MECPer-3D

MECPer-3D is an International project born in Siena. It is a pre-clinical study focused on Rett syndrome (RTT). The main purpose of the project is the validation of gene therapy, in the form of CRISPR/Cas9-based gene editing, as an innovative treatment for the Syndrome.

The project will provide a proof-of concept of the efficacy and safety of gene editing for the correction of MECP2 mutations, paving the way toward the clinical application of this approach for the treatment of RTT.  Specifically, project objectives are:

• Collection of biospeciments for the generation of patient-specific cellular models for 4 recurrent MECP2 mutations: c.473C>T - (p.(T158M)), c.502C>T (p(R168X)), c.763C>T (p.(R255X)), c.916C>T (p.(R306C)). 

• Design and in vitro validation (efficiency, specificity, safety) of a gene editing-based correction approach for the 4 recurrent mutations in patient fibroblasts.  

• Validation in a relevant human cellular model, namely neurons differentiated from patient-specific induced Pluripotent Stem cells 9iPSCs), in 2D and 3D (organoids) culture  conditions.

• Validation of efficiency and specificity in vivo in mouse models with the 4 selected mutations.

• Development of new hybrid Adeno-Associated (AAV) capsids with increased capacity to cross the Blood Brain Barrier and good tropism for the CNS in order to efficiently target the primarily affected tissue.

• Confirmation of the effectiveness of MECP2 gene editing in reversing Rett phenotypes, both in vitro and in vivo.  

The consortium brings together 5 groups with complementary expertise and AIRETT association. Their concerted efforts will allow gaining a comprehensive view of the relevance of AAV-CRISPR/Cas9 therapy for RTT, by testing efficiency and specificity in relevant human and mouse models. 

• Prof. Ilaria Meloni is a geneticist with a long-lasting expertise on the study of the molecular bases of RTT. She has given a relevant contribution to the identification of the 3 genes commonly associated with RTT (MECP2, CDKL5 and FOXG1). To study disease mechanisms, her group has established and characterized a patient-specific iPSC-based human cellular model, identifying common players in disease pathogenesis. Using this model, they are now exploring the potential of CRISPR/Cas9 gene editing as a therapeutic approach. 

• Prof. Miguel Chillon has more than 25 years of expertise in vector production and administration in animal models for the study of the CNS. He has participated in international Networks of Excellence in gene therapy strategies for neurodegenerative diseases as well as in vector production. He is also the Director of the Vector Production Unit, a Vector Core from a public Institution, which produces and characterizes high quality AAV vectors for preclinical assays for more than 150 research groups around the world.

• Dott. Julia Ladewig has more than 15 years of experience in human iPSCs culture and differentiation. She has contributed to the field of neuronal programming, neural stem cell engineering, neural differentiation, stem cell-based disease modeling and cell-based therapy. She is a leading scientist in the field of cerebral organoids and their application to study neurodevelopment and associated disorders. 

• Dott. Yann Herault is a leading mouse geneticist who will provide with his colleagues at the Mouse Clinical Institute the expertise necessary for in vivo experiments in the mouse models: stereotactic surgery, behavioral testing, biochemical analysis (immunohistochemistry and western blotting), and confocal microscopy. 

• Dott. Mario Chiariello is a recognized expert in the study of autophagy and its contribution to cancer as well as genetic diseases. As such he has contributed to the most updated guidelines for the use and interpretation of assays for monitoring autophagy, guaranteeing the use of most acceptable and appropriate methods to score this phenomena in cells and tissues. 

• AIRETT: Italian patient association

The MECPer-3D project is a pre-clinical study whose main purpose is to validate gene editing as a therapeutic approach in patient cells and animal models. For this purpose it is essential to have a statistically significant number of samples.

In particular, two types of samples are required: a peripheral blood sample and a skin biopsy from which lymphocytes and fibroblasts will be obtained, respectively. Lymphocytes are used to generate iPSCs essential for differentiation into neurons, the cellular model of primary interest for the project. Fibroblasts are used to validate the in vitro correction system and to perform further functional analyses.
The reference contacts for Italian and foreign families, hospitals and research groups are: 

• Prof. Ilaria Meloni: ilaria.meloni@dbm.unisi.it 

• Dott. Susanna Croci susanna.croci@dbm.unisi.it