Nucleic acid-based therapies using living myocardial slices

Abstract

Background Gene therapy based on viral vectors is a promising new approach and offers great potential for the study and the treatment of cardiac diseases. Nucleic acid-based transfection of human heart tissue could be considered a valid and translational alternative to the use of transgenic animal models in cardiovascular research. Purpose Here we explore the use of Living Myocardial Slices (LMS) as a platform for nucleic acid-based therapies. Rat LMS and Adeno-Associated viruses (AAV) were used to optimise and analyse gene transfer efficiency, viability, tissue functionality, and cell tropism in cardiac tissue. Human cardiac samples from failing (dilated cardiomyopathy) hearts were also used to validate the human model. In addition, we transfected and tested for the first time the regenerative effect of AAV-miR-199a in both healthy and pathologically overloaded LMS. Methods LMS (300 μm thick) were prepared and glued to PTFE-coated holders, mounted on custom stretchers, and cultured at physiological sarcomere length for 72-hours under electrical stimulation. Two recombinant AAV serotypes (AAV-6 and AAV-9) at different multiplicity of infection (MOI) expressing green fluorescent protein (GFP) or the miR-199 transgene were added to the surface of the LMS. Results AAV6 proved to be the most suitable serotype and 20,000 MOI the most efficient dose, as these conditions were proven not to affect LMS contractility and to have the highest transduction and penetrability efficiency. This serotype exhibited preferential cell tropism to cardiomyocytes and stromal cells (40% of transduction in both) in rat LMS, with lower efficiency in endothelial cells, (20% of transduction). In contrast, AAV6 in human failing LMS showed higher transduction efficiency in endothelial cells and myofibroblasts. Treatment with miR-199a significantly increased the active force of pathologically overstretched LMS, downregulated the expression of its target genes, and affected the proliferative capacity of LMS. Conclusion LMS can be used as a model for nucleic acid-based therapies and their transfection protocol can be adapted to samples obtained from failing human hearts. With great translational relevance, this model would accelerate preclinical studies of novel nucleic acid-based therapies for heart failure. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation