“Gene Modulation in Human iPSC-Cardiomyocytes for Control of Excitation-Contraction Coupling Using CRISPRi, Optogenetic and Sonogenetic Actuators”
Monday, April 3rd, 2023 at 11:30 p.m. - 1:30 p.m.
SEH 2000
WebEx Meeting Link
JULIE LEANN HAN
The George Washington University
Advisor: Dr. Emilia Entcheva
ABSTRACT
The heart is a complex multicellular system, whose reliable functionality is critical for sustaining life. Gaining mechanistic understanding of heart’s function and resilience to inform new cardiac therapies has been particularly challenging due to the limited sources of human primary cardiomyocytes and lack of tools to probe gene function and dynamically monitor responses. However, advancements in stem cell technology, genetic engineering, optogenetics, and sonogenetics have yielded more refined tools to not only visualize responses with high spatial and temporal resolution, but also to actively interrogate and control biological function. Here, we leverage these approaches in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to gain mechanistic insights and inform more effective treatments for cardiac arrhythmias.
The rapid progress of CRISPR-based technologies has facilitated the manipulation of the human genome, epigenome, and transcriptome that is both scalable and more specific compared to traditional methods of gene modulation. Combining these tools, e.g. interference CRISPR (CRISPRi), in particular, with high-throughput all-optical platforms for cardiac electrophysiology allow us to perturb gene transcription in a controlled manner and to correlate RNA, DNA, protein content with features of the measured electrophysiological phenotype, thus establishing a methodology of performing human functional genomics in a dish. In this work, in depth validation and characterization of these tools (CRISPRi and comprehensive scalable all-optical electrophysiology) is presented, illustrated by perturbations of genes involved in cardiac excitability, repolarization and conduction in hiPSC-CM syncytia. Furthermore, we present initial efforts in extending optogenetic approaches to control of ultrastructure and interactions of calcium release stores in myocytes.
While optogenetics has already made an impact on the development of high-throughput phenotyping platforms for hiPSC-CMs in vitro, for future in vivo work, it is important to explore actuation strategies with potential for deep-tissue control of heart function. Here, we develop hybrid optogenetic-sonogenetic approaches to control cardiomyocyte responses through the application of light and ultrasound after genetic modifications. To accomplish this, we developed specialized molecular tools (delivery of light- and mechanosensitive ion channels) and experimental platforms for gated light-ultrasound delivery in 2D and 3D cardiac cell constructs. Taken together, these tools represent important technological developments towards improved personalized medicine and drug screening using iPSC-CM models of the human heart, but also exploration of potential new gene therapy approaches to rescue or augment cardiac function in vivo.