Periodic Reporting for period 1 - Edit-hCOs (Precise Genome Editing to Correct Cardiomyopathies in Human Cardiac Organoids)
Reporting period: 2022-10-01 to 2024-09-30
Inherited cardiovascular diseases (CVDs) represent a significant health concern, contributing to sudden cardiac deaths and imposing a substantial burden on healthcare systems across Europe. These conditions, predominantly encompassing cardiomyopathies and channelopathies, affect approximately 1 in every 200 adults. While a few medications exist to slow disease progression, no cure currently exists for inherited CVDs. These diseases primarily stem from individual point mutations scattered throughout various genes, with multiple pathological mutations often identified within a single gene.
Over the past two decades, significant advancements have occurred in genome editing, offering powerful tools to correct disease-causing mutations at the DNA level, particularly for translational research. Among these tools, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing has emerged as a simple and cost-effective approach for potentially treating previously untreatable conditions, such as inherited CVDs. The recent development of base editing technology has further enhanced the precision of editing, allowing for the correction of point mutations without DNA cleavage. Base editors can induce specific base pair transitions within a defined editing window, guided by a single guide RNA (sgRNA). Despite the growing recognition of base editing’s potential in the field of cardiology, its efficacy in rectifying mutations causing cardiomyopathies and the promising therapeutic benefits remain underexplored. This is partially due to the absence of a suitable in vitro model of mature human cardiomyocytes that accurately replicates the intricate cellular makeup of the human heart.
Recent advancements in tissue engineering, coupled with a deeper understanding of the interactions between noncardiomyocytes and cardiomyocytes, have led to the development of three-dimensional (3D) human cardiac organoids (hCOs) derived from human induced pluripotent stem cells (hiPSCs). hCOs represent a cutting-edge in vitro model that closely mimics the human heart’s in vivo environment, overcoming previous limitations related to cardiomyocyte immaturity and the inability to replicate adult heart characteristics. These hCOs are constructed as 3D scaffold-free cardiac microtissues, composed of three key cell types: hiPSC-derived cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. This approach offers a straightforward and versatile platform for modeling inherited CVDs.
The “Precise Genome Editing to Correct Cardiomyopathies in Human Cardiac Organoids” (Edit-hCOs) project was initiated with the aim of establishing an innovative research avenue for developing genome editing-based therapies to address inherited CVDs. The overarching objective of the project was to integrate the precision genome editing capabilities of CRISPR-Cas9 base editing with 3D hCOs, creating a platform to showcase the potential applications of base editing in an advanced in vitro model that faithfully recapitulates the complex cellular landscape of the human heart. Leveraging base editing within hCOs allows for the rapid and robust assessment of the effectiveness, delivery systems, and safety profiles of genome editing components. This represents a crucial pre-clinical step toward the therapeutic genome editing of CVDs.
The project’s objectives were initially planned to be completed within a 2-year timeframe. However, after the first year, the Fellow received a career advancement opportunity, accepting a position as a Professor of Genetics at the University of Bologna. Despite this change, the project’s major objectives were successfully achieved within the initial 12 months, demonstrating that base editing could be effectively used to introduce genetic disease-causing mutations in cardiac organoids and, more importantly, to correct genetic mutations identified in patients affected by CVDs. Future collaboration between the Fellow and Supervisor will ensure the completion of the functional characterization of these cardiac organoids.
Over the past two decades, significant advancements have occurred in genome editing, offering powerful tools to correct disease-causing mutations at the DNA level, particularly for translational research. Among these tools, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing has emerged as a simple and cost-effective approach for potentially treating previously untreatable conditions, such as inherited CVDs. The recent development of base editing technology has further enhanced the precision of editing, allowing for the correction of point mutations without DNA cleavage. Base editors can induce specific base pair transitions within a defined editing window, guided by a single guide RNA (sgRNA). Despite the growing recognition of base editing’s potential in the field of cardiology, its efficacy in rectifying mutations causing cardiomyopathies and the promising therapeutic benefits remain underexplored. This is partially due to the absence of a suitable in vitro model of mature human cardiomyocytes that accurately replicates the intricate cellular makeup of the human heart.
Recent advancements in tissue engineering, coupled with a deeper understanding of the interactions between noncardiomyocytes and cardiomyocytes, have led to the development of three-dimensional (3D) human cardiac organoids (hCOs) derived from human induced pluripotent stem cells (hiPSCs). hCOs represent a cutting-edge in vitro model that closely mimics the human heart’s in vivo environment, overcoming previous limitations related to cardiomyocyte immaturity and the inability to replicate adult heart characteristics. These hCOs are constructed as 3D scaffold-free cardiac microtissues, composed of three key cell types: hiPSC-derived cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. This approach offers a straightforward and versatile platform for modeling inherited CVDs.
The “Precise Genome Editing to Correct Cardiomyopathies in Human Cardiac Organoids” (Edit-hCOs) project was initiated with the aim of establishing an innovative research avenue for developing genome editing-based therapies to address inherited CVDs. The overarching objective of the project was to integrate the precision genome editing capabilities of CRISPR-Cas9 base editing with 3D hCOs, creating a platform to showcase the potential applications of base editing in an advanced in vitro model that faithfully recapitulates the complex cellular landscape of the human heart. Leveraging base editing within hCOs allows for the rapid and robust assessment of the effectiveness, delivery systems, and safety profiles of genome editing components. This represents a crucial pre-clinical step toward the therapeutic genome editing of CVDs.
The project’s objectives were initially planned to be completed within a 2-year timeframe. However, after the first year, the Fellow received a career advancement opportunity, accepting a position as a Professor of Genetics at the University of Bologna. Despite this change, the project’s major objectives were successfully achieved within the initial 12 months, demonstrating that base editing could be effectively used to introduce genetic disease-causing mutations in cardiac organoids and, more importantly, to correct genetic mutations identified in patients affected by CVDs. Future collaboration between the Fellow and Supervisor will ensure the completion of the functional characterization of these cardiac organoids.
The Edit-hCOs project successfully achieved its objectives through the synergistic integration of various components: 1. Expertise in Genome Editing: The project capitalized on my extensive knowledge and experience in genome editing, specifically in correcting disease-causing mutations across various models, including cardiomyocytes. 2. Cutting-edge Environment for hCO Generation: The laboratory of Supervisor Prof. Milena Bellin at the Department of Biology (DiBio), University of Padua (UNIPD), provided an advanced setting for the generation and characterization of human cardiac organoids (hCOs) derived from human induced pluripotent stem cells (hiPSCs). 3. Cardiac Cell Biology Expertise: The EU Host Institution, the University of Padua, contributed long-standing and multifaceted expertise in the field of cardiac cell biology.
The project utilized base editors within hCOs to accomplish the following: A. Introducing Patient-Specific Point Mutations: To investigate the molecular pathways leading to dilated or hypertrophic cardiomyopathy, we efficiently introduced patient-specific point mutations in the FLNC gene, which encodes the sarcomeric protein filamin C. This gene has gained particular significance, with recent studies associating FLNC mutations with various cardiomyopathy phenotypes. B. Correction of LQT1 Syndrome Mutation: Long QT Syndrome (LQTS) is a genetically diverse arrhythmogenic disorder, and LQT1 represents the most common subtype. The project aimed to develop a base editing system to correct a single nucleotide LQT1 mutation associated with patients. This proof-of-concept demonstrated the feasibility of base editing in correcting LQT1 mutations in patient-specific hiPSC-cardiomyocytes. LQT1 patients are at risk of severe cardiac events, and our work lays the groundwork for potential therapeutic strategies targeting the genetic basis of the disease. C. Universal Strategy for DMD Cardiomyopathy: Duchenne Muscular Dystrophy (DMD) is a devastating muscular dystrophy primarily affecting boys, leading to cardiac and respiratory failure. DMD results from mutations in the dystrophin gene, and more than 7,000 mutations have been identified. Utrophin, a related protein, can function similarly but is poorly expressed in the heart due to translational silencing mediated by miRNAs. The project employed base editing to introduce precise modifications in the utrophin gene within hCOs, disrupting the miRNA target sites and thereby preventing translational silencing. This approach enhances utrophin protein expression in cardiac cells, potentially improving cardiac muscle function.
In summary, the Edit-hCOs project successfully harnessed base editing technology to advance our understanding of inherited cardiovascular diseases, paving the way for potential therapeutic interventions. It demonstrated the capability to introduce and correct disease-causing mutations, laying the foundation for future research and potential clinical applications in the treatment of these debilitating conditions.
