Paradigms for conditional expression of RNA interference molecules for use against viral targets.
The rapid increase in the study of small interfering RNA (siRNA) as a means to decrease expression of targeted genes has led to concerns about possible unexpected consequences of constitutive siRNA expression. We therefore devised a conditional siRNA expression system in which siRNA targeting hepatitis C virus (HCV) would be produced in response to HCV. We found that HCV acts via NFkappaB to stimulate the HIV long terminal repeat (LTR) as a promoter. We exploited this observation by designing conditional siRNA transcription constructs to be triggered by HCV-induced activation of NFkappaB. These were delivered by using highly efficient recombinant Tag-deleted SV40-derived vectors. Conditional activation of HIV-LTR and consequent siRNA synthesis in cells expressing HCV were observed. HCV-specific RNAi decreased HCV RNA greatly within 4 days, using transient transfection of the whole HCV genome as a model of acute HCV entry into transduced cells. We then tested the effectiveness of rSV40-delivered anti-HCV siRNA in cells stably transfected with the whole HCV genome to simulate hepatocytes chronically infected with HCV. There is considerable need for regulated production of siRNAs activated by a particular set of conditions (HCV in this case) but quiescent otherwise. Approaches described here may serve as a paradigm for such conditional siRNA expression.
Strayer
David S
DS
Department of Pathology and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107-5587, USA.
Feitelson
Mark
M
Sun
Bill
B
Matskevich
Alexey A
AA
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D016174:Hepacivirus / Q000235:genetics; D010957:Plasmids; D034622:RNA Interference; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral