Periodic Reporting for period 2 - TRAIN-HEART (TRAIN-HEART)
Okres sprawozdawczy: 2021-06-01 do 2023-08-31
Heart failure (HF) is a widespread and burdensome condition characterized by pathological changes in the myocardium, resulting in pump failure and potential sudden death. Despite its prevalence, our understanding of the disease mechanisms remains limited, leading to a one-size-fits-all approach in HF therapy, which overlooks the various disease subtypes and individual differences.
In the Western world, the primary cause of HF is ischemic heart disease, often stemming from conditions like myocardial infarction (MI). To address this, a paradigm shift in HF research is required, focusing on groundbreaking concepts that can pave the way for an entirely new class of therapeutics targeting specific molecules and mechanisms within cardiomyocytes. Advances in cardiovascular biology have ushered in a gene-level approach to cardiac repair, known as gene therapy. Within this framework, non-coding RNAs (ncRNAs), including small ncRNAs (<200 nt, such as microRNAs) and long non-coding RNAs (lncRNAs; >200 nt), have emerged as potent regulators of cellular and tissue function at the post-transcriptional level. Notably, alterations in local ncRNA levels have been linked to disruptions in normal cellular processes and the development of conditions like cardiac remodeling and hypertrophy, highlighting their potential as therapeutic targets in HF patients.
The TRAIN-HEART program sought to broaden the scope of scientific inquiry and expedite the development of RNA-based therapeutics for the treatment of ischemic heart failure. MicroRNAs and long non-coding RNAs, in particular, play pivotal roles as regulators of cardiac tissue remodeling under chronic cardiac stress. Innovative therapies that target these RNA molecules hold significant promise in addressing the root causes of ischemic heart failure. Currently, aside from heart transplantation, no curative therapy exists for this debilitating disease. However, advancements in chemically modified oligonucleotides and drug delivery strategies are making substantial progress, addressing challenges related to delivery, specificity, and tolerability of RNA therapeutics.
In the Western world, the primary cause of HF is ischemic heart disease, often stemming from conditions like myocardial infarction (MI). To address this, a paradigm shift in HF research is required, focusing on groundbreaking concepts that can pave the way for an entirely new class of therapeutics targeting specific molecules and mechanisms within cardiomyocytes. Advances in cardiovascular biology have ushered in a gene-level approach to cardiac repair, known as gene therapy. Within this framework, non-coding RNAs (ncRNAs), including small ncRNAs (<200 nt, such as microRNAs) and long non-coding RNAs (lncRNAs; >200 nt), have emerged as potent regulators of cellular and tissue function at the post-transcriptional level. Notably, alterations in local ncRNA levels have been linked to disruptions in normal cellular processes and the development of conditions like cardiac remodeling and hypertrophy, highlighting their potential as therapeutic targets in HF patients.
The TRAIN-HEART program sought to broaden the scope of scientific inquiry and expedite the development of RNA-based therapeutics for the treatment of ischemic heart failure. MicroRNAs and long non-coding RNAs, in particular, play pivotal roles as regulators of cardiac tissue remodeling under chronic cardiac stress. Innovative therapies that target these RNA molecules hold significant promise in addressing the root causes of ischemic heart failure. Currently, aside from heart transplantation, no curative therapy exists for this debilitating disease. However, advancements in chemically modified oligonucleotides and drug delivery strategies are making substantial progress, addressing challenges related to delivery, specificity, and tolerability of RNA therapeutics.
The TRAIN-HEART program was divided into three parts:
In part 1, mechanisms of action of pre-discovered non-coding RNAs were extensively explored. This part started with the establishment of the functional relevance of the candidate ncRNAs by manipulating their expression in primary myocardial cells in culture and analyzing alterations of the transcriptome through RNA-seq. The expression of the most differentially and abundantly expressed target messenger RNAs (targetome) of leading non-coding RNAs was studied and manipulated in fractionated cardiac cell types isolated from adult hearts of wildtype mice. Genomic regions associated with the found changes in the specific ncRNA species were mapped by interrogating pre-existing human and mouse SNP/epigenetic datasets. Bioinformatics approaches were implemented to understand the mechanistic basis and uncover the cell-type specific targetome of therapeutically relevant ncRNA interactomes. In this part, five research fellows collaborated intensively to advance the fundamental research work.
In part 2, another group of five research fellows was dedicated to the successful pre-clinical application of existing non-coding RNAs by improving their efficacy and delivery systems. In particular, therapeutic effects of antagomirs were extensively investigated in both small (mice) and large (pigs) animal models. These activities included toxicity testing and the pharmacological delivery of RNA therapeutics. Further optimization of antagomirs or RNA therapeutics was achieved through additional chemical modifications to improve binding affinity and nuclease resistance. Another remarkable achievement in this part was the development of a novel and clinically relevant culture platform for a heart failure model to assess non-coding RNAs (ncRNAs)-based therapeutics. Importantly, the complex multicellular network was successfully recapitulated in these innovative platforms, such as LMS and engineered heart tissue (EHT), which was proposed as a powerful tool to bridge from preclinical to clinical translation.
