Mongersen,an oral Smad7 antisense oligonucleotide,in patients with active Crohn’s disease

Introduction

Crohn’s disease (CD) is a chronic relapsing inflammatory disorder of the alimentary tract characterized by segmental and transmural inflammation, which can lead to the development of local complications (i.e. strictures, abscesses and fistulae) and extraintestinal manifestations [Xavier and Podolsky, 2007]. The cause of CD remains unknown, even though there is evidence that the pathologic process arises from the interaction between genes and environmental factors, which ultimately triggers an immunological response against components of the luminal flora [Xavier and Podolsky, 2007; MacDonald et al. 2012; Maloy and Powrie, 2011; Bouma and Strober, 2003; Strober and Fuss, 2011; MacDonald et al. 2011]. Perpetuation and amplification of the tissue-damaging immune-inflammatory response are also sustained by defects in counter-regulatory mechanisms. One of such mechanism involves transforming growth factor (TGF)-β1, a pleiotropic cytokine with potent immunoregulatory properties. Early studies in mice showed that deficient TGF-β1 production/activity led to the development of pathology in the colon [Kulkarni and Karlsson, 1993; Gorelik and Flavell, 2000] while protection or amelioration of experimental colitis was seen following induction of TGF-β1 expression/activity [Neurath et al. 1996; Kitani et al. 2000]. Taken together these findings indicate that TGF-β1 triggers anti-inflammatory signals in the gut and plays a major role in the control of mucosal homeostasis.

In this article, we review the available data showing that defective TGF-β1 activity marks the pathological process in CD and discuss the involvement of Smad7 in the negative control of TGF-β1-mediated immunosuppression and the recent data on the clinical benefit seen in CD patients treated with mongersen, an oral Smad7 antisense oligonucleotide.

Smad7-dependent block of TGF-β1 signalling occurs in CD

TGF-β1 is produced by many immune and nonimmune cells in the gut of mice and humans [Letterio and Roberts, 1998] and the two TGF-β1 receptors (type I and type II) are expressed on virtually all intestinal cells [Gorelik and Flavell, 2012]. TGF-β1 suppresses the activation and function of effector T cells and macrophages, and contributes to the peripheral differentiation of both regulatory Foxp3-expressing T cells [Fantini et al. 2004] and T helper (Th)-type 17 cells [Mangan et al. 2006]. It also provides a chemotactic gradient for leukocytes and other cells participating in inflammatory responses and inhibits these cells once they have become activated [Letterio and Roberts, 1998]. TGF-β1 inhibits the production of extracellular matrix-degrading proteases by stromal cells and at the same time stimulates these cells to make collagen and promotes margination of epithelial cells [Ciacci et al. 1993]. Moreover, TGF-β1 is the major cytokine involved in the production of mucosal immunoglobulin A (IgA) [Cerutti and Rescigno, 2008; Mestecky et al. 1999; Fagarasan et al. 2002; Peterson et al. 2007; Phalipon et al. 2002; Kadaoui and Corthesy, 2007].

Consistent with data generated in mouse models of inflammation, blockade of endogenous TGF-β1 activity in cultures of normal, intestinal mucosal cells or explants with a neutralizing antibody enhances induction of inflammatory molecules [Di Sabatino et al. 2008] while stimulation of normal intestinal immune cells with recombinant TGF-β1 abrogates inflammatory signals [Monteleone et al. 2004]. The anti-inflammatory function of TGF-β1 relies mostly on the phosphorylation and activation of two intracellular proteins, termed Smad2 and Smad3 [Derynck et al. 1998]. Following the phosphorylation, these two proteins interact with a common partner, termed Smad4, and the complex translocates to the nucleus where it regulates transcription of a wide spectrum of target genes (Figure 1a) [Abdollah et al. 1997; Dennler et al. 1998]. In inflamed CD tissue, TGF-β1 RNA transcripts are highly expressed and both immune and nonimmune cells express high levels of TGF-β1 receptor type I and type II as compared with uninflamed intestinal mucosa [Babyatsky et al. 1996]. However, TGF-β1 is unable to activate the TGF-β1 receptor-associated Smad pathway and inhibit inflammatory signals [Heldin et al. 1997]. Such a defective TGF-β1 activity is due to high levels of Smad7, a protein that binds to TGF-β1 receptor type I and suppresses TGF-β1/Smad-associated signalling (Figure 1b) [Monteleone et al. 2001]. In IBD, overexpression of Smad7 associates with diminished phosphorylation of Smad3 and occurs in both immune and nonimmune cells as a result of post-transcriptional mechanisms that enhance protein stability [Monteleone et al. 2005]. Indeed there is evidence that p300-dependent Smad7 acetylation on lysine residues prevents ubiquitination-driven proteosomal degradation of Smad7 [Monteleone et al. 2005]. In CD, increased acetylation status of Smad7 could also depend on defective expression or activity of SIRT1, a component of the mammalian sirtuin family of proteins that deacetylates the lysine residues of Smad7 [Kume et al. 2007; Zhang et al. 2009; Caruso et al. 2014].

