Exploiting KCNQ2 upstream open reading frames for therapeutic opportunity

PROJECT SUMMARY Heterozygous loss-of-function (LOF) is a common genetic mechanism among many monogenic epilepsy genes including KCNQ2, which encodes a brain voltage-gated potassium channel (Kv7.2). Pathogenic KCNQ2 variants are associated with severe developmental and epileptic encephalopathies that lack targeted treatments. We identified an upstream open reading frame (uORF) within the 5’-untranslated region (5’-UTR) of the KCNQ2 mRNA transcript that competes with the canonical reading frame for translation and attenuates Kv7.2 functional expression in non-neuronal cells. Because suppressing uORF translation could be an effective strategy for boosting KCNQ2 expression in the setting of heterozygous LOF, we seek to determine the physiological relevance of the KCNQ2 uORF (Aim 1), and to target the uORF with antisense oligonucleotides (ASO) using a structure-driven approach (Aim 2). Our approach to Aim 1 will involve genome editing of human induced pluripotent stem cells (iPSC) to inactivate the uORF start codon and to compare uORF usage, Kv7.2 translation, and electrophysiological properties of the channel in neurons differentiated from edited and non-edited iPSC lines. For Aim 2, we will experimentally deduce the secondary structure of the KCNQ2 5’-UTR, then design a series of ASOs targeting the least structured regions proximal to the uORF. We will then confirm binding of candidate ASOs to the KCNQ2 mRNA transcript and determine if an ASO strategy will boost Kv7.2 translation and function. These studies will clarify the physiological relevance of the uORF and identify a potential therapeutic strategy to correct heterozygous LOF in this epilepsy gene.