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

Hubert Denise, Sterghios Moschos, Benjamin Sidders, Frances Burden, Hannah Perkins, Nikki Carter, Tim Stroud, Michael Kennedy, Sally-Ann Fancy, Cris Lapthorn, Helen Lavender, Ross Kinloch, David Suhy, Romu Corbau

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

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).
Original languageEnglish
Pages (from-to)e145
JournalMolecular Therapy - Nucleic Acids
Volume3
DOIs
Publication statusPublished - 4 Feb 2014

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