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Figure 1.

Schematic illustration showing the TGF-β1-associated Smad pathway in normal (a) and inflamed (b) intestine. Physiologically active TGF-β1 binds to the TGF-β receptor subunit II (RII) and promotes phosphorylation (p) and activation of TGF-β receptor subunit I (RI), thus leading to phosphorylation of Smad2 and Smad3. Once phosphorylated, Smad2 and Smad3 interact with Smad4 and the complex translocates to the nucleus, where it controls the transcriptional activity of multiple genes, including those encoding for inflammatory molecules (a). In the gut of patients with inflammatory bowel diseases (b) there is a defective TGF-β1-associated Smad pathway, because cells express elevated levels of Smad7, an intracellular protein whose expression is regulated by molecules that enhance its acetylation status (i.e. p300). Smad7 binds to TGF-βRI and prevents phosphorylation of Smad2 and Smad3, with the downstream effect of suppressing TGF-β1-driven counter-regulatory properties, thus amplifying inflammatory gene expression.

Knockdown of Smad7 with a specific antisense oligonucleotide restores responsiveness of CD mucosal cells to TGF-β1 thereby leading to a robust suppression of inflammatory genes [Boirivant et al. 2006]. Upregulation of Smad7 is also seen in inflamed colon of patients with ulcerative colitis [Heldin et al. 1997] as well as in the stomach of patients with Helicobacter pylori infection [Monteleone et al. 2004].

Smad7 controls gut inflammation in mice

There is no animal model that faithfully recapitulates the major features of CD. Therefore most studies aimed at exploring pathogenic events are performed using the trinitrobenzene sulfonic acid (TNBS)-induced colitis model, in which rectal administration of this haptenating reagent causes colitis that exhibits immunological and morphological similarities with CD [Neurath et al. 1995; Boirivant et al. 1998]. Like CD, TNBS-induced colitis is characterized by elevated production of TGF-β1, reduced Smad3 phosphorylation and high Smad7 [Boirivant et al. 2006]. Oral administration of Smad7 antisense oligonucleotide to colitic mice restores TGF-β1-associated Smad signalling thereby promoting resolution of intestinal inflammatory lesions [Boirivant et al. 2006]. Since upregulation of Smad7 during intestinal inflammation occurs in both T and non-T cells, [Boirivant et al. 2006] we generated a T cell-specific Smad7 transgenic (Tg) mouse on a C57B6 genetic background in order to evaluate the specific role of Smad7 in this cell type [Fantini et al. 2009]. Smad7 Tg mice exhibited no sign of colitis over a period of 8 weeks, but Smad7-overexpressing CD4+ T cells showed a high proliferation rate, produced a huge amount of inflammatory cytokines following activation and induced severe colitis when transferred in immunodeficient mice. Strikingly, such a colitis is resistant to regulatory T cell (Tregs)-mediated immunosuppression [Fantini et al. 2009]. The susceptibility of Smad7 Tg mice to colitis was also investigated by treating mice with three cycles of 2.5% dextran sodium sulphate to mimic human chronic-relapsing colitis. Smad7 Tg mice developed a more severe colitis compared to wild-type mice and produced high levels of interferon-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-6, and IL-17A [Rizzo et al. 2011].

