ReGeNeRETT

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily affecting girls, with an incidence of 1 in 10,000 births and accounting for 10% of profound genetic intellectual disabilities in women. RTT patients seem healthy at birth but regress between 6 to 18 months, leading to severe neurological symptoms and autistic traits. RTT is caused by mutations in the MeCP2 gene coding for a key regulator of gene expression. Mutations For instance, the T158M, within the Methyl Binding Domain are frequent and severe. RTT is incurable but researchers demonstrated its reversibility in mice. In this context, gene replacement therapy offered the promise to restore a copy of the gene but this approach has been fraught with difficulties. Major challenges remain, such as achieving accurate MeCP2 rescue, without causing over-expression reminiscent of MeCP2 duplication syndrome. Current gene therapies struggle with controlling the expression strength, timing, and cellular context. Synthetic biology provides programmable gene networks for precise transgene regulation. Although complex synthetic gene circuits are powerful, they are difficult to deliver and impose a high metabolic load. In contrast, simpler gene regulatory networks might reduce such issues while keeping key advantages, including low basal noise and gene dosage compensation. Our project aims to overcome the problem of uncontrolled MeCP2 expression by developing a simple yet effective synthetic biology approach. We will design an elementary gene cascade to regulate MeCP2 re-expression through a synthetic transcription factor on a “sensor” vector, which will then control the expression of the MeCP2 gene on a “regulator” vector. This Cascade-Regulated MeCP2 (CR-MeCP2) will contain specific and scalable binding sites and negative feedback elements, while maintaining a small footprint compatible with AAV genome packaging for neuronal transduction and in vivo experiment. After selecting the most promising CR-MeCP2 systems by microscopy-based cytometry in HEK cells, we will transfer them into AAV-PhP.eB vectors for efficient co-delivery into mouse hippocampal neurons. Finally, we will assess the ability of the optimal CR-MeCP2 systems to rescue key RTT-like behavioral and neuronal morphology phenotypes in a RTT mouse model featuring the T158M mutation. The development of a precise and scalable gene therapy, offering the potential to restore normal neurological function in RTT patients, will ultimately extend the range of therapeutic options for devastating neurodevelopmental disorders.