In part 3, the final section of TRAIN-HEART, five research fellows actively worked together on developing and improving RNA drug efficacy and delivery. Together, they established two different approaches to encapsulate non-coding RNA molecules in either Chitosan or calcium-phosphate (CaP) nanoparticles (NPs). It was demonstrated that single-stranded DNA binds to chitosan-based NPs, and the internalization of CaP-NPs was evaluated in unpolarized, inflammatory, or reparative macrophages. One fellow characterized the functional role of microRNA-27b (miR-27b) in cardiac macrophages and initiated a study using a miR-27b inhibitor in the murine cardiac pressure overload model of transverse aortic constriction (TAC). It was also observed that Gapmer treatment against a specific RNA target significantly reduced hypertrophy after TAC in male mice. In this part, an aortic banding model in a porcine model was also established, providing a perspective for testing the best candidates in a large animal model. In the second program period, NPs encapsulating the above-mentioned non-coding RNA therapeutics were tested in combination with the established aortic banding model.
In part 1, mechanisms of action of pre-discovered non-coding RNAs were extensively explored. This part started with the establishment of the functional relevance of the candidate ncRNAs by manipulating their expression in primary myocardial cells in culture and analyzing alterations of the transcriptome through RNA-seq. The expression of the most differentially and abundantly expressed target messenger RNAs (targetome) of leading non-coding RNAs was studied and manipulated in fractionated cardiac cell types isolated from adult hearts of wildtype mice. Genomic regions associated with the found changes in the specific ncRNA species were mapped by interrogating pre-existing human and mouse SNP/epigenetic datasets. Bioinformatics approaches were implemented to understand the mechanistic basis and uncover the cell-type specific targetome of therapeutically relevant ncRNA interactomes. In this part, five research fellows collaborated intensively to advance the fundamental research work.
In part 2, another group of five research fellows was dedicated to the successful pre-clinical application of existing non-coding RNAs by improving their efficacy and delivery systems. In particular, therapeutic effects of antagomirs were extensively investigated in both small (mice) and large (pigs) animal models. These activities included toxicity testing and the pharmacological delivery of RNA therapeutics. Further optimization of antagomirs or RNA therapeutics was achieved through additional chemical modifications to improve binding affinity and nuclease resistance. Another remarkable achievement in this part was the development of a novel and clinically relevant culture platform for a heart failure model to assess non-coding RNAs (ncRNAs)-based therapeutics. Importantly, the complex multicellular network was successfully recapitulated in these innovative platforms, such as LMS and engineered heart tissue (EHT), which was proposed as a powerful tool to bridge from preclinical to clinical translation.
In part 3, the final section of TRAIN-HEART, five research fellows actively worked together on developing and improving RNA drug efficacy and delivery. Together, they established two different approaches to encapsulate non-coding RNA molecules in either Chitosan or calcium-phosphate (CaP) nanoparticles (NPs). It was demonstrated that single-stranded DNA binds to chitosan-based NPs, and the internalization of CaP-NPs was evaluated in unpolarized, inflammatory, or reparative macrophages. One fellow characterized the functional role of microRNA-27b (miR-27b) in cardiac macrophages and initiated a study using a miR-27b inhibitor in the murine cardiac pressure overload model of transverse aortic constriction (TAC). It was also observed that Gapmer treatment against a specific RNA target significantly reduced hypertrophy after TAC in male mice. In this part, an aortic banding model in a porcine model was also established, providing a perspective for testing the best candidates in a large animal model. In the second program period, NPs encapsulating the above-mentioned non-coding RNA therapeutics were tested in combination with the established aortic banding model.
TRAIN-HEART is grounded in the recognition that addressing heart failure demands pioneering concepts to drive the development of an entirely new class of therapeutics. Our proposal centers on non-coding RNA (ncRNA) molecules and the intricate ncRNA-based mechanisms that offer precision in targeting essential, highly localized biological processes within cardiomyocytes. This approach minimizes interference from distant systemic effects, providing a clearer path forward. We aim to shift the conventional emphasis from protein-coding genes (constituting <2% of the human genome) to the regulatory non-coding regions of the genome, which play a pivotal role in human diversity and contribute significantly to the development of ischemic heart failure.
Non-coding RNAs are integral to the fundamental mechanisms that underlie a substantial portion of human phenotypic diversity and, in all likelihood, disease susceptibility. A deeper understanding of this influential genome segment is expected to enhance our comprehension of the development of ischemic heart failure. Among ncRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have gained particular recognition for their role in the pathogenesis of ischemic heart failure, and they constitute the core focus of this research network. TRAIN-HEART stands as the pioneering multidisciplinary program, spanning from fundamental research to late-stage preclinical development. It offers invaluable insights into the genomic pathophysiology of ischemic heart failure while exploring the therapeutic potential of existing RNA therapeutics, both in their naked form and in nanoformulations.
Non-coding RNAs are integral to the fundamental mechanisms that underlie a substantial portion of human phenotypic diversity and, in all likelihood, disease susceptibility. A deeper understanding of this influential genome segment is expected to enhance our comprehension of the development of ischemic heart failure. Among ncRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have gained particular recognition for their role in the pathogenesis of ischemic heart failure, and they constitute the core focus of this research network. TRAIN-HEART stands as the pioneering multidisciplinary program, spanning from fundamental research to late-stage preclinical development. It offers invaluable insights into the genomic pathophysiology of ischemic heart failure while exploring the therapeutic potential of existing RNA therapeutics, both in their naked form and in nanoformulations.