Improved Small Interfering RNA Therapy Targeting the Long, Non-Coding RNA SMILR For Therapeutic Intervention in Coronary Artery Bypass Graft Failure

Coronary artery bypass graft (CABG) surgery remains the gold standard of care to prevent myocardial ischaemia in patients with advanced atherosclerosis, however long-term graft patency remains a considerable and long-standing problem. Excessive vascular smooth muscle cell (vSMC) proliferation in the grafted tissue is recognised as central to late CABG failure. We previously showed that siRNA-mediated silencing of SMILR, a human-specific SMC-enriched lncRNA, blocks vSMC proliferation, suggesting that targeting SMILR expression could be a novel way to prevent neointima formation, and thus CABG failure. Here, we describe the design and synthesis of a library of 76 chemically-enhanced SMILR-targeting siRNA. From this library, we identify a lead siRNA, BHF7, which demonstrates potent and reproducible silencing of SMILR expression and which robustly blocks vascular smooth muscle cell proliferation, both in vitro and up to 14 days in the ex vivo human saphenous vein model. We further demonstrate using RNA-sequencing that BHF7 down-regulates the expression of genes associated with proliferation and does not induce the expression of interferon or apoptosis genes, suggesting it has an acceptable safety profile both on and off-target. Finally, we performed TUNEL staining and measured the levels of cleaved caspase-3 after BHF7 treatment which demonstrated that BHF7 does not induce a cytotoxic response either in vitro or ex vivo. Collectively, this data represents a pre-clinical package into the function and specificity of BHF7 which warrants further investigation into the possibility of utilising BHF7 as a novel, ex vivo RNA therapeutic for the prevention of CABG failure.