Deep sequencing insights in therapeutic shRNA processing and siRNA target cleavage precision.

Denise, Hubert, Moschos, Sterghios, Sidders, Benjamin, Burden, Frances, Perkins, Hannah, Carter, Nikki, Stroud, Tim, Kennedy, Michael, Fancy, Sally-Ann, Lapthorn, Cris, Lavender, Helen, Kinloch, Ross, Suhy, David and Corbau, Romu (2014) Deep sequencing insights in therapeutic shRNA processing and siRNA target cleavage precision. Molecular Therapy - Nucleic Acids, 3. e145. ISSN 2162-2531

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Official URL: http://dx.doi.org/10.1038/mtna.2013.73

Abstract

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).

Item Type: Article
Subjects: C700 Molecular Biology, Biophysics and Biochemistry
Department: Faculties > Health and Life Sciences > Applied Sciences
Depositing User: Sterghios Moschos
Date Deposited: 24 Oct 2016 13:05
Last Modified: 24 Oct 2017 11:24
URI: http://nrl.northumbria.ac.uk/id/eprint/28132

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