Locked Nucleic Acid gapmers and conjugates induce potent silencing of ADAM33, an asthma-associated metalloprotease with nuclear-localized mRNA

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 RNA interference (RNAi) approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We now compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four different classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded siRNAs (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 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT 2 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 antisense oligonucleotide-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.