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.
Highlights
Asthma is a chronic respiratory disease involving airway inflammation and structural changes in the form of remodeling of the conducting airways.[1,2] It causes more than 345,000 deaths annually and affects more than 340 million people worldwide.[3]
ADAM33 Silencing by small interfering RNAs (siRNAs) and ss-siRNAs We designed and synthesized a panel of 13 duplex siRNAs targeting different regions of the ADAM33 transcript (Figure 2A)
We tested the efficacy of these siRNAs in Medical Research Council (MRC)-5 human lung fibroblast cells, transfecting the siRNAs with a lipid transfection reagent and measuring silencing by qPCR
Summary
Asthma is a chronic respiratory disease involving airway inflammation and structural changes in the form of remodeling of the conducting airways.[1,2] It causes more than 345,000 deaths annually and affects more than 340 million people worldwide.[3] ADAM33 is the first asthma susceptibility gene to be identified by positional cloning.[4]. Expression of human sADAM33 in transgenic mice causes pathological airway remodeling and makes airways more susceptible to allergen-induced inflammatory responses.[7] Promisingly, when induction of human sADAM33 is arrested, this leads to reversal of the remodeling and reduced sensitivity to inflammatory responses.[7] in Adam33-null mice challenged with allergen, both airway remodeling and inflammation are suppressed. Inhibition of ADAM33 represents an attractive disease-modifying therapeutic strategy for treating the root cause of asthma in many patients
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