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Mol Ther Nucleic Acids. 2014 Feb 4;3:e145. doi: 10.1038/mtna.2013.73.

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

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Metagenomics, European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory, Wellcome Trust Genome Campus, Hinxton, UK.
Department of Molecular and Applied Biosciences, Faculty of Science and Technology, University of Westminster, London, UK.
Neusentis, Pfizer Global Research & Development, Cambridge, UK.
New Opportunities Unit, Pfizer Global Research and Development, Sandwich, UK.
Analytical Sciences, Pfizer Global Research and Development, Sandwich, UK.
Tacere Therapeutics Inc, Pleasanton, CA.


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).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

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