MicroRNA-sensing plasmid system for dynamic control of functional ion channel expression

Abstract Gene therapy offers the potential for long-term treatment or cures for a range of chronic diseases. However, permanent gene therapy expression may not be desirable. Efforts have been made to create systems which can be switched on/off by stimuli including light, designer drugs, or cellular contexts such as increased electrical activity. Here, we designed a novel plasmid system in which ion channel expression, and therefore function, is regulated by microRNA (miR) – an endogenous class of short noncoding RNAs which negatively regulate gene expression via binding the 3’ untranslated region of target transcripts. We modified an existing voltage-gated potassium channel gene therapy with a binding cassette for miR-193a-3p, and transfected this ‘miR-193-OFF’ system in neuro2A cells. Co-transfection with an inhibitor or mimic of miR-193a-3p respectively enhanced or repressed expression of our transgene, assessed using a GFP marker. Using whole-cell voltage clamp, we observed enhanced voltage-gated potassium currents in cells co-transfected with a miR-193a-3p inhibitor, compared with a non-targeting control oligonucleotide. Together, this demonstrates the concept of a novel miR-mediated molecular switch which can bias therapeutic ion channel expression based on a specific miR signal. As miRs are a ubiquitous molecular mechanism, our approach could be applied to a wide range of cellular and disease contexts, potentially expanding gene therapy to new patient populations.