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

Link to Article in PubMed

PubMed ID

28918018

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.