Mechanisms of focal epileptogenesis in mTORopathies - PROJECT SUMMARY The goal of this R35 application is to capitalize on new mouse models of epilepsy that my lab has developed to determine whether, when and under what conditions epileptogenesis can be treated and reversed. Studies are designed to address fundamental questions about the basic mechanisms of epileptogenesis and to advance the development of AAV-mediated gene therapy approaches to bring new treatments to patients with intractable epilepsy caused by mTOR pathway mutations. The present proposal is a culmination of almost two decades of research examining basic mechanisms of epileptogenesis in mouse mTORopathy models. The mTORopathies are a class of developmental diseases caused by mutations in the mTOR pathway. Disease-causing germline and somatic mutations have been identified in more than a dozen mTOR pathway genes, causing focal brain malformations, autism and epilepsy. Tuberous sclerosis complex (TS) and focal cortical dysplasia type II (FCD), the focus of the current proposal, are among the most common conditions. Studies take advantage of key features of the mouse mTORopathy models we have developed. Notably, AAVs are used to experimentally control the epilepsy-causing mutation (deletion of an mTOR pathway gene), allowing us to regulate the number, position and cell type specificity of mutant cells, thus modeling the genetics, focality and cellular mosaicism characteristic of mTORopathies. Moreover, we can combine these models with other technical innovations, such as optogenetics, chemogenetics, calcium imaging and diphtheria toxin-mediated cell ablation. These capabilities put us in position to answer fundamental questions in the epilepsy field. Specifically, we will examine epilepsy reversibility by determining whether and when ablating the mutant cells that cause epilepsy will cure the disease. While focal brain lesions cause epilepsy in mTORopathies, it is not known whether the lesions alone drive the seizures or whether epilepsy can be sustained by recruitment of cells outside the focal lesions. Time course experiments with this ablation approach will reveal if there is a critical period for efficacy, while real-time imaging experiments will examine recruitment of surrounding tissues. Manipulating the cellular distribution of mTOR mutations in the models will reveal whether this feature of the disease is clinically relevant. Mouse experiments will utilize well validated – but non translatable – approaches to set the bar for therapeutic interventions, while new – translatable – AAV vector gene-therapy constructs will be developed and tested in animal models and resected patient tissues. At the completion of the proposed studies, we plan to have developed optimized AAV vectors for treating mTORopathies, and generated data to inform when treatments need to be given, the extent of brain tissue targeted, and how treatments need to be adapted for different patient subclasses.
