Gene therapy represents a powerful therapeutic tool to treat diseased tissues and provide a durable and effective correction. The central nervous system (CNS) is the target of many gene therapy protocols, but its high complexity makes it one of the most difficult organs to reach, in part due to the blood-brain barrier that protects it from external threats. Focused ultrasound (FUS) coupled with microbubbles appears as a technological breakthrough to deliver therapeutic agents into the CNS. While most studies focus on a specific targeted area of the brain, the present work proposes to permeabilize the entire brain for gene therapy in several pathologies. Our results show that, after i.v. administration and FUS sonication in a raster scan manner, a self-complementary AAV9-CMV-GFP vector strongly and safely infected the whole brain of mice. An increase in vector DNA (19.8 times), GFP mRNA (16.4 times), and GFP protein levels (17.4 times) was measured in whole brain extracts of FUS-treated GFP injected mice compared to non-FUS GFP injected mice. In addition to this increase in GFP levels, on average, a 7.3-fold increase of infected cells in the cortex, hippocampus, and striatum was observed. No side effects were detected in the brain of treated mice. The combining of FUS and AAV-based gene delivery represents a significant improvement in the treatment of neurological genetic diseases.

Mutations in the X‐linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder for which there is no treatment. Several studies have linked the loss of MeCP2 function to alterations of brain‐derived neurotrophic factor (BDNF) levels, but non‐specific overexpression of BDNF only partially improves the phenotype of Mecp2 ‐deficient mice. We and others have previously shown that huntingtin (HTT) scaffolds molecular motor complexes, transports BDNF‐containing vesicles, and is under‐expressed in Mecp2 knockout brains. Here, we demonstrate that promoting HTT phosphorylation at Ser421, either by a phospho‐mimetic mutation or inhibition of the phosphatase calcineurin, restores endogenous BDNF axonal transport in vitro in the corticostriatal pathway, increases striatal BDNF availability and synaptic connectivity in vivo, and improves the phenotype and the survival of Mecp2 knockout mice—even though treatments were initiated only after the mice had already developed symptoms. Stimulation of endogenous cellular pathways may thus be a promising approach for the treatment of RTT patients.

Rett syndrome: think outside the (skull) box

Mutations in the X-linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder. MECP2 is a transcriptional modulator that finely regulates the expression of many genes, specifically in the central nervous system. Several studies have functionally linked the loss of MECP2 in astrocytes to the appearance and progression of the RTT phenotype in a non-cell autonomous manner and mechanisms are still unknown. Here, we used primary astroglial cells from Mecp2-deficient (KO) pups to identify deregulated secreted proteins. Using a differential quantitative proteomic analysis, twenty-nine proteins have been identified and four were confirmed by Western blotting with new samples as significantly deregulated. To further verify the functional relevance of these proteins in RTT, we tested their effects on the dendritic morphology of primary cortical neurons from Mecp2 KO mice that are known to display shorter dendritic processes. Using Sholl analysis, we found that incubation with Lcn2 or Lgals3 for 48 h was able to significantly increase the dendritic arborization of Mecp2 KO neurons. To our knowledge, this study, through secretomic analysis, is the first to identify astroglial secreted proteins involved in the neuronal RTT phenotype in vitro, which could open new therapeutic avenues for the treatment of Rett syndrome.

Le syndrome de Rett est une maladie génétique rare, causée par des mutations du gène Mecp2 situé sur le chromosome X, et caractérisée par une régression du développement des patientes âgées de 6 à 18 mois. Si la pathologie est caractérisée principalement par une altération du fonctionnement cérébral associé à une déficience intellectuelle sévère, de nombreuses comorbidités sont observées et réduisent la qualité et l'espérance de vie des patientes. Cette thèse a pour objectifs d'étudier l'implication des organes périphériques (hors système nerveux central) dans la physiopathologie du syndrome de Rett mais aussi dans les stratégies thérapeutiques comme la thérapie génique. Pour répondre à ces objectifs, trois projets ont été menés chez un modèle murin de la pathologie. Le premier projet a permis d'étudier les symptômes gastro-intestinaux majeurs liés à la pathologie grâce à l'étude de la neurotransmission cholinergique dans le système nerveux entérique à la fois d’un point de vue moléculaire et fonctionnel. Le deuxième projet a porté sur le rôle du foie dans l'apparition des effets secondaires liés à la thérapie génique avec un vecteur AAV9-Mecp2. En effet, l’administration intra veineuse de ce dernier a montré des effets hépatotoxiques chez les souris modèles. Afin de limiter les effets secondaires liés à des organes non-ciblés, le troisième projet s'est intéressé à l'application d'une technologie utilisant des ultrasons focalisés afin d'augmenter la perméabilité de la barrière hémato-encéphalique et faciliter l'accès des AAV9 dans le cerveau.