a. Boosting the brain delivery of viral vectors using focused ultrasound (fUS) technology.

Over the past years, several MeCP2 gene replacement strategies were tested in RTT mouse models with varying degrees of success (Jagadeeswaran et al., 2024).

Unfortunately, the number of transduced brain cells remained limited due to the low permeability of the blood-brain barrier (BBB) to AAVs. To overcome this limitations, intracerebral injections are often used, but this approach appears much harder to translate in clinics given the risks for the patients.

Focused ultrasound (fUS) coupled with circulating microbubbles (MB), is an innovative technology enabling temporary disruption of the BBB. MB are sensitizers of mechanical stress under bulk-pressures of varying amplitude. These are gas-filled and their size ranges from 1–5 μm. MB can repeatedly expand and compress when they are exposed to low acoustic pressures. This oscillation leads to tight junctions opening via mechanical stress on the endothelium. Many groups, validated this method to deliver AAVs in the brain and transduce a large number of brain cells in models of several pathologies such as RTT, ALS, AD, HD (Ye et al., 2024).

Goals: Increase brain delivery of the AAV-driven MeCP2 gene therapy vectors; maximize the transduction of disease-relevant brain cells

b. Manipulation of importin α-dependent transport in MeCP2 related disorders.

We identified two functionally relevant importin-transcription factors (TF) interacting pairs (α5-MeCP2 and α3-c-Fos). However, to date, the transport mechanisms controlling the abundance of TFs in the nucleus, and the transcriptional pathways at the core of neurodevelopmental disorders, have not been targeted in a systematic manner.

Thus, the main aim of our lab is to study the role of nuclear transport factors in neurogenetic brain disorders. Given the key role of MeCP2 in gene expression, both Rett syndrome (RTT) and MeCP2 fields tackled questions fundamental to the understanding of neuronal functions, gene expression, and brain disorders. MeCP2 mutations cause RTT, a devastating and progressive neurodevelopmental disorder while duplication of the MeCP2 locus cause severe cases of intellectual disabilities in MeCP2 Duplication Syndrome (MDS) patients.

Our priority in this aim will be to:

• Test how modulations of MeCP2 transport can rescue phenotypes associated with MeCP2-related disorders (Lombardi et al., 2015; Lyst and Bird, 2015). We will then expand the study to additional TFs and cellular/mouse models of genetic disorders.

• Characterize the basic rules of importin α-MeCP2’s interaction and transport dynamics using:
  • Behavioral analyses,
  • In vivo fUS imaging,
  • live imaging of MeCP2 nuclear shuttling,

Goals: Hijack the importin-dependent transport of MeCP2 to titrate the level of the MeCP2 protein in the nucleus and avoid the potential overexpression phenotypes caused by untuned gene therapeutic strategies.

Using a battery of approaches from behavior to molecular mechanisms, we discovered the role of importin α5 in anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons (Panayotis et al., 2018) and the role of importin α3 in the transport of c-Fos in dorsal root ganglia (DRG) neurons during chronic pain (Marvaldi et al., 2020). Our previous extensive behavioral screening of importin α single knockouts also revealed specific and intriguing phenotypes from alteration of spatial memory, reduced sensorimotor coordination, and increased dominance/aggression in mice.

Aside from launching experiments aimed at the dissection of the precise anatomical locus of the importin α5-anxiolysis, our team will perform in-depth analyses of the different models using advanced imaging and RNA-seq, in cell and animal models. This research is expected to generate exciting insights on importin control of transcription and neurobehavioral mechanisms.

Goals: Define the specific contributions of Importin-dependent subcellular transport in neuronal functions and behaviors.

a. In silico drug screening

As demonstrated in our recent works (Marvaldi et al. , 2020; Panayotis et al., 2021), the proposed research will also make the most of RNA-seq and in silico approaches to screen drugs that can present new therapeutics properties (e.g., anxiolytics, painkillers, etc.)

b. Preclinical characterization of newly synthetized molecules.

To date, Rett syndrome remains an incurable pathology. For a long time, the only available treatments aimed at alleviating some symptoms in order to improve the patients’ quality of life. Several pharmacological interventions have been proposed, thanks to the discovery of targets of interest in RTT mouse models. We are collaborating with experts in Medicinal Chemistry and Pharmacology in order to synthetize, characterize, and validate the therapeutic potential of new candidate drugs.

Goals: Identify and validate new pharmacological options to ameliorate anxiety, pain, and comorbidities in neurodevelopmental disorders.