Abstract |
The use of RNA interference (RNAi) to inhibit gene expression is potentially applicable in the treatment of viral infections such as hepatitis B virus (HBV) persistence. Although efficient HBV gene silencing by short hairpin RNA ( shRNA) expressed from RNA polymerase (Pol) III promoters has been reported, constitutive high-level transcription may cause harmful side effects. Here, we report an approach that allows the use of a Pol II promoter to improve transcription regulation of expressed RNAi effecters. Pol II [cytomegalovirus (CMV)] or Pol III (U6) promoter cassettes that transcribe anti-HBV primary microRNA (pri-miR)-122 and pri-miR-31 shuttles were generated. In cultured cells both types of pri-miR-like sequences effected knockdown of markers of viral replication (>80%) and were processed to form intended 21-nucleotide guides. The concentration of CMV-expressed miRs was approximately 85-fold lower than the U6 shRNA-derived guide RNA. When cells were co-transfected with pri-miR expression cassettes, attenuation of independent RNAi-mediated gene silencing was not observed, which is in contrast to the action of U6 shRNA expression cassettes. The efficacy of the anti-HBV pri-miR shuttles in vivo was verified using the murine hydrodynamic injection model. Employing Pol II-expressed pri-miR mimics may be useful in the treatment of HBV infection, and potentially also for generic application in RNAi-based therapy.
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Authors | Abdullah Ely, Tanusha Naidoo, Steven Mufamadi, Carol Crowther, Patrick Arbuthnot |
Journal | Molecular therapy : the journal of the American Society of Gene Therapy
(Mol Ther)
Vol. 16
Issue 6
Pg. 1105-12
(Jun 2008)
ISSN: 1525-0024 [Electronic] United States |
PMID | 18431360
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
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Topics |
- Animals
- Cell Line
- Cells, Cultured
- Cytomegalovirus
(genetics)
- Gene Silencing
- Genetic Therapy
(methods)
- Hepatitis B virus
(genetics)
- Humans
- In Vitro Techniques
- Mice
- MicroRNAs
- Models, Genetic
- Plasmids
(metabolism)
- RNA Interference
- Transfection
- Virus Replication
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