Drugs (2015) 75:723–730 DOI 10.1007/s40265-015-0391-0


Antisense Approach to Inflammatory Bowel Disease: Prospects and Challenges Irene Marafini1 • Davide Di Fusco1 • Emma Calabrese1 • Silvia Sedda1 Francesco Pallone1 • Giovanni Monteleone1

Published online: 25 April 2015 Ó Springer International Publishing Switzerland 2015

Abstract Despite the great success of anti-tumour necrosis factor-based therapies, the treatment of Crohn’s disease (CD) and ulcerative colitis (UC) still remains a challenge for clinicians, as these drugs are not effective in all patients, their efficacy may wane with time, and their use can increase the risk of adverse events and be associated with the development of new immune-mediated diseases. Therefore, new therapeutic targets are currently being investigated both in pre-clinical studies and in clinical trials. Among the technologies used to build new therapeutic compounds, the antisense oligonucleotide (ASO) approach is slowly gaining space in the field of inflammatory bowel diseases (IBDs), and three ASOs have been investigated in clinical trials. Systemic administration of alicaforsen targeting intercellular adhesion molecule-1, a protein involved in the recruitment of leukocytes to inflamed intestine, was not effective in CD, even though the same compound was of benefit when given as an enema to UC patients. DIMS0150, targeting nuclear factor (NF) jBp65, a transcription factor that promotes pro-inflammatory responses, was very promising in pre-clinical studies and is currently being tested in clinical trials. Oral mongersen, targeting Smad7, an intracellular protein that inhibits transforming growth factor (TGF)-b1 activity, was safe and well tolerated by CD patients, and the results of a phase II clinical trial showed the efficacy of the drug in inducing clinical remission in patients with active disease. In this leading article, we review the rationale and the clinical data

& Giovanni Monteleone [email protected] 1

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy

available regarding these three agents, and we discuss the challenge of using ASOs in IBD.

Key Points Three antisense oligonucleotides are currently being tested for the treatment of human inflammatory bowel diseases. Alicaforsen, targeting intercellular adhesion molecule-1, is effective in ulcerative colitis patients but not in Crohn’s disease patients. Oral mongersen, targeting Smad7, is effective in inducing clinical remission and clinical response in patients with active Crohn’s disease.

1 Introduction Crohn’s disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD) in human beings, are chronic, relapsing, inflammatory disorders of the gastrointestinal tract, the aetiology of which is far from being completely understood [1, 2]. IBDs arise from an interaction between genetic and environmental factors, which ultimately promotes an excessive immune response against components of the normal microflora [3–7]. In CD, the inflammation is transmural and segmental, and it may involve any part of the alimentary tract, from the mouth to the anus, whereas in UC, the inflammatory process is confined to the mucosal layer of the rectum and may extend


proximally to the whole colon. Although the exact pathogenesis of IBDs remains unknown, over the last two decades the advent of molecular and cellular biology techniques has largely contributed to advancing our understanding of the inflammatory pathways that drive tissue damage in these disorders [8]. This progress has paved the way for the development of compounds that have already been tested in IBD patients or are now ready to move into clinical practice. Blockade of cytokine activity and recruitment of inflammatory cells from the blood to the gut is at the forefront of this new era, with the success of monoclonal antibodies against tumour necrosis factor (TNF)-a, the p40 subunit shared by interleukin (IL)-12, IL23 and a4b7 integrin. These drugs are, however, not effective in all patients, their efficacy may wane with time, and their use can enhance the risk of adverse events and can be associated with development of new immune-mediated diseases [8–10]. Therefore, a new challenge is to identify new targets for therapeutic purposes. In this context, it has been demonstrated that integrin-targeting therapies and adhesion molecule-targeting therapies are useful to limit recruitment of effector leukocytes in inflamed tissue, thereby attenuating the detrimental inflammatory response in some subsets of IBD patients [11]. Amplification and perpetuation of the pathogenic response in IBD rely also on the activity of many transcription factors [e.g. nuclear factor (NF) jB]—which control both activation of many immune and non-immune cell types and production/activity of effector cytokines—as well as on the expression of intracellular proteins that restrain immunesuppressive mechanisms (e.g. Smad7). Inhibition of such molecules can be accomplished with the use of antisense oligonucleotides (ASOs), and such an approach has already been adopted to dampen mucosal inflammation in murine models of colitis and in IBD patients. In this leading article, we review the currently available data on the use of ASOs in the treatment of IBD and discuss the prospects connected to this approach.

