A Novel Smad7 Genetic Variant Mapping on the Genomic Region Targeted by Mongersen Is Associated with Crohn's Disease

Abstract

Background: Down-regulation of Smad7 with a specific Smad7 antisense (AS) oligonucleotide-containing oral drug (Mongersen) was effective in pre-clinical studies and initial clinical trials in Crohn's disease (CD) patients. A recent phase 3 trial was discontinued due to an apparent inefficacy of the drug, but factors contributing to the failure of this study remain unknown. Here, we analysed the frequency in CD of rs144204026 C/T single nucleotide polymorphism (SNP), which maps on the corresponding region targeted by the Smad7 AS contained in the Mongersen formulation and examined whether such a variant allele affects the ability of Smad7 AS to knockdown Smad7.

Methods: rs144204026 SNP frequency was evaluated in two independent Italian cohorts of Crohn's disease patients and normal controls. Genotyping was performed by allelic discrimination assay. Smad7 expression was evaluated in wild-type or heterozygous PBMCs treated with Smad7 AS.

Results: No TT genotype was seen in CD patients and controls. Heterozygous genotype was more frequent in CD patients of both cohort 1 (11/235, 4.68%) and cohort 2 (8/122, 6.56%) as compared to controls (6/363, 1.65%; p = 0.029 and p = 0.01 respectively). Overall, a statistically significant association was observed between the T variant allele and CD patients' susceptibility (p = 0.008; OR = 3.28, 95%CI: 1.3-8.3). Smad7 AS down-regulated Smad7 RNA independently of the presence of the variant allele.

Conclusions: This is the first study to show an association between Smad7 rs144204026 SNP and CD patients. Data indicate that such a variant does not negatively influence the in vitro inhibitory effect of Smad7 AS on Smad7.

Keywords: Crohn’s disease; Smad7; inflammatory bowel disease; single nucleotide polymorphisms; ulcerative colitis.

Figure 1

Smad7 antisense oligonucleotide does not induce cell death and inhibits Smad7 expression independently of the presence of rs144204026 SNP. (A) PBMCs were isolated from 3 wt (CC) and 3 heterozygous (CT) healthy individuals and were either left untreated (Unst) or treated with Smad7 sense or antisense oligonucleotides by Amaxa Nucleofector Tecnology. Histograms show the percentage of viable cells considered as AnnexinV−/PI−cells assessed by flow cytometry after 18 h. Data are expressed as mean ± SD. (B) PBMCs from 3 wt (CC) and 3 heterozygous (CT) healthy individuals were transfected with Smad7 AS or sense oligonucleotide (both at final concentration 2 μg/mL) by Amaxa Nucleofector Tecnology and Smad7 transcripts were evaluated by Real-time PCR. PBMCs were cultured for 18 h and rhTGF-β1 was added to the cell cultures 30 min before the end of the treatment. Levels are normalized to β-actin. Each point indicates the value of Smad7 in a single cell sample.