MECPer-3D: Personalized MECP2 gene therapy using CRISPR/Cas9 technology coupled to AAV-mediated delivery in 3D cell culture and KI mice

Rett syndrome is one of the most common causes of intellectual disability in girls, resulting in severe cognitive and physical disabilities. The classic form is caused by mutations in the transcriptional regulator MECP2. Effective therapies are not available and gene editing based on CRISPR/Cas9 combined with Homology Directed Repair appears an appealing option for the development of new therapies. We already engineered a gene editing toolkit and demonstrated its ability to efficiently correct the most common MECP2 mutation, c.473C>T – p.T158M, in patient cells. Based on these results, in this project we will further validate constructs for this mutation and validate toolkits for other MECP2 mutation hotspots. To characterize the potential of our approach in a relevant context and define its efficiency in a human 3D model, we will employ cerebral organoids differentiated from patient-derived induced pluripotent stem cells. Thanks to the ability of some AAV serotypes to cross the Blood Brain Barrier (BBB) following intravenous injection, we will test our system in KI mice to validate efficacy and safety in vivo. Moreover, since available AAV serotypes have an imperfect brain tropism, with significant distribution to other organs, new serotypes will be developed and validated for their ability to cross the BBB in the mouse and their efficacy and specificity in human cells. These experiments will allow us demonstrating the full potential of gene editing as a therapeutic option for Rett and for other neurodevelopmental disorders currently lacking an effective treatment.