2 Antisense Oligonucleotide Strategy ASOs are unmodified or chemically modified, relatively short (13–25 nucleotides), single-stranded nucleotides, which target single messenger RNA (mRNA) or DNA sequences, with limited off-target effects [12]. It has been proposed that at relatively low concentrations, ASO internalization occurs via interaction with a membrane-bound receptor, while at relatively high concentrations, these receptors are saturated and the pinocytotic process assumes greater importance [13, 14]. Cellular uptake of ASOs can be increased by using various transporters and vectors, such as liposomes, poly-L-lysine and polyalkylcyanoacrylate

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nanoparticles [15]. On the basis of the mechanism of action, two classes of ASOs can be considered: RNase H-dependent ASOs, which induce degradation of mRNA, and stericblocker oligonucleotides, which physically prevent or inhibit progression of splicing or the translational machinery. RNase H is a ubiquitous enzyme that hydrolyses the RNA strand of an RNA/DNA duplex, thereby blocking key steps in transcription and translation processes. The majority of the antisense drugs investigated in clinical practice act via an RNase H-dependent mechanism [12]. ASOs have a short in vivo half-life and poor biological stability, as they are rapidly degraded by intracellular endonucleases and exonucleases. The degradation products of ASOs [i.e. deoxynucleoside monophosphate (dNMP) mononucleotides] may be cytotoxic and also exert antiproliferative effects. To overcome these problems, many chemical modifications have been developed. For example, replacement of the native phosphates in the backbone with phosphorothioates diminishes ASO degradation by plasma and tissue nucleases, thus increasing their stability [16]. Moreover, phosphorothioate oligonucleotides are highly soluble, can be easily administered via different routes (e.g. subcutaneous, intravenous, intravitreal, topical, aerosol, oral or enema) and are also capable of activating RNase H activity [13]. These properties of phosphorothioate ASOs have boosted their use in the clinical trial setting, and promising results have been documented in patients with various neoplasias (i.e. melanoma, chronic lymphocytic leukaemia) following treatment with G3139 [17–19], which targets the initiation codons of bcl-2 mRNA, or Isis 3521 (i.e. non-small cell lung cancer), which targets the 30 untranslated region of the protein kinase C-a isoform [20]. It is, however, worth noting that the phosphorothioate backbone can induce sequence-independent biological effects, which are mostly dependent on its high affinity for various cellular proteins, especially heparin-binding growth factors (e.g. acidic fibroblast growth factor, basic fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor) and many heparin-binding molecules (e.g. laminin, fibronectin and Mac-1) [21]. Paradoxically, however, such a property of phosphorothioate ASOs could actually contribute to observed clinical responses by synergizing with the downregulation of the specific target rather than limiting their efficacy. Additional chemical modification of ASOs involves replacement of the hydrogen at the 20 -position of ribose by an O-alkyl group, most frequently methyl. These ‘second-generation’ ASOs are resistant to degradation by cellular nucleases, hybridize specifically to their target RNA with higher affinity than phosphorothioate ASOs and induce an antisense effect via a non-RNase H-dependent mechanism. For a better description of additional second-generation ASOs, the reader is directed towards previous excellent reviews [22, 23].

