Therapeutic Targeting of KCNQ3 in KCNQ3 Gain-of-Function Disorder - Recurrent de novo missense mutations affecting a particular arginine codon (R230) in the KCNQ3 gene are responsible for an electroclinical syndrome that accounts for as many as 1 in 1,000 individuals with intellectual disability and autism. KCNQ3 encodes a voltage-gated potassium channel that is active at subthreshold potentials and concentrated at the axon initial segment, where it opposes depolarizing currents that drive neurons to fire action potentials. We recently reported that R230 variants cause gain-of-function (GoF) effects in an in vitro heterologous system, suggesting that KCNQ3 may be an effective therapeutic target for this disorder. Indeed, as current standard of care is limited to symptom management, the development of effective disease modifying therapy for KCNQ3 GoF Neurodevelopmental Disorder (NDD) represents a critical unmet need. We hypothesize that inhibiting KCNQ3 in the central nervous system (CNS) will provide effective treatment for KCNQ3 GoF NDD. Thus, our long-term goal is to develop therapeutic approaches for the KCNQ3 GoF NDD through inhibition of KCNQ3. We intend to accomplish this goal by reducing expression of KCNQ3 with RNAi, using a gene therapy approach in which KCNQ3-targeted microRNAs (termed miKCNQ3) are delivered to the CNS by adeno-associated viral vectors (AAV). Our study will leverage two different novel mouse models of KCNQ3 GoF NDD. These models are based on two different human disease alleles of KCNQ3 GoF NDD and display shared electroclinical phenotypes (absence seizures and lowered threshold for convulsive seizures) that parallel the human condition and provide robust outcome measures for use in preclinical studies. Preliminary data offers proof-of-principle for this strategy, but our first-generation system is not optimized for clinical use. We believe that optimization of our vector for efficacy and safety at an early pre-clinical stage will provide the most straightforward path forward towards translational application. In this proposal, we will refine our approach with the goals of enhancing safety and specificity of the system. To do this, in the R61 phase, we will develop novel miKCNQ3 expression systems to restrict expression to the target tissue (neuronal populations) and inhibit KCNQ3 without causing toxicity. Upon completion of the R61 phase, we expect to prepare and characterize our lead therapeutic AAV.miKCNQ3 agents, characterize AAV.miKCNQ3 tissue tropism, and estimate sample sizes for use in the R33 phase of the proposal. The goal of the R33 phase is to test the in vivo safety and efficacy of our vectors developed in the R61 phase. To demonstrate efficacy, we will determine the ability of our new KCNQ3-targeted AAV vectors to inhibit KCNQ3 and the electroclinical phenotypes associated with KCNQ3 GoF in vivo leveraging two novel Kcnq3 knock-in mouse models, bearing R231H (Frankel lab) and R231C (Tzingounis lab), representing two different human KCNQ3 GoF NDD disease allele orthologues. Concerns related to off- target effects will also be addressed. Upon completion of the R33 phase of this proposal, we expect to produce data that will support further translation of this strategy toward our end goal of clinical application.
