Focused molecular understanding of hypertrophic cardiomyopathy using CRISPR-engineered human pluripotent stem cell-derived cardiomyocytes
Lead Research Organisation:
University of Nottingham
Department Name: School of Medicine
Abstract
Overview
This proposal aims to investigate a prevalent and complex cardiac disease using a human cell model in order to promote future drug discovery strategies towards its treatment. It builds on previous work focused on the development of the model as well as the molecular tools needed to interrogate its disease features.
Health need
Heart failure represents a major cause of mortality and morbidity worldwide and remains the leading cause of global suffering. In the UK ~900,000 people are affected by heart failure and each year there are 20 million deaths worldwide. Treatment of the disease uses 2% of the UK healthcare budget and worldwide costs will reach 250 billion dollars by 2030. A major contributor is Hypertrophic Cardiomyopathy (HCM) which is the most prevalent cardiac disease, affecting 1:500 people and often leading to sudden cardiac death at a young age. Few drug treatments exist, with the only definite solution being heart transplantation. The major current benefits come from indirect treatment, such as reducing heart rate, blood pressure or risk of stroke. Greater understanding of the molecular mechanisms of HCM would provide a route for much needed development of new therapies. However, mechanisms of HCM are poorly understood, often because data obtained from animal models is frequently misinterpreted due to species-differences (e.g. mouse heart beat rates are 10-fold higher than humans).
Progress
During 2018, I was involved in pioneering work to show that new approaches could be used to model HCM. The emphasis was on the MYH7 gene, which encodes the contractile protein, beta myosin heavy chain. CRISPR/Cas9 engineering was used to make genetic variants of MYH7 in heart cells produced from human pluripotent stem cells - these 'cardiomyocytes' are abbreviated to hPSC-CMs. By conducting very detailed analysis, many of the features of HCM could be reproduced in hPSC-CMs carrying mutant variants of MYH7. Importantly, this included the finding that altered expression of many novel long non-coding RNAs (lncRNAs) appeared to be associated with HCM. This enables hypothesis-driven questions to be asked about their relative importance in heart disease.
Hypothesis and plans
I hypothesise that unveiling the function of these lncRNAs will provide new mechanistic insight and drug targets for HCM. To test this hypothesis, I propose to perform focused experiments to reduce or increase the levels of the lncRNAs in hPSC-CMs and then assess their impact on cellular behaviour and function. This will help clarify their role in the disease progression - whether as disease contributors, cardioprotective or as gene modifiers. Finally, the identification and validation of direct interacting proteins with these lncRNAs will shed light into their molecular mechanism and pave the way for future therapies.
If replacement of 50% of animals for HCM research could be achieved by validating the human model, this will equate to sparing 2100-5000 animals (of all species) in severe procedures in the next 10 years, and would instead generate human-relevant data to further tackle this disease.
This proposal aims to investigate a prevalent and complex cardiac disease using a human cell model in order to promote future drug discovery strategies towards its treatment. It builds on previous work focused on the development of the model as well as the molecular tools needed to interrogate its disease features.
Health need
Heart failure represents a major cause of mortality and morbidity worldwide and remains the leading cause of global suffering. In the UK ~900,000 people are affected by heart failure and each year there are 20 million deaths worldwide. Treatment of the disease uses 2% of the UK healthcare budget and worldwide costs will reach 250 billion dollars by 2030. A major contributor is Hypertrophic Cardiomyopathy (HCM) which is the most prevalent cardiac disease, affecting 1:500 people and often leading to sudden cardiac death at a young age. Few drug treatments exist, with the only definite solution being heart transplantation. The major current benefits come from indirect treatment, such as reducing heart rate, blood pressure or risk of stroke. Greater understanding of the molecular mechanisms of HCM would provide a route for much needed development of new therapies. However, mechanisms of HCM are poorly understood, often because data obtained from animal models is frequently misinterpreted due to species-differences (e.g. mouse heart beat rates are 10-fold higher than humans).
Progress
During 2018, I was involved in pioneering work to show that new approaches could be used to model HCM. The emphasis was on the MYH7 gene, which encodes the contractile protein, beta myosin heavy chain. CRISPR/Cas9 engineering was used to make genetic variants of MYH7 in heart cells produced from human pluripotent stem cells - these 'cardiomyocytes' are abbreviated to hPSC-CMs. By conducting very detailed analysis, many of the features of HCM could be reproduced in hPSC-CMs carrying mutant variants of MYH7. Importantly, this included the finding that altered expression of many novel long non-coding RNAs (lncRNAs) appeared to be associated with HCM. This enables hypothesis-driven questions to be asked about their relative importance in heart disease.
