Development of Self Regulating non-Viral Vector to Restore MeCP2 Activity to Treat Rett Syndrome

Abstract

Rett syndrome (RTT), a debilitating neurological disorder primarily affecting females, is characterised by profound cognitive decline and arises from mutations in the MECP2 gene. While several gene therapy studies on mouse models have extended lifespan, they have achieved only marginal improvements in RTT phenotypes. An additional complication is that elevated levels of MeCP2 protein lead to MECP2 duplication syndrome, which presents symptoms similar to RTT. Consequently, any therapeutic intervention aimed at restoring MECP2 function necessitates meticulous regulation. The growing field of regulated transgene therapies has garnered significant interest due to their robustness and safety profile. A notable development in this area is the translational regulatory system devised by the Wiss’s group, which employs CRISPR technology to achieve high-level regulation of gene expression, independent of the plasmid copy number. Our research focuses on developing a regulated gene therapy that maintains MeCP2 protein levels within therapeutic levels. We have employed the CasE ribonuclease system to facilitate MECP2 expression independent of copy number variations. Additionally, we have incorporated upstream open reading frames (uORFs) to fine-tune the control exerted by CasE. The experimental designs included separate, combined, and bicistronic systems. We have demonstrated that CasE can regulate transgene expression consistently, even when transfected into different plasmids carrying the regulated gene. By varying the repeats of uORFs, we achieved differentiated regulatory levels, further modulating CasE activity. We observed that positioning the plasmid in the opposite orientation diminished expression in non-neuronal cell lines without impeding expression in neuronal cells. The bicistronic system, in particular, exhibited heightened regulatory control and reduced fluctuations in transgene expression. Moreover, CasE successfully decreased the dependency of gene expression on plasmid copy number and consistently reduced the variability of MECP2 expression in subsequent transfections into MECP2-null mouse embryonic fibroblasts. The capability of CasE to minimize expression variability and regulate gene expression irrespective of copy number is particularly promising for RTT treatments, especially in circumventing the complications associated with MECP2 Duplication syndrome. However, future studies should aim towards a more controlled model that mimics the mosaic expression pattern of MECP2 characteristic of RTT, to better reflect the complexities of the disease in therapeutic approaches.