Antisense Oligonucleotide-Based Therapy in Inflammatory Bowel Diseases

2.1 Alicaforsen In IBD, tissue damage occurs in areas massively infiltrated with activated leukocytes, which produce a vast array of inflammatory cytokines. Such a phenomenon is in part due to the continuous recruitment of cells from the bloodstream to the gut, a carefully orchestrated process involving both soluble molecules (i.e. chemokines) and cell-associated molecules (i.e. adhesion molecules and integrins) [24]. Adhesion molecules include members of the selectin family (i.e., E-, L- and P-selectin), which mediate the initial rolling of leukocytes on vascular endothelium, and members of the immunoglobulin family [i.e. intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule (VCAM)-1] interacting with integrins expressed on leukocytes, thus resulting in firm adhesion and transmigration [25]. ICAM-1 is constitutively expressed at low levels on leukocytes and vascular endothelial cells and is upregulated in inflamed tissue in IBD patients [26]. Both ICAM-1 blockade with neutralizing antibodies and knockdown with ASOs are associated with reduced leukocyte trafficking to the gut and attenuation of experimental colitis in mice, thus suggesting that inhibitors of endothelial-leukocyte interactions could have a place in IBD therapy. To this end, ISIS 2302 (alicaforsen), an RNase H-dependent, 20-baselong phosphorothioate ASO, designed to inhibit human ICAM-1, was the first ASO used in IBD. After a phase I clinical trial in which alicaforsen was administered intravenously to healthy volunteers [27] and proved to be well tolerated, a pilot study was carried out in 20 active CD patients, who received 13 infusions of the active drug at different doses, or placebo, over 26 days. Alicaforsen was superior to placebo in inducing clinical remission [28]. Unfortunately, however, these promising results were not confirmed by two double-blind, placebo-controlled, multicentre clinical trials, in which alicaforsen, given either intravenously or subcutaneously to patients with active steroid-dependent or resistant CD, failed to meet the primary end point, defined as steroid-free remission at week 14 [29, 30]. The reason why these studies failed in CD remains unclear. One possibility is that the amount of the drug reaching the gut following systemic administration was not sufficient to suppress ICAM-1 expression. Appropriate pharmacodynamic studies could help address this issue. It is also worth noting that recruitment of leukocytes to inflamed tissue in CD patients is mediated by additional molecules other than ICAM-1. Later on, the ISIS pharmaceutical company announced that in two phase III clinical trials, alicaforsen had not been superior to placebo in inducing remission in CD, and therefore the company had decided to discontinue the therapeutic development of this compound.


An open-label, uncontrolled study was conducted to assess the effect of rectal administration of alicaforsen in 12 patients with chronic, unremitting pouchitis. Per protocol, patients received a 240 mg alicaforsen enema nightly for 6 weeks. The drug was safe and effective in reducing clinical and endoscopic signs of the disease, and, at the end of treatment, seven patients were in clinical remission [31]. In 2006, Van Deventer et al. [32] tested the efficacy of an alicaforsen rectal enema in patients with mild to moderate left-sided UC. The study failed to reach the primary end point, which was a statistically significant percentage reduction in the Disease Activity Index (DAI) at week 6, relative to the baseline DAI, between treatment arms and placebo. However a prolonged reduction in the mean DAI relative to baseline was observed in the 240 mg alicaforsen enema arm, compared with placebo, from week 18 to week 30 [32]. A similar result was obtained in another study in which an alicaforsen enema was compared with a mesalazine enema. In this study, the two treatments had a similar rate of response in the acute phase (the primary end point), but the alicaforsen enema had a more durable effect than the mesalazine enema [33]. Pharmacokinetic studies have shown reduced systemic absorption following rectal administration of alicaforsen, raising the intriguing possibility that topical treatment with ASOs associates therapeutic benefit with low systemic exposure [34]. A subsequent post hoc meta-analysis of four phase II clinical trials confirmed the efficacy of the alicaforsen enema in patients with active UC, especially in those with distal lesions and moderate-to-severe activity [32–36]. The fact that alicaforsen was of benefit in UC but not in CD could depend on the route of administration of the drug or could reflect the different contributions of ICAM-1 in the pathogenesis of the two IBDs. 2.2 Antisense Oligonucleotide Targeting NFjB-p65 NFjB belongs to a family of transcription factors that regulate the promoters of a variety of genes whose products are involved in many biological processes [37, 38]. In trinitrobenzene sulfonic acid (TNBS)-induced colitis and in IL-10 deficient mice, two murine models of colitis showing immunological similarities with CD, the p65 subunit of NFjB was strongly activated and involved in the upregulation of pro-inflammatory cytokines [39]. A specific ASO targeting p65, which was administered to mice either intravenously or intra-rectally, abrogated experimental colitis without inducing hepatic or renal toxicity. NFjB-p65 is also upregulated in intestinal macrophages and endothelial cells in CD patients, and treatment of CD mucosal cells with p65 ASO has been shown to significantly reduce the production of pro-


