TY - JOUR
T1 - Deep sequencing insights in therapeutic shRNA processing and siRNA target cleavage precision
AU - Denise, Hubert
AU - Moschos, Sterghios
AU - Sidders, Benjamin
AU - Burden, Frances
AU - Perkins, Hannah
AU - Carter, Nikki
AU - Stroud, Tim
AU - Kennedy, Michael
AU - Fancy, Sally-Ann
AU - Lapthorn, Cris
AU - Lavender, Helen
AU - Kinloch, Ross
AU - Suhy, David
AU - Corbau, Romu
PY - 2014/2/4
Y1 - 2014/2/4
N2 - 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).
AB - 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).
UR - https://www.scopus.com/pages/publications/84893825866
U2 - 10.1038/mtna.2013.73
DO - 10.1038/mtna.2013.73
M3 - Article
SN - 2162-2531
VL - 3
SP - e145
JO - Molecular Therapy - Nucleic Acids
JF - Molecular Therapy - Nucleic Acids
ER -
