UMMS Affiliation
RNA Therapeutics Institute; Department of Biochemistry and Molecular Pharmacology
Publication Date
2017-09-15
Document Type
Article
Disciplines
Biochemistry, Biophysics, and Structural Biology | Respiratory Tract Diseases | Therapeutics
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.
Keywords
ADAM33, antisense, gene silencing, nuclear RNA, siRNAs
Rights and Permissions
Copyright © 2017 The Author(s). Open Access funded by Engineering and Physical Sciences Research Council
DOI of Published Version
10.1016/j.omtn.2017.06.012
Source
Mol Ther Nucleic Acids. 2017 Sep 15;8:158-168. doi: 10.1016/j.omtn.2017.06.012. Epub 2017 Jun 21. Link to article on publisher's site
Journal/Book/Conference Title
Molecular therapy. Nucleic acids
Related Resources
PubMed ID
28918018
Repository Citation
Pendergraff HM, Krishnamurthy PM, Debacker AJ, Moazami MP, Sharma VK, Niitsoo L, Yu Y, Tan YN, Haitchi HM, Watts JK. (2017). Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA. Open Access Articles. https://doi.org/10.1016/j.omtn.2017.06.012. Retrieved from https://escholarship.umassmed.edu/oapubs/3163
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Included in
Biochemistry, Biophysics, and Structural Biology Commons, Respiratory Tract Diseases Commons, Therapeutics Commons