Therapeutic preclinical development of novel RNA drug for scleroderma heart disease - PROJECT SUMMARY/ABSTRACT Systemic sclerosis (SSc), a lethal complication of the autoimmune disease scleroderma, is complicated by a distinctive cardiomyopathy. Fibrosis of the heart leads to diastolic dysfunction, arrhythmias and premature death. Here we investigate the mechanisms whereby TY1, a synthetic noncoding RNA (ncRNA), reverses fibrosis in mouse models of SSc and in human SSc cells. TY1, bioinspired by the contents of extracellular vesicles, is a 24 nucleotide new chemical entity (NCE), chemically-modified for stability. Dissection of TY1’s cardioprotective benefits of TY1 in myocardial infarction revealed an unprecedented mechanism of action: TY1 upregulates the DNA exonuclease TREX1, thereby attenuating the DNA damage response (cGAS/STING) pathway of innate immunity. If so, there is reason to believe TY1 might benefit SSc, an autoimmune disorder with underlying DNA damage. Preliminary data show that TY1 inhibits pro-fibrotic pathways in macrophages, and reverses pathology in murine models of scleroderma with SSc. Remarkably, TY1, when formulated in casein/chitosan micelles, works orally. In the bleomycin mouse model of scleroderma, which we have found to manifest SSc-like heart disease, oral TY1 decreases cardiac fibrosis and improves diastolic function. Similar benefits are seen in a genetic model of SSc (tsk-1 mice). The effects are specific: they are not mimicked by ncRNA of the same nucleotide content in scrambled order. We have discovered that the antifibrotic effects of TY1 on the heart in the bleomycin model are abrogated by clodronate-induced macrophage depletion, consistent with the hypothesis (which we will test further here) that macrophages mediate the benefits of TY1 in SSc. As a small chemically- modified orally-effective NCE, TY1 has key features desirable in a synthetic RNA drug for SSc, a deadly illness with little by way of effective therapeutic options. The focus here is on better understanding how TY1 decreases cardiac fibrosis in SSc. We will investigate the following mechanistic hypothesis: By augmenting TREX1, TY1 inhibits the DNA damage response in macrophages, thereby priming the macrophages to attenuate organs fibrosis and improve cardiac function. We will address the following specific questions: 1) In the context of TY1’s unique ability to upregulate TREX-1 and attenuate the DNA damage response, what are the anti-fibrotic mechanisms of TY1 in human SSc and control macrophages? 2) When human macrophages have been primed with TY1, does their conditioned media decrease the activation of human SSc fibroblasts? 3) What are the paracrine factors that mediate macrophage-fibroblast crosstalk in SSc? 4) Are the therapeutic benefits of TY1 in tsk-1 mice mediated by macrophages, despite the inflammation-independent nature of the fibrosis in this model? 5) Are macrophages necessary and sufficient for TY1 efficacy in mice with SSc induced by bleomycin? 6) What are the relative roles of tissue-resident versus circulating monocyte-derived macrophages in the anti-fibrotic effects of TY1 in SSc? Beyond the new mechanistic insights, a novel, orally-bioavailable ncRNA drug that attenuates the DNA damage response opens up new therapeutic options to this deadly illness.