Hypothesis and plans
I hypothesise that unveiling the function of these lncRNAs will provide new mechanistic insight and drug targets for HCM. To test this hypothesis, I propose to perform focused experiments to reduce or increase the levels of the lncRNAs in hPSC-CMs and then assess their impact on cellular behaviour and function. This will help clarify their role in the disease progression - whether as disease contributors, cardioprotective or as gene modifiers. Finally, the identification and validation of direct interacting proteins with these lncRNAs will shed light into their molecular mechanism and pave the way for future therapies.
If replacement of 50% of animals for HCM research could be achieved by validating the human model, this will equate to sparing 2100-5000 animals (of all species) in severe procedures in the next 10 years, and would instead generate human-relevant data to further tackle this disease.
Technical Summary
Rationale and Timeliness:
Further molecular insight of the pathways involved in hypertrophic cardiomyopathy (HCM) is warranted for deeper understanding of the disease and drug development efforts. My 2018 publications in European Heart Journal and Circulation Research showed HCM could be phenocopied by a set of 11 isogenic CRISPR/Cas9 variants of the MYH7 gene (beta myosin heavy chain) of human pluripotent stem cell-cardiomyocytes (hPSC-CMs). Twelve different phenotyping methods were used to interrogate the generated human cell lines, often showing contradictory results relative to animal models. Importantly, a cohort of long non-coding RNAs (lncRNAs) potentially linked to HCM was identified by RNA sequencing. This collection of cell resources, underpinning technologies and putative lncRNA pathway controllers form the basis of this proposal.
Hypothesis and plans:
I hypothesise that unveiling the function of these novel lncRNAs will provide new mechanisms and drug targets for HCM. To test this hypothesis, I propose to modulate the expression of these lncRNAs in hPSC-CMs by harnessing the dead Cas9 technology. I will then assess the phenotype of up/down-regulating these lncRNAs in the context of HCM, by employing the methodologies previously developed (molecular and functional assays). This will enable determining the role of lncRNAs in HCM - either as disease-causing or cardioprotective per se, or as gene modifiers regulating the primary disease effects caused by the structural defect. Finally, prediction and validation of lncRNA-interacting proteins by coupling in silico tools with molecular biology methods will result in the identification of new pathways involved in HCM, shedding light into the development of future drug treatments.
If replacement of 50% of animals for HCM research could be achieved by further validating the human model, this will equate to sparing of 2100-5000 animals (of all species) in severe procedures in the next 10 years.
Further molecular insight of the pathways involved in hypertrophic cardiomyopathy (HCM) is warranted for deeper understanding of the disease and drug development efforts. My 2018 publications in European Heart Journal and Circulation Research showed HCM could be phenocopied by a set of 11 isogenic CRISPR/Cas9 variants of the MYH7 gene (beta myosin heavy chain) of human pluripotent stem cell-cardiomyocytes (hPSC-CMs). Twelve different phenotyping methods were used to interrogate the generated human cell lines, often showing contradictory results relative to animal models. Importantly, a cohort of long non-coding RNAs (lncRNAs) potentially linked to HCM was identified by RNA sequencing. This collection of cell resources, underpinning technologies and putative lncRNA pathway controllers form the basis of this proposal.
Hypothesis and plans:
I hypothesise that unveiling the function of these novel lncRNAs will provide new mechanisms and drug targets for HCM. To test this hypothesis, I propose to modulate the expression of these lncRNAs in hPSC-CMs by harnessing the dead Cas9 technology. I will then assess the phenotype of up/down-regulating these lncRNAs in the context of HCM, by employing the methodologies previously developed (molecular and functional assays). This will enable determining the role of lncRNAs in HCM - either as disease-causing or cardioprotective per se, or as gene modifiers regulating the primary disease effects caused by the structural defect. Finally, prediction and validation of lncRNA-interacting proteins by coupling in silico tools with molecular biology methods will result in the identification of new pathways involved in HCM, shedding light into the development of future drug treatments.