inflammatory cytokines. Targeting NFjB-p65 was also effective in dextran sulfate sodium (DSS)-induced colitis [40]. In this study, a confocal laser microscopy analysis showed that the ASO, administered as an enema formulation, was taken up predominantly by lamina propria mononuclear cells (LPMCs). In addition, in a mouse model of colitis-driven intestinal fibrosis induced by weekly intrarectal administration of TNBS, an NFjB-p65 ASO enema, given either prophylactically or therapeutically, counteracted the development of inflammation and fibrosis [41]. In a pilot study, 11 steroid-resistant IBD patients were given a single dose of rectal antisense NFjB-p65 oligonucleotide. Clinical, endoscopic and histologic improvements were seen at day 7 in 71 % of the treatment group compared with 25 % of the placebo group [42]. Moreover, in 2011, Tahara and co-workers [43] tested chitosan-modified poly(D,L-lactide-co-glycolide) nanospheres for delivery of an orally NFjB oligonucleotide in experimental models of colitis. The nanospheres were able to improve the stability of the antisense in an acid medium, and oral administration determined a significant amelioration of DSS colitis in rats. ASOs targeting NFjB-p65 are now being tested in clinical trials, but the results are not yet publicly available. 2.3 Smad7 Antisense Oligonucleotide: From Bench to Bedside In recent years, a large body of evidence has been accumulated to support the hypothesis that, in IBDs, maintenance and amplification of the pathogenic immune response are sustained by defects of counterregulatory mechanisms [7]. One such mechanism involves the immunosuppressive cytokine transforming growth factor (TGF)-b1, a cytokine that is produced by many mucosal cell types and is able to negatively regulate the activation and function of multiple immune cells [44]. In line with this is the demonstration that lack of TGF-b1 expression and/or activity drives or exacerbates experimental colitis and boosting TGF-b1 activity promotes resolution of colitis in mice [45–47]. The immunoregulatory properties of TGF-b1 are mainly mediated by the canonical Smad pathway, which is triggered by binding of the cytokine to its heterodimeric receptor [48, 49]. In inflamed tissue in both CD and UC patients, activity of TGF-b1 is markedly diminished as indicated by reduced phosphorylation of TGF-b receptor-driven Smad2/3 (Fig. 1a). This is due to high Smad7, an inhibitory Smad that binds to the TGF-b receptor and blocks TGF-b1-induced Smad2/3 phosphorylation [50]. In IBD mucosal cells, knockdown of Smad7 with a specific ASO restores Smad2/3 phosphorylation, thus resulting in decreased production of inflammatory cytokines [50] (Fig. 1b). Smad7 is upregulated in the colons of mice with TNBS- and oxazolone-induced colitis,

