and led to the conception of ON analogs with good biological activities. However, in spite of the large number of ONs that are currently under clinical trials, the FDA approved only few of them. Thus, new series still deserve to be discovered regarding the high potential of ON-based therapeutics and the growing economical market of this field. Our project deals with the design and synthesis of new ON series that would be able to counteract the main limitations of current therapeutic ONs. In front of the importance of the fluorine atom in medicinal chemistry, we will be particularly interested in the preparation of fluorinated nucleic acids that have been less explored nowadays. Indeed, several organofluorinated groups will be introduced onto the 2’-position of nucleosides to increase both the thermal stability of RNA duplex, resistance towards nucleases as well as cellular uptake. To access to these target molecules, automated solid-phase phosphoramidite-based ON synthesis will be privileged. Indeed, new synthetic methodologies based on ionic and radical approaches will be first developed to synthesize the different fluorinated phosphoramidites in each nucleobase series (A, C, G, U). These laters will be then incorporated in various model sequences (siRNA, miRNA) to study their physico-chemical and biological properties.