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

Lookup NU author(s): Professor Yen Nee Tan

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

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.

Publication metadata

Author(s): Pendergraff HM, Krishnamurthy PM, Debacker AJ, Moazami MP, Sharma VK, Niitsoo L, Yu Y, Tan YN, Haitchi HM, Watts JK

Publication type: Article

Publication status: Published

Journal: Molecular Therapy Nucleic Acids

Year: 2017

Volume: 8

Pages: 158-168

Print publication date: 15/09/2017

Online publication date: 21/06/2017

Acceptance date: 16/06/2017

Date deposited: 21/06/2019

ISSN (electronic): 2162-2531

Publisher: Cell Press

URL: https://doi.org/10.1016/j.omtn.2017.06.012

DOI: 10.1016/j.omtn.2017.06.012

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