Pharmacological read-through of R294X Mecp2 in a novel mouse model of Rett syndrome

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in Methyl-CpG-binding Protein 2 (MECP2). More than 35% of affected individuals have nonsense mutations in MECP2. For these individuals, nonsense suppression has been suggested as a possible therapeutic approach. To assess the viability of this strategy, we created and characterized a mouse model with the common p.R294X mutation introduced into the endogenous Mecp2 locus (Mecp2R294X). Mecp2R294X mice exhibit phenotypic abnormalities similar to those seen in complete null mouse models; however, these occur at a later time point consistent with the reduced phenotypic severity seen in affected individuals containing this specific mutation. The delayed onset of severe phenotypes is likely due to the presence of truncated MeCP2 in Mecp2R294X mice. Supplying the MECP2 transgene in Mecp2R294X mice rescued phenotypic abnormalities including early death and demonstrated that the presence of truncated MeCP2 in these mice does not interfere with wild-type MeCP2. In vitro treatment of a cell line derived from Mecp2R294X mice with the nonsense suppression agent G418 resulted in full-length MeCP2 protein production, demonstrating feasibility of this therapeutic approach. Intraperitoneal administration of G418 in Mecp2R294X mice was sufficient to elicit full-length MeCP2 protein expression in peripheral tissues. Finally, intracranial ventricular injection of G418 in Mecp2R294X mice induced expression of full-length MeCP2 protein in the mouse brain. These experiments demonstrate that translational read-through drugs are able to suppress the Mecp2 p.R294X mutation in vivo and provide a proof of concept for future preclinical studies of nonsense suppression agents in RTT.

MeCP2: The Genetic Driver of Rett Syndrome Epigenetics

“…There are two main approaches, firstly that of addressing downstream effects of the MECP2 mutation, for instance attempting to upregulate genes under MeCP2’s control, such as BDNF or IGF1 [reviewed in Vashi and Justice (2019) ], or neurotransmitter pathways such as NMDA receptors, or downstream target K + /Cl – co-transporter 2 (KCC2) ( Tang et al, 2019 ). The second approach is to target MECP2 directly, either through gene therapy, delivering a functional version of the MECP2 gene exogenously, for example using adenoviral delivery systems [e.g., ( Tillotson et al, 2017 )], through correcting the mutation at the level of genomic DNA, for example using CRISPR/cas9 editing, or at the RNA level by programmable RNA editing (e.g., Sinnamon et al, 2020 ), or using compounds such as aminoglycosides to enable “read-through” of MECP2 nonsense mutations [e.g., ( Merritt et al, 2020 )].…”

Expanding theMECP2network using comparative genomics reveals potential therapeutic targets for Rett syndrome

“…In murine models, reintroduction of MECP2 to adult animals led to vast phenotypic improvements (13) . In parallel with approaches aimed at increasing MECP2 levels directly (10, 11, 68) , accurate mapping of the MECP2 gene network is important to provide additional avenues for intervention, and avoid the toxicity associated with overexpression of MECP2 (17) . Here, we have presented a framework for identifying potential drug-gene interactions based on comparative genomics and validate these interactions in human immortalized primary neural cells.…”

“…Therapies aiming to restore MECP2 loss of function are in early development stages and are not approved for patients. These include attempts at read-through inducing drugs to overcome nonsense mutations (10, 11) , X reactivating therapies to allow expression of silenced endogenous MECP2 (12) , and gene therapy treatments to introduce wild-type MECP2 copies intro the brain (13, 14) . Furthermore, current gene therapy methods show limited transduction efficiency and are unable to modify all cells in the target tissue (15) .…”

ROP16-Mediated Activation of STAT6 Suppresses Host Cell Reactive Oxygen Species Production, Facilitating Type III Toxoplasma gondii Growth and Survival

“…005977) and bred in the University of Arizona BIO5 Animal Facility in accordance with protocols approved by the University of Arizona Institutional Animal Care and Use Committee. Murine ear tip fibroblasts (mETFs) were generated according to previously described protocols ( 72 ). Briefly, 2-mm ear punch biopsy specimens were collected from the pinna of adult mice, submerged in 70% ethanol, and then transferred to mETF culture medium on ice for dissection.…”