In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries' may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

Use of gene expression profiling to identify a novel glucocorticoid sensitivity determining gene, BMPRII.

Wide variation in glucocorticoid (Gc) sensitivity exists between individuals which may influence susceptibility to, and treatment response of, inflammatory diseases. To determine a genetic fingerprint of Gc sensitivity 100 healthy human volunteers were polarized into the 10% most Gc-sensitive and 10% most Gc-resistant following a low dose dexamethasone (0.25 mg) suppression test. Gene expression profiling of primary lymphocytes identified the 98 most significantly Gc regulated genes. These genes were used to build a subnetwork of Gc signaling, with 54 genes mapping as nodes, and 6 non-Gc regulated genes inferred as signaling nodes. Twenty four of the 98 genes showed a difference in Gc response in vitro dependent on the Gc sensitivity of their donor individuals in vivo. A predictive model was built using both partial least squares discriminate analysis and support vector machines that predicted donor glucocorticoid sensitivity with 87% accuracy. Discriminating genes included bone morphogenetic protein receptor, type II (BMPRII). Transfection studies showed that BMPRII modulated Gc action. These studies reveal a broad base of gene expression that predicts Gc sensitivity and determine a Gc signaling network in human primary T lymphocytes. Furthermore, this combined gene profiling, and functional analysis approach has identified BMPRII as a modulator of Gc signaling.

Gene expression changes caused by the p38 MAPK inhibitor dilmapimod in COPD patients: analysis of blood and sputum samples from a randomized, placebo-controlled clinical trial.

The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway responds to a variety of extracellular stimuli, including cytokines, Toll-like receptor agonists, and components of cigarette smoke to influence the expression of proinflammatory mediators. Activation of p38 MAPK is increased within the lungs of chronic obstructive pulmonary disease (COPD) patients. In clinical trials, treatment of COPD patients with p38 MAPK inhibitors has been shown to reduce systemic inflammation plasma biomarkers C-reactive protein (CRP) and fibrinogen. As CRP and fibrinogen have been associated with poor clinical outcomes in COPD patients, such as mortality, exacerbation, and hospitalization, we analyzed gene expression data from COPD subjects treated with dilmapimod with the aim of understanding the effects of p38 MAPK inhibition on the inflammatory genome of immune cells within the systemic circulation. Whole blood and induced sputum samples were used to measure mRNA levels by gene array and PCR. Pathway and network analysis showed STAT1, MMP-9, CAV1, and IL-1ß as genes regulated by dilmapimod that could also influence fibrinogen levels, while only IL-1ß was identified as a gene regulated by dilmapimod that could influence CRP levels. This suggests that p38 MAPK inhibits specific inflammatory pathways, leading to to differential effects on CRP and fibrinogen levels in COPD patients.

Progression and variability of TNBS colitis-associated inflammation in rats assessed by contrast-enhanced and T2-weighted MRI.

BACKGROUND: A common feature of preclinical models of colitis is that the time-course, magnitude, and persistence of inflammation vary considerably within the experimental animal group. Accordingly, noninvasive, serial quantification of colonic inflammation could advantageously guide dosing regimens and assess drug efficacy, thus enhancing the value of colitis models in research. This investigation using magnetic resonance imaging (MRI) was therefore undertaken to objectively determine inflammatory progression, variability, and response to therapy associated with trinitrobenzene sulfonic acid (TNBS)-induced colitis in Wistar rats. METHODS: Rats underwent TNBS treatment on Day 0 and received sulfasalazine or vehicle (methylcellulose) orally, daily, from Day -1 (prophylactically) or Day 2 (therapeutically). T2-weighted and semidynamic T1-weighted contrast-enhanced MRI (CE-MRI) was repeated over 7-10 days to measure colon wall thickness and perfusion-related aspects of inflammation. Rectal bleeding, stool consistency, and disease activity were scored throughout and colon pathology determined terminally. RESULTS: Principal component analysis of the CE-MRI time-series highlighted colon wall and mesenteric inflammation, which increased by 6-8x naïve values. Peristaltic artifacts were distinguished from perfusion changes using the normalized temporal standard deviation. MRI correlated strongly with terminal colon weight (mean correlation r = 0.8), well with body weight change (r = -0.7), but little with conventional clinical scores. Sulfasalazine reduced inflammation administered prophylactically and therapeutically. CONCLUSIONS: Inflammation and therapeutic efficacy can be sensitively quantified noninvasively using MRI in TNBS-treated rats. This methodology provides unique and objective in vivo measures of inflammation that can guide dosing strategies, enhancing colitis research effectiveness and the assessment of potential IBD therapeutics.

Potential novel biomarkers of disease activity in rheumatoid arthritis patients: CXCL13, CCL23, transforming growth factor alpha, tumor necrosis factor receptor superfamily member 9, and macrophage colony-stimulating factor.

OBJECTIVE: To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients. METHODS: Plasma proteins (n = 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 age- and sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial least-squares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor alpha (anti-TNFalpha). RESULTS: The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < or = 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colony-stimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor alpha, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity (kappa = 0.64). In anti-TNFalpha-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score. CONCLUSION: This exploratory study for biomarker discovery led to the identification of several proteins predictive of RA disease activity that may be useful in the definition of disease subphenotypes and in the measurement of response to therapy in clinical studies.