AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes.

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.

Development and characterization of anti-fibrotic natural compound similars with improved effectivity.

Cardiac fibroblasts constitute the major cell type of the murine and human heart. Once activated, they contribute to an excessive deposition of extracellular matrix (ECM) leading to cardiac fibrosis and subsequently organ dysfunction. With the exception of the pulmonary drugs, nintedanib and pirfenidone, drugs specifically targeting anti-fibrotic pathways are scarce. We recently performed large library screenings of natural occurring compounds and identified first lead structures with anti-fibrotic properties in vitro and in vivo. In line, we now aimed to improve efficacy of these anti-fibrotic lead structures by combining in vitro validation studies and in silico prediction. Next to this combined approach, we performed large OMICs-multi-panel-based mechanistic studies. Applying human cardiac fibroblasts (HCF), we analysed 26 similars of the initially identified anti-fibrotic lead molecules bufalin and lycorine and determined anti-proliferative activity and potential toxicity in an array of in vitro and ex vivo studies. Of note, even at lower concentrations, certain similars were more effective at inhibiting HCF proliferation than nintedanib and pirfenidone. Additionally, selected similars showed low cytotoxicity on human iPS-derived cardiomyocytes and anti-fibrotic gene regulation in human ex vivo living myocardial slices. Further, array and RNA sequencing studies of coding and non-coding RNAs in treated HCFs revealed strong anti-fibrotic properties, especially with the lycorine similar lyco-s (also known as homoharringtonine), that led to a nearly complete shutdown of ECM production at concentrations 100-fold lower than the previously identified anti-fibrotic compound lycorine without inducing cellular toxicity. We thus identified a new natural compound similar with strong anti-fibrotic properties in human cardiac fibroblasts and human living heart tissue potentially opening new anti-fibrotic treatment strategies.

Non-coding RNAs: emerging players in cardiomyocyte proliferation and cardiac regeneration.

Soon after birth, the regenerative capacity of the mammalian heart is lost, cardiomyocytes withdraw from the cell cycle and demonstrate a minimal proliferation rate. Despite improved treatment and reperfusion strategies, the uncompensated cardiomyocyte loss during injury and disease results in cardiac remodeling and subsequent heart failure. The promising field of regenerative medicine aims to restore both the structure and function of damaged tissue through modulation of cellular processes and regulatory mechanisms involved in cardiac cell cycle arrest to boost cardiomyocyte proliferation. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are functional RNA molecules with no protein-coding function that have been reported to engage in cardiac regeneration and repair. In this review, we summarize the current understanding of both the biological functions and molecular mechanisms of ncRNAs involved in cardiomyocyte proliferation. Furthermore, we discuss their impact on the structure and contractile function of the heart in health and disease and their application for therapeutic interventions.