Abstract 14298: Basic Cardiovascular Science Best Abstract Award: Allele-specific Disruption Rescues Electrophysiological Abnormalities in Human iPS Cell Model of Long-QT Syndrome With a CALM2 Mutation
Background: Calmodulin is a ubiquitous Ca2+-sensor molecule and an identical calmodulin protein is encoded by the 3 distinct genes, CALM1-3. Recently, mutations in CALM are reported to be associated with severe early-onset long-QT syndrome (LQT). We have established CALM2-related LQT patient-derived iPSC model which exhibited prolonged action potential duration (APD) due to impaired inactivation of L-type Ca2+ currents (ICaL). It has been suggested that heterozygous CALM mutations could exert a dominant-negative effect, therefore, we aimed to assess the mutant allele-specific disruption by genome editing for the treatment of CALM-related LQT.
Methods: We generated hiPSC from a 12-year-old boy with LQT carrying a CALM2-N98S mutation. In order to disrupt mutated calmodulin, we designed guide RNA for targeting the mutant allele sequence. The Cas9/guide RNA expression vectors were transfected in LQT-hiPSCs and genome-edited clones were confirmed by sequencing. LQT- and LQT-KO-hiPSCs were differentiated into cardiomyocytes (CMs), and action potentials (APs) and ICaL were analyzed by patch-clamp technique.
Results: We successfully obtained mutant allele-specific knockout clones using the CRISPR-Cas9 system. As a result of electrophysiological analyses, the decreased beating rate and prolonged APDs in CALM2-related hiPSC-CMs were rescued in LQT-KO-hiPSC-CMs (Fig. A). The parameters in LQT-KO clones were similar to those in control generated by a healthy volunteer. In addition, the impaired inactivation of L-type Ca2+ channels in CALM2-related LQT was ameliorated in LQT-KO clones (Fig. B).
Conclusion: Mutant allele-specific disruption using CRISPR-Cas9 system rescued the electrophysiological abnormalities in CALM2-related LQT iPSC model, which provides us new insights into a promising therapeutic approach using the latest genome-editing technology for inherited cardiac diseases especially caused by dominant-negative mechanism.
Author Disclosures: Y. Yamamoto: None. T. Makiyama: None. T. Harita: None. K. Sasaki: None. M. Hayano: None. S. Nishiuchi: None. Y. Wuriyanghai: None. H. Kohjitani: None. S. Hirose: None. J. Chen: None. T. Ishikawa: None. H. Motomura: None. S. Ohno: None. K. Chonabayashi: None. Y. Yoshida: None. M. Horie: None. N. Makita: None. T. Kimura: None.
- © 2016 by American Heart Association, Inc.