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AAV vectors are promising delivery tools for human gene therapy. However, broad tissue tropism and pre-existing immunity against natural serotypes limit their clinical use. We identified two AAV capsid variants, AAV2-THGTPAD and AAV2-NLPGSGD, by in vivo AAV2 peptide display library screening in a murine model of pressure overload-induced cardiac hypertrophy. Both variants showed significantly improved efficacy in in vivo cardiomyocyte transduction compared with the parental serotype AAV2 as indicated by a higher number of AAV vector episomes in the nucleus and significant improved transduction efficiency. Both variants also outcompeted the reference serotype AAV9 regarding cardiomyocyte tropism, reaching comparable cardiac transduction efficiencies accompanied with liver de-targeting and decreased transduction efficiency of non-cardiac cells. Capsid modification influenced immunogenicity as sera of mice treated with AAV2-THGTPAD and AAV2-NLPGSGD demonstrated a poor neutralization capacity for the parental serotype and the novel variants. In a therapeutic setting, using the long non-coding RNA H19 in low vector dose conditions, novel AAV variants mediated superior anti-hypertrophic effects and revealed a further improved target-to-noise ratio, i.e., cardiomyocyte tropism. In conclusion, AAV2-THGTPAD and AAV2-NLPGSGD are promising novel tools for cardiac-directed gene therapy outperforming AAV9 regarding the specificity and therapeutic efficiency of in vivo cardiomyocyte transduction., Competing Interests: Declaration of interests L.R., C.B., T.T., and H.B. have filed and/or been granted patents in the field of AAV evolution for clinical applications. T.T. and J.V. have filed and been licensed a patent on the use of H19 in cardiovascular disease. T.T. is a founder of and holds shares in Cardior Pharmaceuticals GmbH., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: Pathological cardiac remodelling and subsequent heart failure represents an unmet clinical need. Long non-coding RNAs (lncRNAs) are emerging as crucial molecular orchestrators of disease processes, including that of heart diseases. Here, we report on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy., Method and Results: Pressure overload-induced left ventricular cardiac remodelling revealed an up-regulation of H19 in the early phase but strong sustained repression upon reaching the decompensated phase of heart failure. The translational potential of H19 is highlighted by its repression in a large animal (pig) model of left ventricular hypertrophy, in diseased human heart samples, in human stem cell-derived cardiomyocytes and in human engineered heart tissue in response to afterload enhancement. Pressure overload-induced cardiac hypertrophy in H19 knock-out mice was aggravated compared to wild-type mice. In contrast, vector-based, cardiomyocyte-directed gene therapy using murine and human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. Mechanistically, using microarray, gene set enrichment analyses and Chromatin ImmunoPrecipitation DNA-Sequencing, we identified a link between H19 and pro-hypertrophic nuclear factor of activated T cells (NFAT) signalling. H19 physically interacts with the polycomb repressive complex 2 to suppress H3K27 tri-methylation of the anti-hypertrophic Tescalcin locus which in turn leads to reduced NFAT expression and activity., Conclusion: H19 is highly conserved and down-regulated in failing hearts from mice, pigs and humans. H19 gene therapy prevents and reverses experimental pressure-overload-induced heart failure. H19 acts as an anti-hypertrophic lncRNA and represents a promising therapeutic target to combat pathological cardiac remodelling., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Myocardial ischemia induces a multifaceted remodeling process in the heart. Novel therapeutic entry points to counteract maladaptive signalling include the modulation of non-coding RNA molecules such as long non-coding RNA (lncRNA). We here questioned if the lncRNA candidate H19 exhibits regulatory potential in the setting of myocardial infarction. Initial profiling of H19 expression revealed a dynamic expression profile of H19 with upregulation in the acute phase after murine cardiac ischemia. In vitro, we found that oxygen deficiency leads to H19 upregulation in several cardiac cell types. Repression of endogenous H19 caused multiple phenotypes in cultivated murine cardiomyocytes including enhanced cardiomyocyte apoptosis, at least partly through attenuated vitamin D signalling. Unbiased proteome analysis revealed further involvement of H19 in mRNA splicing and translation as well as inflammatory signalling pathways. To study H19 function more precisely, we investigated the phenotype of systemic H19 loss in a genetic mouse model of H19 deletion (H19 KO). Infarcted heart tissue of H19 KO mice showed a massive increase of pro-inflammatory cytokines after ischemia-reperfusion injury (I/R) without significant effects on scar formation or cardiac function but exaggerated cardiac hypertrophy indicating pathological cardiac remodeling. H19-dependent changes in cardiomyocyte-derived extracellular vesicle release and alterations in NF-κB signalling were evident. Cardiac cell fractionation experiments revealed that enhanced H19 expression in the proliferative phase after MI derived mainly from cardiac fibroblasts. Here further research is needed to elucidate its role in fibroblast activation and function. In conclusion, the lncRNA H19 is dynamically regulated after MI and involved in multiple pathways of different cardiac cell types including cardiomyocyte apoptosis and cardiac inflammation., Competing Interests: Declaration of Competing Interest TT and JF have filed patents in the field of cardiovascular non-coding RNA diagnostics and therapeutics. TT is founder and shareholder of Cardior Pharmaceuticals GmbH., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rationale: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing., Objective: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction., Methods and Results: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 ( Meg3 ) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp -2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-β I. Chromatin immunoprecipitation showed that further induction of Mmp -2 expression by transforming growth factor-β I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance., Conclusions: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling., (© 2017 American Heart Association, Inc.)

A novel long noncoding RNA Chaer acts as noncoding epigenetic regulator at the onset of cardiac hypertrophy and enables an improved understanding about the complex mechanisms in cardiovascular disease., (© 2017 American Heart Association, Inc.)

Advances in RNA-sequencing techniques have led to the discovery of thousands of non-coding transcripts with unknown function. There are several types of non-coding linear RNAs such as microRNAs (miRNA) and long non-coding RNAs (lncRNA), as well as circular RNAs (circRNA) consisting of a closed continuous loop. This review guides the reader through important aspects of non-coding RNA biology. This includes their biogenesis, mode of actions, physiological function, as well as their role in the disease context (such as in cancer or the cardiovascular system). We specifically focus on non-coding RNAs as potential therapeutic targets and diagnostic biomarkers., (Copyright © 2016 the American Physiological Society.)

Recent studies highlighted long noncoding RNAs (lncRNAs) to play an important role in cardiac development. However, understanding of lncRNAs in cardiac diseases is still limited. Global lncRNA expression profiling indicated that several lncRNA transcripts are deregulated during pressure overload-induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy-associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)-operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell-derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain-containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases., (Copyright © 2016, American Association for the Advancement of Science.)