Chronic inflammatory diseases are debilitating lifelong illnesses, whose development and emergence mechanisms are strongly correlated with dysregulation of cytokine signaling network. It has been shown that IL-6 trans-signaling is a major inflammation mediator involved in multiple pathogeneses, including development, progression and maintenance of many chronic inflammatory diseases and various types of cancer. Therefore, IL-6 trans-signaling represents an ideal target for new anti-inflammatory and anti-cancer remedies. The scope of this study was to induce exon skipping in exons 9 and 15 of Gp130 pre-mRNA by a splice-switching gene therapy, thus generating soluble Gp130 protein isoforms sGp130-RAPS and sGp130-Sharkey, respectively. Soluble Gp130 variants, which lack transmembrane and cytoplasmic domains, are natural antagonists of the IL-6 trans-signaling, and therefore constitute a promising therapeutic modality for the treatment of IL-6 induced ailments. By altering alternative splicing using specially designed 2′O-Me-PS SSOs, targeted against 5′ss, 3′ss, and ESE of Gp130 exon 9 and exon 15, we were able to obtain sGp130-RAPS and sGp130-Sharkey mRNA in different human and mouse cells. At the protein level, soluble isoforms could not be detected, since mRNA sequences with altered alternative splicing are frequently a substrate of NMD and do not translate into proteins. Furthermore, a double downregulating effect was seen upon SSO treatment, indicating that SSOs impact both soluble and full-length membrane bound Gp130 receptor productions. From a therapeutic point of view, double downregulation of Gp130 is a positive effect, since the disease pathogenesis can be suppressed while vital IL-6 functions remain intact. Alongside classical exon skipping, alternative intronic polyadenylation represents additional mechanism for obtaining protein isoforms, moreover mRNAs arose from usage of the intronic PAS are naturally immune to NMD. To avoid the mRNAs undergoing NMD, Gp130 gene was screened for intronic PASs. Presumably, intronic PASs were found in human Gp130 introns 9, 10 and 11. Based on the results, SSO treatment holds a great promise as a novel therapy against chronic inflammatory diseases. The potential also lies in the usage of naturally present intronic PAS in Gp130 gene. In the future, both approaches for production of soluble protein isoforms could be used as an anti-inflammation remedy.
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