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the latter resembling UC, and treatment of mice with Smad7 ASO inhibits production of inflammatory cytokines and ameliorates experimental colitis [51]. Since the murine and human Smad7 genes are homologous, knockdown of Smad7 in both models of IBD was obtained using the same ASO that was effective in in vitro studies with human IBD mucosal cells. For these studies, Smad7 ASO was administered orally to mice in order to deliver the active compound in the gut, limit systemic distribution of the ASO and therefore prevent adverse events. Following oral administration, Smad7 ASO was taken up by both epithelial cells and LPMCs in the small intestine and colon, while no uptake was seen in the stomach, liver, kidney or spleen. These results led to development of a pharmaceutical compound containing the specific Smad7 ASO, termed GED0301 and, more recently, mongersen. Mongersen is formulated as a solid oral dosage form, protected by an external coating made of methacrylic polymers at pH 6.6–7.2, which allows the compound to transit through the upper part of the gastrointestinal tract and to be released only in the lumen of the terminal ileum and right colon—the intestinal sites that are primarily affected by CD. Mongersen is a synthetic single-stranded DNA oligonucleotide in which the internucleotide linkages are modified to O,O-linked phosphorothioates to increase stability. The ASO contains two cytosine–phosphate–guanine (CpG) motifs, which are chemically modified to avoid immune-stimulatory effects. To assess the safety of the compound, a phase I, open label, dose-escalating study was conducted in active, steroid-dependent/resistant CD patients [52]. Fifteen patients were divided into three cohorts and received mongersen 40, 80 or 160 mg/day for 7 days. The treatment was well tolerated, and no serious adverse events were documented. Twenty-five adverse events occurred in 11 patients and were classified as mild and unrelated to treatment. Mongersen was barely detectable in the plasma of one sample from a single patient, suggesting that systemic absorption is limited. Mongersen treatment was effective in reducing clinical scores of disease activity in all of the patients and was associated with a significant reduction in the fraction of circulating cytokine-producing, C-C chemokine receptor type 9 (CCR9)—expressing T cells—a subset of CD4? T cells with gut properties. Since TGF-b1 stimulates fibroblasts to produce collagen and TGF-b1 activity is strictly linked to development of fibrosis in many organs, mongersen-treated patients were followed up for 6 months and monitored for the development of intestinal strictures by ultrasonography [53]. At the end of follow-up, no patient had developed strictures and no change in the blood concentrations of fibrosis-related proteins had been documented. Interestingly, more than 50 % of the patients maintained clinical remission at month 6. These results encouraged us to carry out a

Antisense Oligonucleotide-Based Therapy in Inflammatory Bowel Diseases


Fig. 1 a Inflamed intestine in a patient with inflammatory bowel disease: high Smad7 binds to transforming growth factor (TGF)-b1 receptor type I (RI), prevents TGF-b1-driven Smad2/3 phosphorylation (p) and abrogates TGF-b1-mediated inhibition of inflammatory genes. This results in uncontrolled production of pro-inflammatory mediators and activation and recruitment of effector cells to the inflamed gut and epithelial damage. b Administration of Smad7 antisense oligonucleotide (ASO) reduces Smad7, thereby allowing

endogenous TGF-b1 to phosphorylate Smad2/3. Phosphorylated Smad2/3 interacts with Smad4, and the complex Smad2/3/4 translocates to the nucleus and binds to DNA, with the downstream effect of controlling expression of target genes and promoting resolution of mucosal inflammation and epithelial barrier repair. CCL chemokine C-C motif ligand, IFN interferon, IL interleukin, RII receptor type II, TNF tumour necrosis factor

phase II, multicentre, double-blind, placebo-controlled study, in which steroid-dependent and/or resistant CD patients were allocated to receive placebo or mongersen 10, 40 or 160 mg/day for 2 weeks. The main goal of this study was to determine the clinical efficacy of mongersen [54]. The rates of clinical remission were greater in the groups of patients treated with mongersen 40 or 160 mg/day (55 and 65 %, respectively) than in those receiving mongersen 10 mg/day (12 %) or placebo (9.5 %). Moreover, all three mongersen groups had significantly greater rates of clinical response than the placebo group, thus suggesting that even the 10 mg/day dose may be therapeutically useful in a subset of patients. Oral administration of mongersen was safe, and no drug-related side effect was documented during the trial. The clinical response and remission rates obtained with mongersen are remarkable, considering other

