Combinatorial Genetic Screening for New Cardiac Therapies

Abstract

As the leading global cause of morbidity and mortality, heart disease represents one of the greatest challenges in medicine. Of note, heart failure (HF) afflicts over 6 million Americans with an anticipated 46% increase in prevalence by 2030. Prognosis remains poor for many HF patients, with ≤50% surviving 5 years after an index HF hospitalization despite the best available therapies. Despite the large and growing unmet need for improved HF treatments, the development of new HF therapeutics has been slow. Moreover, no FDA-approved therapies target one of the key underlying factors in heart disease development and progression: loss of cardiomyocytes. In an effort to identify potential new therapeutics for the prevention and treatment of HF, we employed massively parallel combinatorial genetic screening to search for combinations (pairwise or higher order) of miRNAs with the ability to promote the proliferation and/or survival of cardiac myocytes (CMs). These screens and follow-up validation experiments identified multiple miRNA combinations which stimulated CM proliferation in vitro, as well as combinations which protected CMs from the chemotherapeutic drug doxorubicin (Dox), the cardiotoxicity of which is the main dose-limiting factor in its clinical use. The most effective miRNA combination at stimulating CM proliferation in vitro (rno-miR-295 + rno-miR-139) was also tested in adult mice using adeno-associated virus (AAV)-mediated overexpression and found to stimulate adult CM cell cycle activity when used in the context of cardiac injury, despite having no apparent effect in healthy hearts. The most effective miRNA combination at protecting CMs in vitro against Dox (mmu-miR-222 + mmu-miR-455) is being tested for a protective effect in vivo using a developing zebrafish Dox cardiotoxicity model, with promising initial results. Bioinformatic analysis (including RNA-seq and in silico miRNA target prediction) is being used to identify the genes and pathways responsible for these effects. The results of these experiments further our knowledge of the cardiac-relevant effects of individual miRNAs and their combinations, shed light on longstanding issues in the field such as the postnatal restriction of mammalian CM proliferation, and raise questions including whether stimulating adult CM proliferation is necessarily beneficial in the context of cardiac injury.