The project utilized base editors within hCOs to accomplish the following: A. Introducing Patient-Specific Point Mutations: To investigate the molecular pathways leading to dilated or hypertrophic cardiomyopathy, we efficiently introduced patient-specific point mutations in the FLNC gene, which encodes the sarcomeric protein filamin C. This gene has gained particular significance, with recent studies associating FLNC mutations with various cardiomyopathy phenotypes. B. Correction of LQT1 Syndrome Mutation: Long QT Syndrome (LQTS) is a genetically diverse arrhythmogenic disorder, and LQT1 represents the most common subtype. The project aimed to develop a base editing system to correct a single nucleotide LQT1 mutation associated with patients. This proof-of-concept demonstrated the feasibility of base editing in correcting LQT1 mutations in patient-specific hiPSC-cardiomyocytes. LQT1 patients are at risk of severe cardiac events, and our work lays the groundwork for potential therapeutic strategies targeting the genetic basis of the disease. C. Universal Strategy for DMD Cardiomyopathy: Duchenne Muscular Dystrophy (DMD) is a devastating muscular dystrophy primarily affecting boys, leading to cardiac and respiratory failure. DMD results from mutations in the dystrophin gene, and more than 7,000 mutations have been identified. Utrophin, a related protein, can function similarly but is poorly expressed in the heart due to translational silencing mediated by miRNAs. The project employed base editing to introduce precise modifications in the utrophin gene within hCOs, disrupting the miRNA target sites and thereby preventing translational silencing. This approach enhances utrophin protein expression in cardiac cells, potentially improving cardiac muscle function.
In summary, the Edit-hCOs project successfully harnessed base editing technology to advance our understanding of inherited cardiovascular diseases, paving the way for potential therapeutic interventions. It demonstrated the capability to introduce and correct disease-causing mutations, laying the foundation for future research and potential clinical applications in the treatment of these debilitating conditions.
The Edit-hCOs project stands as a pioneering endeavor, bridging the divide between genome editing and therapies for inherited cardiovascular diseases. Several groundbreaking achievements and advancements distinguish this project.
Integration of Cutting-edge Genome Editing with Advanced Pre-clinical Models. This project represents the first of its kind, combining the latest genome editing technologies with state-of-the-art pre-clinical human cardiac organoid (hCO) models. The development of these advanced in vitro models, harboring mutations identical to those found in human patients, represents a significant leap forward. This breakthrough enhances our understanding of the role of filamin C in cardiomyopathy, shedding new light on the disease mechanisms.
Correction of LQT1 Mutation. By demonstrating the correction of point mutations causing Long QT Type 1 syndrome (LQT1) in patient-specific hiPSC-derived cardiomyocytes, this project showcases the transformative potential of base editing. This achievement is pivotal in addressing LQT1, a common and life-threatening cardiovascular disorder. The correction of these mutations brings us closer to the development of potential therapies targeting the genetic basis of LQT1.
Universal Gene Editing Strategy for DMD. The project introduces a novel and universal gene editing strategy to treat dilated cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients, regardless of the specific mutation type. This approach holds promise for enhancing the quality of life and extending the lifespan of all DMD patients. The utilization of base editing components in hCOs derived from DMD patient-specific hiPSCs represents a significant step towards achieving this goal.
In essence, the Edit-hCOs project has not only pushed the boundaries of current research but has also paved the way for transformative advancements in understanding, diagnosing, and potentially treating inherited cardiovascular diseases. The integration of genome editing with cutting-edge pre-clinical models holds immense potential for the future of cardiovascular disease research and therapy development.
Integration of Cutting-edge Genome Editing with Advanced Pre-clinical Models. This project represents the first of its kind, combining the latest genome editing technologies with state-of-the-art pre-clinical human cardiac organoid (hCO) models. The development of these advanced in vitro models, harboring mutations identical to those found in human patients, represents a significant leap forward. This breakthrough enhances our understanding of the role of filamin C in cardiomyopathy, shedding new light on the disease mechanisms.
Correction of LQT1 Mutation. By demonstrating the correction of point mutations causing Long QT Type 1 syndrome (LQT1) in patient-specific hiPSC-derived cardiomyocytes, this project showcases the transformative potential of base editing. This achievement is pivotal in addressing LQT1, a common and life-threatening cardiovascular disorder. The correction of these mutations brings us closer to the development of potential therapies targeting the genetic basis of LQT1.
Universal Gene Editing Strategy for DMD. The project introduces a novel and universal gene editing strategy to treat dilated cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients, regardless of the specific mutation type. This approach holds promise for enhancing the quality of life and extending the lifespan of all DMD patients. The utilization of base editing components in hCOs derived from DMD patient-specific hiPSCs represents a significant step towards achieving this goal.
In essence, the Edit-hCOs project has not only pushed the boundaries of current research but has also paved the way for transformative advancements in understanding, diagnosing, and potentially treating inherited cardiovascular diseases. The integration of genome editing with cutting-edge pre-clinical models holds immense potential for the future of cardiovascular disease research and therapy development.