Modulation of Pro-Inflammatory IL-6 Trans-Signaling Axis by Splice Switching Oligonucleotides as a Therapeutic Modality in Inflammation

Abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in maintaining normal homeostatic processes under the pathogenesis of various inflammatory and autoimmune diseases. This context-dependent effect from a cytokine is due to two distinctive forms of signaling: cis-signaling and trans-signaling. IL-6 cis-signaling involves binding IL-6 to the membrane-bound IL-6 receptor and Glycoprotein 130 (GP130) signal-transducing subunit. By contrast, in IL-6 trans-signaling, complexes of IL-6 and the soluble form of the IL-6 receptor (sIL-6R) signal via membrane-bound GP130. Various strategies have been employed in the past decade to target the pro-inflammatory effect of IL-6 in numerous inflammatory disorders. However, their development has been hindered since these approaches generally target global IL-6 signaling, also affecting the anti-inflammatory effects of IL-6 signaling too. Therefore, novel strategies explicitly targeting the pro-inflammatory IL-6 trans-signaling without affecting the IL-6 cis-signaling are required and carry immense therapeutic potential. Here, we have developed a novel approach to specifically decoy IL-6-mediated trans-signaling by modulating alternative splicing in GP130, an IL-6 signal transducer, by employing splice switching oligonucleotides (SSO), to induce the expression of truncated soluble isoforms of the protein GP130. This isoform is devoid of signaling domains but allows for specifically sequestering the IL-6/sIL-6R receptor complex with high affinity in serum and thereby suppressing inflammation. Using the state-of-the-art Pip6a cell-penetrating peptide conjugated to PMO-based SSO targeting GP130 for efficient in vivo delivery, reduced disease phenotypes in two different inflammatory mouse models of systemic and intestinal inflammation were observed. Overall, this novel gene therapy platform holds great potential as a refined therapeutic intervention for chronic inflammatory diseases.

Keywords: IL-6 trans-signaling; inflammation; nucleic acid therapies; oligonucleotides.

Figure 1

(A) Brief overview of the Gp130 alternative splicing and known protein isoforms. (B) SSO mediated sGp130 production to inhibit IL-6 trans-signaling specifically.

Figure 2

(A) Design and activity of different SSOs targeting Gp130 exon 9 splicing regulatory elements. End-point RT-PCR of Gp130 exon 9 skipping in HEK293T cells 24 h post-transfection of 100 nM Gp130 exon 9 SSOs by lipofectamine 2000. Splice switching efficiency of different Gp130 SSOs in (B) Huh7 cells, (C) mouse N2a cells, and (D) mouse C2C12 cells 24 h after transfection. The SSOs for mouse Gp130 were designed to bind at the positions corresponding to the those of the human Gp130 SSOs.

Figure 3

(A) Design and activity of different SSOs targeting Gp130 exon 15 splicing regulatory elements. End-point RT-PCR of Gp130 exon 15 skipping in HEK293T cells 24 h post-transfection of 100 nM Gp130 exon 15 SSOs by lipofectamine 2000. Splice switching efficiency of different Gp130 SSOs in (B) Huh7 cells, (C) mouse N2a cells, and (D) mouse C2C12 cells 24 h after transfection. The SSOs for mouse Gp130 were designed to bind at the positions corresponding to the those of the human Gp130 SSOs.

Figure 4

SSO-induced soluble Gp130 downregulates IL-6 trans-signaling in HeLa cells. (A) RLU/mg in HeLa STAT3 Luc cell lysate 24 h after the addition of 10ng/mL hyper IL-6 and conditioned medium from N2A cells transfected with Gp130 SSOs. Statistical analysis was done with two-way ANOVA and Fischer’s LSD test. * = p < 0.05. Data represent mean + SD of three replicates. Only 100 nM 9-3 SSO induced a significant effect compared with untreated. (B) STAT3 phosphorylation as determined by Western blot in HeLa cells 6 h post-treatment with IL-6 + sIL-6R cocktail and conditioned medium from HEK293T cells transfected with Gp130 SSOs. (C) Gp130 exon skipping downregulates full-length Gp130 isoform in mouse N2A cells. Flow cytometry analysis of the mGp130 expression after the treatment with 9-1, 9-3, 15-1, and 15-3 SSOs, and 705 SSO as a negative control.

Figure 5

(A) Covalent conjugation of PMO with Pip6a CPP enhances splice switching in vitro and in vivo. End-point RT-PCR of Gp130 exon 9 skipping in mouse N2A cells treated with varying amounts of Pip6a-Exon 9 PMO SSO. (B) Pip6a-PMO conjugates show efficient in vivo exon skipping upon systemic delivery in wild-type mice. End-point RT-PCR of Gp130 exon 9 skipping in different tissues 72 h post intravenous administration of Pip6a PMO at 5 mg/kg or 15 mg/kg.

Figure 6

(A) Gp130 exon 9 SSOs improve survival and disease phenotype in LPS-induced systemic inflammation. C57Bl6J were injected intravenously either with 15 mg/kg Pip6a-Gp130 PMO or saline, and, 24 h later, systemic inflammation was induced by intraperitoneal administration of 15 mg/kg LPS (n = 5). (A) Kaplan–Meier survival curve of animals with and without SSO treatment post LPS induction. Plasma levels of (B) IL-6 and (C) TNF-alpha as determined by ELISA 24 h post disease induction. Significance calculated by one-way ANOVA. (D) The percentage change in body weight relative to the initial weight over the disease course of mice induced with colitis by intrarectal injection of TNBS and treated intravenously 24 prior with either 15 mg/kg Pip6a-PMO or saline. Significance calculated by two-way ANOVA, data non-significant.