Strategies for In Vivo Screening and Mitigation of Hepatotoxicity Associated with Antisense Drugs.

Antisense oligonucleotide (ASO) gapmers downregulate gene expression by inducing enzyme-dependent degradation of targeted RNA and represent a promising therapeutic platform for addressing previously undruggable genes. Unfortunately, their therapeutic application, particularly that of the more potent chemistries (e.g., locked-nucleic-acid-containing gapmers), has been hampered by their frequent hepatoxicity, which could be driven by hybridization-mediated interactions. An early de-risking of this liability is a crucial component of developing safe, ASO-based drugs. To rank ASOs based on their effect on the liver, we have developed an acute screen in the mouse that can be applied early in the drug development cycle. A single-dose (3-day) screen with streamlined endpoints (i.e., plasma transaminase levels and liver weights) was observed to be predictive of ASO hepatotoxicity ranking established based on a repeat-dose (15 day) study. Furthermore, to study the underlying mechanisms of liver toxicity, we applied transcriptome profiling and pathway analyses and show that adverse in vivo liver phenotypes correlate with the number of potent, hybridization-mediated off-target effects (OTEs). We propose that a combination of in silico OTE predictions, streamlined in vivo hepatotoxicity screening, and a transcriptome-wide selectivity screen is a valid approach to identifying and progressing safer compounds.

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.

Pharmacological Characterization of a Novel ENaCα siRNA (GSK2225745) With Potential for the Treatment of Cystic Fibrosis.

Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC(50) (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5' RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC(50) = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.Molecular Therapy - Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013.

PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms.

Asthma and chronic obstructive pulmonary disease are highly prevalent and economically important inflammatory airway diseases associated with mucus hypersecretion. Considerable additional morbidity and mortality are related to acute exacerbations, which are associated with further mucus hypersecretion. MUC5AC is a prominent airway mucin; however, the signalling pathways regulating MUC5AC hypersecretion are not fully characterised. We investigated the signalling pathway regulating phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC gene and protein expression in human respiratory epithelial cells. Using NCI-H292 cells, we demonstrated that treatment with PMA increased production of total and MUC5AC-specific mucin proteins. This increase was dependent on de novo MUC5AC gene transcription. We identified a short, proximal region of the MUC5AC promoter essential for this activity containing three specificity protein (Sp) 1 transcription factor-binding sites and a single CACCC site. By chemical inhibition, site-directed promoter mutagenesis and electrophoretic mobility-shift assay (EMSA), we demonstrated that PMA induced proteins binding to all three Sp1 sites and that they were all required for full induction of MUC5AC promoter activity. We then demonstrated a Ras-Raf-MEK/ERK signalling pathway was exclusively activated upstream of Sp1 activating the promoter and confirmed the requirement for matrix metalloproteinase activation leading to a ligand-dependent activation of the epidermal growth factor receptor. Finally, we demonstrated that activation of the novel protein kinase C isoforms delta and theta; was required upstream of the metalloproteinase activation. We have characterised a signalling pathway regulating PMA induction of MUC5AC. Studies such as this identify key signalling intermediates as targets for pharmacological intervention to treat mucus hypersecretion.