Establishing the impact of disease-associated KCNH1 mutations on neuronal physiology

About This Grant

ABSTRACT Heterozygous de novo missense variants in KCNH1 are associated with the rare neurodevelopmental disorders Temple‐Baraitser Syndrome (TBS) and Zimmermann‐Laband Syndrome (ZLS), which share features including developmental delay, facial and digital abnormalities, and frequent epileptic seizures. KCNH1 encodes KV10.1, which is a voltage-gated potassium channel (Kv) widely expressed in central neurons. To date, more than 40 pathogenic KCNH1 variants have been reported. Preliminary evidence supports a gain-of-function (GOF) effect for most variants. The mechanistic insight we have on these mutations is limited to patch-clamp recording experiments with heterologous cells. The cellular mechanisms responsible for KCNH1-associated neuronal defects, particularly how intrinsic ion channel dysfunction causes abnormal human neuron excitability, remain completely unexplored. Additionally, there are no effective disease-modifying therapies for KCNH1-related disorders. Selective small molecule treatment is challenging given the high degree of conservation among related KV channels especially hERG, which in human heart is the major cause of life-threatening proarrhythmic off-target drug effects. Alternative approaches for targeting KCNH1, such as antisense oligonucleotides (ASOs) may be more selective and avoid off-target effects that compromise safety. However, proof-of-concept evidence that an ASO can reverse KCNH1 GOF defects in neuronal excitability does not exist, nor do any human-based model systems of KCNH1-associated disease. Here, we will use our expertise in ion channel biology, CRISPR/Cas9 gene editing and induced pluripotent stem cell (iPSC) technologies to perform a systematic characterization of KCNH1 mutations in human iPSC-derived excitatory and inhibitory neurons. In Aim 1 we will study excitatory and inhibitory neurons differentiated from a cohort of iPSC lines heterozygous for 3 distinct and functionally diverse KCNH1 mutations to determine their neurophysiological properties. We will evaluate expression of KV10.1 channels and measure single cell and population-based firing properties using whole-cell patch clamp and multi-electrode array (MEA) based recordings, respectively in cultured neurons. In Aim 2 we will evaluate the ability of a KCNH1 targeting ASO to normalize activity of iPSC-neurons heterozygous for pathogenic KCNH1 variants. We will assess potency, efficacy and time-dependence of gene suppression by monitoring the reduction in KCNH1 mRNA and protein expression following ASO treatment. We will use single cell (patch clamp recording) and population (MEA) electrophysiology to test the hypothesis that ASO treatment will normalize the resting potential and firing rate. We will optimize methods to isolate KV10.1 current in ASO- treated iPSC-neurons and assess functional knockdown of the current by the ASO. The goals of this exploratory R21 proposal are to: a) establish the first human iPSC-based models of KCNH1-associated diseases, b) provide a systematic evaluation of the impact of disease-associated mutations on neuronal physiology, and c) take the first step of developing a rational therapeutic for these devastating diseases.