induction studies in patients with active CD. In the SONIC trial, treatment with infliximab was associated with a 32.5 % rate of glucocorticoid-free remission at week 6, while in the CLASSIC-1 trial, adalimumab was associated with a 36 % clinical remission rate at week 4; in the GEMINI II trial, vedolizumab induced remission in 14.5 % of patients at week 6 and in 39 % of patients at week 54. 2.4 Use of Other ASOs in Experimental Models of IBD There are other ASOs that are currently being investigated in vitro and in vivo in experimental models of IBD. An anti-CD40 ASO, which blocks the interaction between CD40 and CD154, attenuates TNBS-induced colitis in rats [55]. Improvements in the delivery of the anti-CD40 ASO


were made using amphoteric liposomes administered systemically to mice [56]. ASO targeting either mucosal addressin cellular adhesion molecule (MAdCAM)-1, an adhesion protein expressed on endothelial cells that mediate leukocyte trafficking to the gut [57], or macrophage migration inhibitory factor (MIF), a protein with pro-inflammatory properties, ameliorated clinical signs of colitis in mice [58–60]. The ASO targeting MIF was administered together with schizophyllan, a polysaccharide of the glucan family, which enhances the stability of the ASO and facilitates phagocyte uptake by dectin-1 [61]. Given the fact that development of antibodies against TNF-a blockers is associated with loss of response to these drugs [62], ASOs inhibiting TNF-a, which are now in development, could represent an intriguing alternative to anti-TNF-a-antibody-based therapy in IBD [63]. The possibility of delivering an antisense to specific cell types has been explored using two possible vehicles, either a galactosylated low molecular weight chitosan or a cationic glucomannan phytagel, both combined with an ASO against TNF-a. These complexes specifically deliver the ASO into activated macrophages and are effective in various experimental models of colitis [64, 65]. Intra-colonic administration of the complexes, using both vehicles, was associated with successful accumulation of the ASO in colonic activated macrophages and a consequent reduction in TNF-a levels. Despite the interesting possibility of specifically delivering an ASO to a particular cell subtype, macrophages do not represent the only cell source of TNFa in IBD; thus these compounds are unlikely to have a better clinical outcome than monoclonal anti-TNF-a antibodies currently used in clinical practice.

3 Conclusions In recent years, data obtained from high-throughput technologies have deepened our understanding of the molecular and gene networks in immune and non-immune cells in IBD patients. It is also becoming evident that antisense agents could be used as inhibitors of the expression of specific targets, with the downstream effect of modulating IBD-associated mucosal inflammation. However, clinical trials have provided conflicting results in IBD patients. For example, knockdown of ICAM-1 was effective in UC but not in CD. Although these results could depend on the different contribution of ICAM-1 in the pathogenesis of UC and CD, it is worth noting that ICAM-1 ASO was administered rectally in UC and intravenously in CD. This raises the possibility that the success of ASO-based therapy in IBD requires delivery of the compound directly to the inflamed tissue. Indeed, it is known that compared with systemic delivery, localized delivery of ASOs into target tissues and

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cells offers high bioavailability and efficiency and reduced side effects. Consistent with this is the demonstration that oral mongersen, an ASO targeting Smad7, has been shown to be safe and well tolerated and has provided clinical benefit in CD patients [52]. Future studies are needed to confirm these results and to determine the real effect of such therapies on the natural history of both CD and UC, and to understand if there are specific subsets of patients who could greatly benefit from such treatment. Declaration of personal interests GM has filed a patent related to the treatment of IBD with Smad7 ASOs. The remaining authors have no conflicts of interest. No funding was used to support the writing of this manuscript.

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Antisense approach to inflammatory bowel disease: prospects and challenges.

Despite the great success of anti-tumour necrosis factor-based therapies, the treatment of Crohn's disease (CD) and ulcerative colitis (UC) still rema...
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