The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.

MicroRNA 122 (miR-122) is liver specific, fine-tunes lipid metabolism, and is required for hepatitis C virus (HCV) abundance. Miravirsen, an oligonucleotide with locked nucleic acid, binds to miR-122, potently inhibiting its activity. We aimed at determining the safety of the miR-122 antagonism in vivo in 6 to 10 cynomolgus monkeys/group intravenously treated with a range of dose levels twice weekly for 4 weeks. Survival, body weights, clinical signs, and cardiovascular and ophthalmologic parameters were unaffected. Anticipated hypolipidemia due to the inhibition of miR-122 was observed in all treated animals. Only the highest dose level produced distinct transient prolongations of clotting times, slight alternative complement pathway activation, and a reversible increase of hepatic transaminases. Distribution half-life was 10-20 minutes, and accumulation was mainly in the kidney and liver with slow elimination. Microscopic examinations revealed granulated Kupffer cells and lymph node macrophages, cytoplasmic vacuolation in proximal renal tubules, and hepatocytes. The granules were most likely phagolysosomes containing miravirsen. A slightly increased incidence of hepatocyte apoptosis was observed in some monkeys given the highest dose; otherwise, there was no evidence of treatment-related degenerative changes in any organ. In conclusion, the maximal inhibition of miR-122 was associated with limited phenotypic changes, indicating that the clinical assessment of miravirsen as host factor antagonist for treatment of HCV infections is warranted.

Background High‐intensity focused ultrasound (HIFU) operating at 20 MHz is new and applicable to skin. Details of use and instrumentation are not documented. Materials and Methods A GLP compliant 12‐week study of Göttingen minipigs (n = 3) was undertaken. Effects of HIFU treatment at different focal depths, energy levels and field size (single shot vs 5 × 5 multiple shots) were studied. Clinical scoring and histology of treated sites were made. Results High‐intensity focused ultrasound showed instant and initial effects with wheal and flare responses followed by delayed inflammatory reactions associated with outer skin necrosis, depending on energy dose. HIFU treatment was tunable in the range 0.3‐1.5 J, ablative at higher energy level. Transducers with deeper focal points gave more profound effects, while epidermal effects were comparable. Multiple doses of 5 × 5 shots produced stronger reactions than single dose indicating that nearby applied shots were synergistic. Recovery from single doses was faster than in multidose areas. Clinical scarring at the end point was not seen despite occasional fibrous change of dermis. Findings illustrated intended therapeutic use; no special safety issues of concern were raised. Conclusion The new 20 MHz HIFU was reproducible, tunable and produced targeted effects in the outer skin, for example instant wheal and flare followed by inflammation and possibly necrosis depending on energy setting. Reactions recovered during the study with only minor findings at study end. No special safety concerns were raised. The method can be controlled and modulated, and it is ready for clinical testing of dermatological disease indications including conditions presently treated with lasers.