Targeting myocardial fibrosis with a novel noncoding RNA in Duchenne muscular dystrophy

PROJECT SUMMARY This proposal seeks to understand the anti-fibrotic mechanisms of a new noncoding RNA (ncRNA) entity, TY4, that is bioinspired by the cargo constituents of extracellular vesicles (EVs). Our focus is on one class of small ncRNA, not previously recognized as particularly relevant to muscular dystrophy. Y-RNAs, which were known to influence DNA replication (but not gene expression), captured our attention because molecules encoded by the YRNA4 gene are exceptionally plentiful in EVs secreted by cardiosphere-derived cells (CDCs). The predominant such ncRNA, EV-YF1, has salutary properties when delivered on its own. Here, we present preliminary data on TY4, a new chemical entity (NCE) bioinspired by EV-YF1. Importantly, TY4 exerts potent disease-modifying bioactivity in mdx mice, a mouse correlate of Duchenne muscular dystrophy (DMD). Remarkably, TY4, when formulated with the breast milk protein, casein, works when administered orally to mdx mice. In terms of mechanism, preliminary data show that TY4 targets macrophages, inducing robust transcriptomic changes that underlie its bioactivity. As a small chemically-modified mutant NCE, TY4 has key features desirable of a synthetic RNA drug. Other RNA drugs, whether FDA-approved or in development, all have known mechanisms of action: they are antisense oligos, small interfering RNAs, aptamers, or miR-modifiers. TY4 does not fit into any of those classes and, thus is the prototype for a new class of RNA drugs. The focus here is on understanding the mechanisms whereby TY4 targets fibrosis in the hearts of mdx mice. We will test the following mechanistic hypothesis: TY4 is anti-fibrotic in DMD hearts by reversing pathological chromatin remodeling (i.e., hyper- inflammatory and pro-fibrotic gene expression) in macrophage precursor cells of the bone marrow. We will pursue the following Aims: 1) Investigate the mechanism(s) whereby TY4 alters gene expression. Sub-aims seek to: a) characterize the effects of TY4 on chromatin remodeling in the bone marrow niche, b) evaluate the contribution of Prdm2 and Smyd4 to pathological chromatin remodeling, c) characterize the transcriptomic changes induced by TY4, and d) prioritize biological targets. 2) Examine the anti-fibrotic basis of TY4. Sub-aims seek to: a) compare fibrogenic outcomes with or without Arg-1, b) characterize proteomic changes in cardiac fibroblasts induced by macrophages with or without TY4, and c) investigate the mechanism of TY4-mediated suppression of Arg-1. 3) Test whether manipulation of mdx macrophages confers anti-fibrotic actions in vivo. Sub-aims seek to: a) determine if adoptive transfer of TY4-conditioned mdx macrophages attenuates myocardial fibrosis using a novel transgenic macrophage-depletable mdx mouse model, b) characterize the effects of adoptive transfer of arginase-1 silenced mdx macrophages in macrophage-depleted mdx mice, and c) investigate whether long-term oral administration of TY4 prevents myocardial fibrosis. Discoveries made here will also provide valuable insights to facilitate future development of anti-fibrotic agents for a variety of heart diseases.