tRNA Lys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs.
Using exogenous sequences to express RNA interference (RNAi) activators has potential for the treatment of chronic viral infections. However, availability of a variety of suitable of promoter elements is important to optimize transcription control of silencing sequences and facilitate multitargeting. Recent demonstration that tRNA miR genes occur naturally has prompted investigating the incorporation these tRNA Pol III promoters into exogenous RNAi-activating cassettes. We have assessed efficacy of Pol III tRNA(Lys3) short hairpin RNA (shRNA) sequences that target hepatitis B virus (HBV). These cassettes achieved good silencing at low concentrations, and efficacy compared favorably to that of equivalent U6, H1 and CMV expression cassettes. HBV replication in cell culture was inhibited and northern blot hybridization analysis confirmed processing of the tRNA(Lys3) transcripts to form intended antiviral guide sequences. Importantly effects were observed without evidence of disruption of endogenous miR function. Analysis in a murine hydrodynamic injection model of HBV replication confirmed that the tRNA(Lys3) expression cassettes are also effective in vivo. Usefulness of tRNA(Lys3) antiviral expression cassettes expands the repertoire of promoters available for RNAi-mediated HBV silencing and advances the application of expressed sequences for therapeutic gene silencing.
Dyer
Victoria
V
Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand Medical School, Private Bag 3, Wits 2050, South Africa.
Ely
Abdullah
A
Bloom
Kristie
K
Weinberg
Marc
M
Arbuthnot
Patrick
P
D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D015316:Genetic Therapy; D006509:Hepatitis B / Q000628:therapy*; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012357:RNA, Transfer, Lys / Q000235:genetics*; D014779:Virus Replication
C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D012357:RNA, Transfer, Lys