Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animalsA series of antisense oligonucleotides (ASOs) containing either 2'-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights. This toxicity was evident for multiple sequences targeting three different biological targets, as well as in mismatch control sequences having no known mRNA targets. Histopathological evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was observed as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.
Short Antisense Oligonucleotides with Novel 2′−4′ Conformationaly Restricted Nucleoside Analogues Show Improved Potency without Increased Toxicity in AnimalsPunit P. Seth, Andrew Siwkowski, Charles Allerson et al.|Journal of Medicinal Chemistry|2008 The potency of second generation antisense oligonucleotides (ASOs) in animals was increased 3- to 5 -fold (ED(50) approximately 2-5 mg/kg) without producing hepatotoxicity, by reducing ASO length (20-mer to 14-mer) and by employing novel nucleoside modifications that combine structural elements of 2'-O-methoxyethyl residues and locked nucleic acid. The ability to achieve this level of potency without any formulation agents is remarkable and likely to have a significant impact on the future design of ASOs as therapeutic agents.
Inhibition of <i>HPV-16 E6/E7</i> immortalization of normal keratinocytes by hairpin ribozymesLuis Marat Álvarez-Salas, Amy Cullinan, Andrew Siwkowski et al.|Proceedings of the National Academy of Sciences|1998 HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6/E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6/E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6/E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6/E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization.
Antisense Oligonucleotide Blockade of Tumor Necrosis Factor-α in Two Murine Models of ColitisKathleen Myers, Sreekant Murthy, Anne Flanigan et al.|Journal of Pharmacology and Experimental Therapeutics|2003 Human RNase H1 Discriminates between Subtle Variations in the Structure of the Heteroduplex SubstrateWalt F. Lima, J. Bart Rose, Josh G. Nichols et al.|Molecular Pharmacology|2006