Identification of Early Changes in Extracellular Microrna Biomarkers of Scleroderma for Point-of-care Diagnosis

Abstract Introduction and aims Scleroderma is a rare autoimmune disease, characterized by excessive extracellular matrix deposition leading to chronic inflammation and fibrosis. As a progressive disease initially affecting the skin, it presents similarly to Raynaud phenomenon, which delays diagnosis and treatment. Clinical diagnostic tests lack sensitivity, therefore novel Methods of detection and sensitive biomarkers are required to improve diagnosis rates. MicroRNAs are short, stable, noncoding RNAs, often present in the extracellular space, and can be sampled from interstitial skin fluid (ISF) through the employment of hydrogel microneedles. We aim to use this technique to improve upon current clinical gold standards of diagnosing scleroderma, at earlier stages of the disease. Methods In this study, we performed next-generation small-RNA sequencing on ISF from healthy individuals, patients prior to a clinical diagnosis of scleroderma [termed very early detection of systemic sclerosis (VEDOSS)], and patients with scleroderma classified as low and high skin score, identifying a panel of microRNA biomarkers for scleroderma diagnosis. Swellable hydrogel microneedles were manufactured in house, applied to excised human tissue and preclinical mouse models to demonstrate the ability to access and quantify extracellular microRNAs in ISF. Results Patients who received a VEDOSS diagnosis have increased ISF abundance of miR-379-5p and miR-125a-5p and decreased miR-30e-3p compared with healthy patients. miR-379-5p, miR-363-3p and miR-221-3p are sensitive to the progression of scleroderma and could be used to track the disease with improved sensitivity to autoantibodies in blood or where clinical practices usually require a skin biopsy. Using preclinical models of skin disease, we could differentiate between conditions, with higher sensitivity than blood sampling using our microneedle technology. Conclusions By identifying extracellular mircoRNAs, we have shown the potential use of these sensitive biomarkers in a liquid biopsy that can be applied at point-of-care. Peptide nucleic acids complementary to our microRNA biomarkers, will be used to provide real-time on-the-chip readouts of these ­biomarkers.