Title | Deep sequencing insights in therapeutic shRNA processing and siRNA target cleavage precision |
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Authors | Denise, H., Moschos, S.A., Sidders, B., Burden, F., Perkins, H., Carter, N., Stroud, T., Kennedy, M., Fancy, S.A., Lapthorn, C., Lavender, H., Kinloch, R., Suhy, D.A. and Corbau, R. |
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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). |
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Journal | Molecular Therapy Nucleic Acids |
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Journal citation | 3, p. e145 |
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ISSN | 2162-2531 |
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Year | 2014 |
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Publisher | Nature Publishing Group |
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Digital Object Identifier (DOI) | https://doi.org/10.1038/mtna.2013.73 |
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Publication dates |
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Published | 2014 |
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