If replacement of 50% of animals for HCM research could be achieved by further validating the human model, this will equate to sparing of 2100-5000 animals (of all species) in severe procedures in the next 10 years.
Planned Impact
This research will directly Replace the use of animals for modeling hypertrophic cardiomyopathy (HCM). Current animal disease models were developed in several species (cats, pigs, dogs, rabbits, zebrafish, Drosophila flies, rats and mice), totalling 4200-10000 animals according to a Pubmed search focused solely on HCM in the past 10 years. Additional estimates were obtained from two collaborating labs where animal models are routinely used for HCM modelling (Carrier, Hamburg; van der Velden, Amsterdam), estimating adult mice usage between 50-100 animals per year per lab. Thus, adoption of the developed human HCM model by research groups national and internationally is expected to replace 50% of the animals used, reducing to 2100-5000 over a similar period of time. This proposal is expected to benefit:
Researchers: in academia and industry, as well as clinician-scientists, in the UK and abroad.
The data obtained by previously developed animal models often contradicts human clinical information due to species-differences (e.g. gene expression and heart physiology). Clinical studies often employ large numbers of patient cohorts with ample variability between patients. The isogenic human HCM model that underpins this proposal would greatly benefit both types of research due to the generation of physiologically-relevant data from genetically-controlled samples. Provision of cell resources to collaborating international groups is already underway, highlighting the ease-of-adoption of this model by other labs (see Letters of Support from collaborators). Upon additional validation provided by gathering data on the mechanism of HCM in this unique human model, the pharmaceutical industry may also potentially benefit in the long term from licensing the gene-edited isogenic cells, thus accelerating drug discovery relative to current animal models.
Patient groups and the Health Services:
The work is designed to improve the health and quality of life for HCM patients, whilst concurrently reducing treatment costs and increasing treatment options. The importance is that HCM is a prevalent cardiac disease. It affects 1:500 people, is a complex chronic condition without a known cure and remains the most common cause of sudden death in younger patients (age <25 years) and athletes. Annual costs of patient management vary by country, hospital and treatment (i.e. surgical intervention vs conventional drug treatment) but are cited to be up to £25,000 per patient per year and expected to collectively reach 250 billion dollars worldwide in 2030.
This proposal aims to provide new mechanistic understanding into the condition, using a human-relevant model of HCM. This information will likely result in the identification of unexplored drug targets, potentially providing routes towards new therapeutics for a currently unresolved disease. This will improve management of HCM, thereby benefitting patients and the clinicians who care for them and the patient groups with which they are associated (e.g. Cardiomyopathy UK; Hypertrophic Cardiomyopathy Association; Cardiac Risk in the Young).
Researchers: in academia and industry, as well as clinician-scientists, in the UK and abroad.
The data obtained by previously developed animal models often contradicts human clinical information due to species-differences (e.g. gene expression and heart physiology). Clinical studies often employ large numbers of patient cohorts with ample variability between patients. The isogenic human HCM model that underpins this proposal would greatly benefit both types of research due to the generation of physiologically-relevant data from genetically-controlled samples. Provision of cell resources to collaborating international groups is already underway, highlighting the ease-of-adoption of this model by other labs (see Letters of Support from collaborators). Upon additional validation provided by gathering data on the mechanism of HCM in this unique human model, the pharmaceutical industry may also potentially benefit in the long term from licensing the gene-edited isogenic cells, thus accelerating drug discovery relative to current animal models.
Patient groups and the Health Services:
The work is designed to improve the health and quality of life for HCM patients, whilst concurrently reducing treatment costs and increasing treatment options. The importance is that HCM is a prevalent cardiac disease. It affects 1:500 people, is a complex chronic condition without a known cure and remains the most common cause of sudden death in younger patients (age <25 years) and athletes. Annual costs of patient management vary by country, hospital and treatment (i.e. surgical intervention vs conventional drug treatment) but are cited to be up to £25,000 per patient per year and expected to collectively reach 250 billion dollars worldwide in 2030.
This proposal aims to provide new mechanistic understanding into the condition, using a human-relevant model of HCM. This information will likely result in the identification of unexplored drug targets, potentially providing routes towards new therapeutics for a currently unresolved disease. This will improve management of HCM, thereby benefitting patients and the clinicians who care for them and the patient groups with which they are associated (e.g. Cardiomyopathy UK; Hypertrophic Cardiomyopathy Association; Cardiac Risk in the Young).