Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.

Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides.

The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.

In Vitro Assessment of the Hepatotoxicity Potential of Therapeutic Oligonucleotides.

The mechanisms of antisense oligonucleotide-induced liver toxicity are still poorly understood. Assessment of the hepatic safety profile is currently mostly investigated directly in rodent studies. A predictive preclinical in vitro model that is capturing liver liabilities of antisense oligonucleotides can be of great help to be used as a first filter in the screening process of therapeutic oligonucleotides. We describe here an in vitro cytotoxicity assay using freshly isolated mouse hepatocytes or cryopreserved human hepatocytes that recapitulates the hepatotoxic profile of antisense oligonucleotides previously observed in rodents and can be used for the prioritization of molecules prior to in vivo testing.

Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.

Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli.

Multi-drug-resistant bacteria can cause serious infections that are extremely difficult to treat. Bacterial efflux pumps are known to contribute to multi-drug resistance and, thus, constitute a promising target for novel antibacterial agents. Resazurin is widely used to monitor bacterial growth because resazurin is reduced to the fluorescent resorufin by live cells. We have shown by flow cytometric analysis and by accumulation studies with wild type and efflux deficient strains that resazurin is a substrate of efflux pumps in Escherichia coli and Pseudomonas aeruginosa. Our investigations showed that the conversion rate of resazurin to resorufin is affected by efflux pumps. This finding was used to design an assay useful to detect efflux pump activity and to find potential efflux-pump inhibitors in a microtiter plate format. Mefloquine was detected as efflux-pump inhibitor when a panel of selected chemical compounds was tested for assay validation purposes.