Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA

Abstract

Two mechanisms dominate the clinical pipeline for oligonucleotide-based gene silencing, namely, the antisense approach that recruits RNase H to cleave target RNA and the RNAi approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded small interfering RNAs (ss-siRNAs), locked nucleic acid (LNA) gapmer antisense oligonucleotides (ASOs), and novel hexadecyloxypropyl conjugates of the ASOs. Of these designs, the gapmer ASOs emerged as lead compounds for silencing ADAM33 expression: several gapmer ASOs showed subnanomolar potency when transfected with cationic lipid and low micromolar potency with no toxicity when delivered gymnotically. The preferential susceptibility of ADAM33 mRNA to silencing by RNase H may be related to the high degree of nuclear retention observed for this mRNA. Dynamic light scattering data showed that the hexadecyloxypropyl ASO conjugates self-assemble into clusters. These conjugates showed reduced potency relative to unconjugated ASOs unless the lipophilic tail was conjugated to the ASO using a biocleavable linkage. Finally, based on the lead ASOs from (human) MRC-5 cells, we developed a series of homologous ASOs targeting mouse Adam33 with excellent activity. Our work confirms that ASO-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.

Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity

“…No significant silencing effect was observed at low concentration (1 μM) without transfection agent. This could be a result of the reported slow and less efficient unassisted gymnotic intracellular delivery of gapmers that requires a relatively high concentration . Accordingly, at a relatively high concentration (10 μM), high levels of HER3 gene silencing effects were observed in both PC‐3 and Caco‐2 cell lines without transfection agent.…”

Oligonucleotides targeting TCF4 triplet repeat expansion inhibit RNA foci and mis-splicing in Fuchs’ dystrophy

“…LNA phosphoramidites were synthesized from the 3′-hydroxyl precursors (Rasayan) and assembled into oligonucleotides as described previously ( 49 ). ASOs were deprotected using concentrated aqueous ammonia for 16 h at 55°C and were characterized by LCMS ( Table 2 ).…”

Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides

“…Single-stranded AONs can bind to and interact with many different target classes. AONs can inhibit miRNA expression [23][24][25], induce alternative splicing [26][27][28][29][30][31], and inhibit mRNA expression by both a steric block [32,33], and an RNase recruiting mechanism [34,35]. Except for miRNA inhibition, it has been shown that AON activity is most effective in the nucleus [36], and in applications involving splice modulation, nuclear entry of AONs is crucial.…”