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Locked Nucleic Acid gapmers and conjugates induce potent silencing of ADAM33, an asthma-associated metalloprotease with nuclear-localized mRNA

Locked Nucleic Acid gapmers and conjugates induce potent silencing of ADAM33, an asthma-associated metalloprotease with nuclear-localized mRNA
Locked Nucleic Acid gapmers and conjugates induce potent silencing of ADAM33, an asthma-associated metalloprotease with nuclear-localized mRNA
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 RNA interference (RNAi) approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We now compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four different classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded siRNAs (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 antisense oligonucleotide-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.
2162-2531
Pendergraff, Hannah M
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Krishnamurthy, Pranathi Meda
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Debacker, Alexandre J.
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Moazami, Michael
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Sharma, Vivek K.
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Niitsoo, Liisa
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Yu, Yong
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Tan, Yen Nee
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Haitchi, Hans Michael
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Watts, Jonathan K.
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Pendergraff, Hannah M
a7eb25e2-f68b-4f4f-b30b-235bedfb1710
Krishnamurthy, Pranathi Meda
e1e25ed6-2e87-4e1a-8ba9-52ec73541156
Debacker, Alexandre J.
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Moazami, Michael
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Sharma, Vivek K.
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Niitsoo, Liisa
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Yu, Yong
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Tan, Yen Nee
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Haitchi, Hans Michael
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Watts, Jonathan K.
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Pendergraff, Hannah M, Krishnamurthy, Pranathi Meda, Debacker, Alexandre J., Moazami, Michael, Sharma, Vivek K., Niitsoo, Liisa, Yu, Yong, Tan, Yen Nee, Haitchi, Hans Michael and Watts, Jonathan K. (2017) Locked Nucleic Acid gapmers and conjugates induce potent silencing of ADAM33, an asthma-associated metalloprotease with nuclear-localized mRNA. Molecular Therapy: Nucleic Acid. (doi:10.1016/j.omtn.2017.06.012). (In Press)

Record type: Article

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 RNA interference (RNAi) approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We now compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four different classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded siRNAs (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 antisense oligonucleotide-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.

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1-s2.0-S2162253117301981-main - Accepted Manuscript
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Accepted/In Press date: 16 June 2017
Organisations: Chemistry, NIHR Southampton Respiratory Biomedical Research Unit, Institute for Life Sciences, Chemical Biology Group, Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 411771
URI: https://eprints.soton.ac.uk/id/eprint/411771
ISSN: 2162-2531
PURE UUID: 7da70ee8-a14a-4f03-bcf8-5e0cfae909bb
ORCID for Hans Michael Haitchi: ORCID iD orcid.org/0000-0001-8603-302X

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Date deposited: 26 Jun 2017 16:30
Last modified: 03 Dec 2019 01:51

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Contributors

Author: Hannah M Pendergraff
Author: Pranathi Meda Krishnamurthy
Author: Alexandre J. Debacker
Author: Michael Moazami
Author: Vivek K. Sharma
Author: Liisa Niitsoo
Author: Yong Yu
Author: Yen Nee Tan
Author: Jonathan K. Watts

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