Mitochondrial Gene Editing in Human Cardiomyocytes

Mitochondria (mt) are regarded as critical components for cardiomyocyte homeostasis and are the nexus of many signaling events that maintain normal cardiac function. As a result, mutations in mitochondrial DNA (mtDNA) have been shown to affect cardiac myocyte function and are associated with the development of cardiomyopathy in ~25% of all mtDNA mutation carriers. However, it is difficult to define mtDNA mutations as the cause of a particular pathology because the penetrance of mtDNA disease is not mendelian and is dependent on the amount of heteroplasmy of mtDNA mutations per cell, the cell type expressing mutations, as well as the specific mutation. Some canonical mtDNA mutations, in particular mt-tRNA mutations, have been shown to be displayed at higher heteroplasmic states and are defined as causative sources for cardiomyopathy, such as in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome caused by a mt-tRNALeu mutation (m.3243A>G). Many mtDNA diseases, such as MELAS, present with aging, yet it’s not completely defined why and how different cardiomyocytes or other cardiac cells i.e., endothelial or fibroblasts, are affected by the presence of MELAS mutations. Understanding of the mechanism of cardiomyopathy by mtDNA mutations would be advanced by useful mtDNA gene editing strategies. In this proposed research plan, we present a novel Cas9 based mitochondrial gene editor system (mEditors) that we designed to introduce mtDNA mutations into human IPSC cardiomyocytes (IPSC-CMs), with the ultimate goal to ascribing pathogenic and potentially causal relationships between specific mtDNA mutations and cardiomyopathy. We hypothesize that mEditors can be used to introduce, rescue, and regulate mtDNA heteroplasmy states of the common MELAS mutation (m.3243A>G), allowing hypothesis-testing on the presence and level of mtDNA mutations with functional effects on cardiomyocytes. The following aims are designed to test these hypotheses: Aim 1: To test the hypothesis that mEditorS can be used to introduce mtDNA mutations in human cardiomyocytes. Aim 2: To test the hypothesis that mEditorS can remove mtDNA mutations in human cardiomyocytes. Aim 3: To test the hypothesis that mtDNA heteroplasmy can be regulated by mEditors in conjunction with functional alterations in human cardiomyocytes. This project will advance a novel mtDNA editing tool with the goal for ascribing causality for mtDNA mutations and possible future therapeutic development for mitochondrial diseases. Project Number: 1R01HL174859-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: RICHARD LEE | Institution: HARVARD UNIVERSITY, CAMBRIDGE, MA | Award Amount: $773,479 | Activity Code: R01 | Study Section: Basic Biology of Blood, Heart and Vasculature Study Section [BBHV] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HL17485901A1