These data indicate that high Smad7 expression contributes to sustain pathogenic responses in the gut.

Mongersen, a Smad7 antisense oligonucleo-tide, is effective in patients with active Crohn’s disease A pharmaceutical compound containing the specific Smad7 antisense oligonucleotide has been recently developed and termed ‘mongersen’ (previously GED0301). Mongersen is formulated as a solid oral dosage form protected by an external coating, which consists of methacrylic acid–ethyl acrylate copolymers. This formulation allows the delivery of the active compound primarily into the lumen of the terminal ileum and right colon [Monteleone et al. 2012]. To determine whether mongersen was safe, a phase I clinical, open-label, dose-escalating study was conducted in patients with active, steroid dependent or steroid resistant CD [Monteleone et al. 2012]. A total of 15 patients were enrolled, divided in three cohorts and treated with mongersen at 40, 80 or 160 mg/day respectively for seven days. No patient experienced serious adverse events (SAEs) and most of the adverse events (AEs) were mild and unrelated to treatment. The drug was detected in the plasma of one patient at a single time point, thus indicating that systemic availability of oral mongersen is very low. Treatment associated with clinical benefit in all the patients and induction of remission was documented in more than two thirds of the patients. Since TGF-β1 is known to stimulate stromal cells to produce collagen and favour fibrogenic processes [Border and Noble, 1994; Di Sabatino et al. 2009] all the patients receiving mongersen were closely monitored for the development of small bowel strictures through small bowel intestine ultrasonography over a period of six months [Zorzi et al. 2012]. No patient developed small bowel strictures or experienced obstructive symptoms during the study. Moreover, no significant change in serum markers of fibrosis (i.e. basic fibroblast growth factor and chitinase 3-like 1) was seen following treatment [Zorzi et al. 2012].

Next, to evaluate the efficacy of mongersen, a phase II, multicentre, double-blind placebo-controlled study was performed [Monteleone et al. 2015]. A total of 166 active, steroid-dependent or steroid-resistant, CD patients were assigned to receive one of three doses of mongersen (10, 40, or 160 mg per day) or placebo daily for 2 weeks. The primary endpoint of the study was the percentage of patients who reached clinical remission at day 15, which was maintained for at least two weeks. Patients receiving the highest doses of mongersen had significantly higher rates of remission (55% and 65%, respectively) than those treated with 10 mg or placebo (12% and 10%, respectively). At entry, 61% of the patients had altered C-reactive protein (CRP) levels. In the four subgroups of patients, the median CRP value was lower than 10 mg/l, perhaps due to the fact that the majority of the patients had lesions confined to the terminal ileum, a condition that is frequently associated with no significant change in the CRP value. In the subgroup of patients with a high CRP level at baseline, the rates of remission in the 160 mg and 40 mg groups were significantly greater than that in the placebo group. Moreover, at the end of follow up (i.e. day 84), the percentage of patients who had a glucocorticoid-free remission was significantly greater in the 160 mg group than in the placebo group. The effect of the treatment on endoscopic lesions was not evaluated in this study and therefore future trials will be needed to determine the impact of mongersen on mucosal healing. A total of nine SAEs were documented in six patients and most of them were related to CD symptoms and complications (e.g. abdominal pain, seton placement for perianal fistula, pyrexia, and surgery for haemorrhoid thrombosis). The rate of AEs did not differ among groups, thus confirming the safety profile of the drug [Monteleone et al. 2015].

Conclusions

The findings discussed in this article suggest that high levels of Smad7 contribute to the propagation of inflammatory signals in the gut, and knockdown of Smad7 with mongersen could help restore TGF-β1-Smad signalling with the downstream effect of dampening mucosal inflammation. This hypothesis is supported by the preliminary results emerging from the phase I and II clinical trials in which treatment of CD patients with mongersen is associated with high rates of clinical remission [Zorzi et al. 2012; Monteleone et al. 2015]. Further work is needed to confirm the efficacy and safety of the drug on a larger population of patients, to optimize the dose of mongersen and the duration of the treatment in inducing and maintaining remission, to ascertain the ideal candidates to the treatment and to determine whether and how mongersen facilitates healing of gut damage. This must however be validated by appropriate phase II and III studies.