Mincle-binding DNA aptamer demonstrates therapeutic potential in a model of inflammatory bowel disease

Abstract

Pattern recognition receptors such as Mincle (Clec4e) play a significant role in the regulation of inflammation. Enhanced signaling of Mincle through the release of damage-associated molecular patterns during sterile inflammation has been shown to be important in the progression and manifestation of several diseases. A limitation to Mincle-targeted therapeutics is the feasibility of human-scale antibody therapy and the lack of alternative small-molecule inhibitors. Herein, we describe a highly specific neutralizing DNA aptamer targeting Mincle and demonstrate its therapeutic potential. Our data demonstrate that AptMincle selectively binds to both human and mouse Mincle with high affinity and is able to directly target and reduce Mincle activation. AptMincle can specifically reduce trehalose-6,6-dibehenate (TDB)-induced Syk and P65 phosphorylation in vitro in a manner comparable to that of the commercially available neutralizing antibody in vitro. Moreover, a bio-stable modified aptamer, AptMincle^DRBL, was successful in reducing disease activity in a dextran sodium sulfate (DSS)-induced model of ulcerative colitis in a dose- and sequence-dependent manner. The results present an alternative, highly specific DNA aptamer with antagonistic function for use in the investigation of Mincle-associated diseases. The data also show the translational potential of Mincle-targeting aptamers as a new category of biologic therapy in the treatment of inflammatory bowel disease (IBD).

Keywords: MT: Oligonucleotides: Therapies and Applications; Mincle; aptamer; inflammation; inflammatory bowel disease; ulcerative colitis.

Graphical abstract

Figure 1

Characterization of the in vitro function of aptamers with Mincle affinity (A) Binding characteristics of AptMincle, and its modified counterparts, with rhMincle as determined by ELONA. (B) Graphical depiction of the modifications made to the aptamer sequences (black, original; white, modified). (C) The predicted secondary and tertiary structures and docking simulation of AptMincle^CORE (purple) with Mincle (white). Box: highlighted predicted region of interaction surrounding a calcium molecule. (D) The predicted secondary and tertiary structures and docking simulation of AptMincle^RND (purple) with Mincle (white). Box: highlighted predicted region of interaction surrounding a calcium molecule. Dissociation constants were calculated on GraphPad Prism 8 using a non-linear regression binding analysis with assumed one-site target parameters.

Figure 2

In vitro binding characteristics of aptamer AptMincle in comparison with antibody (A) Immunofluorescence imaging of the relative expression of pSyk^Y525 and Mincle in unstimulated, TDB-stimulated (50 μM), LPS-primed (10 ng/mL, 24 h), or LPS → TDB (10 ng/mL LPS + 50 μM)-stimulated J774.1 macrophages (left to right). (B) Staining of a heterogeneous population of control (Mincle^low) and LPS-primed (Mincle^high) J774.1 macrophages co-stained with anti-CLEC4E antibody (InvivoGen, USA) and 5′-Cy3-conjugated AptMincle. (C) Dose-dependent inhibition of Syk^Y525 phosphorylation in J774.1 macrophages by anti-Mincle antibody (InvivoGen) compared with AptMincle. (D) Whole-cell ELISA of (i) pSyk^Y525/Syk and (ii) pP65/P65 relative expression in LPS-primed, TDB-treated J774.1 macrophages when challenged with AptMincle (1 μM). (E) Whole-blood assessment of AptMincle^DRBL presence in mice via fully quantitative RT-PCR. IC₅₀ was calculated on GraphPad Prism 8 using a non-linear fit: y = 100/(1 + x/IC₅₀). One-way ANOVA with Tukey’s post hoc test was performed for multiple non-parametric data comparisons. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bar = 10μm.

Figure 3

AptMincle^DRBL suppresses DSS-induced colitis through a dose-dependent inhibition of colonic inflammation (A) Daily changes in disease activity score (DAI) over the onset of DSS-induced colitis. (B) MPO activity within colon. (C) Body weight percentage change at the end of the experiment. (D) Colon length and (E) terminal DAI measured at day 7 with relation to treatment groups with AptMincle^DRBL (0.35, 1, or 3.5 mg/kg) or AptMincle^RND (3.5 mg/kg). Immunoblot analysis of (F) Syk or (G) P65 phosphorylation of total colonic lysates. (H) Representative photographs of colons from mice (DSS + AptMincle^DRBL is 3.5 mg/kg group only). These data are presented as the mean ± SEM and were analyzed by a repeated measures ANOVA with Tukey’s post hoc test. Black asterisks indicate significance against sham group, while red asterisks indicate significance compared with DSS group: ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Mice are a composite of 10-week-old mice, n = 4–12, with experiments performed on separate days.

Figure 4

The protective effect of AptMincle^DRBL in DSS-induced colitis is sequence specific (A) Disease activity score (DAI) during the onset of DSS-induced colitis compared with treatment groups AptMincle^{DRBL/RND/CORE/PORT} (3.5 mg/kg) given at day 3. (B) Presence of detectable AptMincle sequences within the serum of treated mice. (C) Overall body weight percentage change during the experiments. (D) Colon length at termination. (E) DAI measured at termination at day 7. (F) MPO activity in the colon of mice at day 7. (G) The 14-point histological assessment of terminal colon of the groups and (H) the measured villus length of colon samples. These data are presented as the mean ± SEM and were analyzed by a repeated measures ANOVA with Tukey’s post hoc test. Black asterisks indicate significance compared with sham control, while red asterisks are significance against the DSS group: ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Mice are a composite of 10-week-old mice, n = 3, with experiments performed on separate days.