Epilepsy is a devastating neurological channelopathy affecting 2-3 percent of the population. In the face of major breakthroughs in antiepileptic drug development, a large contingent of patients remains pharmacoresistant. Gene therapy is a promising strategy to correct at the molecular level alterations of ion channel responsible for hyperexcitable neuronal and network phenotypes. The current knowledge of epileptogenesis indicate that 'therapeutic antiepileptic genes' need to be specifically transfected to a distinct neuronal phenotype (i.e., such as glutamatergic neurons). Targeted non-viral gene vectors such immunoliposomes have been used to selectively deliver drugs and genetic material to cancerous cells spearing the healthy ones. Immunoliposomes are cationic lipid vesicles with an external targeting moiety (antibodies directed against a cell surface molecule), which may facilitate entry of the complex into neurons via receptor-mediated endocytosis. This application proposes to develop cationic immunoliposomes as gene delivery nanovehicles that can selectively target glutamatergic neurons and deliver 'therapeutic' cDNA for expression and up-regulation of the large conductance calcium-activated potassium (BK) channel (alpha-pore forming subunit). BK channels are located in axon and presynaptic terminals in the hippocampus and it is thought that they control glutamate release. Gene therapy-mediated up-regulation of BK channels may ameliorate excessive release of glutamate in epilepsy.
In Specific Aim 1, we will explore whether targeted immunoliposomes will selectively transfect cDNA coding for BK channel and reporter enhanced green fluorescent protein (EGFP) to hippocampal glutamatergic neurons. Transfections will be characterized 'in vitro' (i.e., primary hippocampal cultures). Selectivity and efficiency of liposomal vectors will be compared with other non-targeted transfection approaches by using immunocytochemistry, viability assays and laser scanning confocal imaging. Functional analysis of immunoliposome-mediated BK channel cDNA transfection will be assessed using visualized patch-clamp recordings to detect and analyze spontaneous miniature postsynaptic excitatory currents (mEPSC). Direct imaging of endocytosis by membrane-selective FM-dyes will assess whether introduction of exogenous BK channels affect synaptic release of glutamate. The long-term goal is to develop innovative targeted non-viral vectors to deliver 'therapeutic genes' for the treatment of neurological disorders as epilepsy. In future studies, the therapeutic potential of targeted immunoliposomes containing 'antiepileptic genes' will be explored 'in vivo' in a model of chronic epilepsy. This multidisciplinary application involves collaborations among junior investigators at The University of Texas at Brownsville with expertise in molecular biology of BK channels, transfection, electrophysiology, epilepsy and liposome technology. The outcome of this exploratory research may lead to a novel non-viral strategy for cell-type specific CNS gene therapy. Relevance: This multidisciplinary nanotechnology project aims to develop an innovative strategy for the gene therapy of pharmacologically resistant epilepsy. The proposed experiments will characterize 'in vitro' a novel delivery system (immunoliposomes) consisting of lipid nanosized vesicles (liposomes) exhibiting a surface recognition molecule (antibody) that can selectively target a specific neuronal population that release the neurotransmitter glutamate (glutamatergic neurons). Completion of this application may provide a revolutionary targeted gene therapy strategy by selectively delivering 'therapeutic' genetic materials to dysfunctional glutamatergic neurons, which are involved in the pathogenesis of major neurological disorders such epilepsy. ? ? ?
