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

• Applicant: Dr. Julia Ladewig
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Experimental Models for the Understanding of Nervous System Diseases
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 460905319

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 currently available and gene editing based on CRISPR/Cas9 combined with Homology Directed Repair appears an appealing option for the development of new therapeutic approaches. We already engineered a gene editing toolkit and demonstrated its ability to efficiently correct one of 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 develop and validate toolkits for three other MECP2 mutation hotspots, namely c.502C>T (p(R168X)), c.763C>T (p.(R255X)), c.916C>T (p.(R306C)). To characterize the potential of our approach in a relevant context and define its efficiency in a human 3D model, we will employ brain organoids differentiated from patient-derived induced pluripotent stem cells. Thanks to the ability of specific 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 to demonstrate the full potential of gene editing as a therapeutic option for Rett and for other neurodevelopmental disorders currently lacking an effective treatment.

• DFG Programme: Research Grants
• International Connection: France, Italy, Spain
• Cooperation Partners: Mario Chiariello, Ph.D.; Miguel Chillon, Ph.D.; Dr. Yann Hérault; Dr. Ilaria Meloni