European Society of Gene and Cell Therapy (ESGCT) 2022

Title: CRISPR/Cas9-based gene editing for Rett syndrome therapy

Italian Society of Human Genetics (SIGU) 2022

Title: A European effort for therapeutic gene editing in Rett syndrome: from organoids to mouse models

Croci S.1,2, Beligni G.1,2, Rossetti A.3, Edo A.4, Sorg T. 5, Manara M. 1,2,6, Lo Rizzo C. 6, Mencarelli M.A.6, Canitano R.7, Sica M. 8., Capitani K.1,2,9, Conticello S.9,10, Molinaro F.8, Chiariello M.9, Chillon M.4,11,12, Ladewig J. 3, Herault Y.5,  Renieri A. 1,2,6, Meloni I 1,2.

1) Medical Genetics, University of Siena, Italy

2) Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Italy

3) Central Institute of Mental Health (ZI), Department for Translational Brain Research, Mannheim, Germany

4) Vall d'Hebron Research Institute (VHIR), Barcelona, Spain

5) Institut Clinique de la Souris, ICS/MCI, PHENOMIN, GIE CERBM, IGBMC, CNRS, INSERM, 1 Rue Laurent    Fries, 67404 Illkirch-Graffenstaden Cedex, France

6) Genetica Medica, Azienda Ospedaliero-Universitaria Senese, Italy

7) Division of Child and Adolescent Neuropsychiatry, University Hospital of Siena, Siena, Italy

8) Pediatric Surgery, Department of Women and Children, S. Maria alle Scotte Hospital, University of Siena, Siena, Italy.

9) Core Research Laboratory, ISPRO Institute for Cancer Research, Prevention and Clinical Network, 50139 Firenze, Italy, 

10) Institute of Clinical Physiology, National Research Council, 56124 Pisa, Italy

11) Catalan Institution for Research and Advanced Studies (ICREA), Spain

12) Institute of Neurosciences (INc) Biochemistry and Molecular Biology Department Autonomous University of Barcelona, Spain

Keywords: Rett syndrome, MECP2, gene therapy

Approximately 95% of patients with Rett syndrome (RTT; OMIM#312750) present pathogenic variants in the X-linked gene MECP2. Reactivation of Mecp2 in symptomatic KO mice can revert disease phenotypes, suggesting that RTT is not irreversible. Gene editing represents an extremely promising therapeutic approach, since it allows maintaining the endogenous regulatory framework. The approach recently made its way to clinical trial for Leber Congenital Amaurosis (NCT03872479), demonstrating its translational potential. To validate its applicability to the 4 most common MECP2 hotspot variants we established an international consortium that was funded from the EU in 2021. We engineered a CRISPR/Cas9 gene editing toolkit composed of a dual plasmid system specific for each MECP2 mutation. We collected RTT samples and reprogrammed iPSCs from 2 patients for each variant to generate brain organoids, 3D structures which closely mimic human brain development. RTT organoids showed significant differences compared to isogenic wild type ones, with more disorganized and less structured architecture. We tested available chimeric AAV7m8 for human models and AAV9P31 for mice with increased brain selectivity, demonstrating their functionality. However, these viruses are still far from optimal since a significant portion spreads to peripheral organs reducing the efficiency of the approach and increasing the risk of toxic effects. For this reason new chimeric serotypes with higher specificity and selectivitychiari will be designed during the project. We validated the correction system efficiency in patient fibroblasts and iPS-derived neurons confirming the functionality of the tested plasmids with a high efficiency. Colonies of KI mice harbouring the 4 variants are also being established to further test the approach in vivo. The phenotyping pipeline  and behavioral characterization has been defined. Our work will represent a real opportunity for the application of gene therapy for RTT, thus providing the first real possibility for a RTT therapy.