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.