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Journal ArticleDOI

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

01 Jan 2018-Intervirology (Intervirology)-Vol. 61, Iss: 2, pp 79-91
TL;DR: A lentiviral vector-based delivery system is a “single-shot” therapeutic strategy that can express duplex shRNA for long-term synergistic inhibition of HCV and qualify as a promising therapeutic approach for sustained inhibition ofHCV replication.
Abstract: Background: The RNAi-based transient therapeutic approach has been well explored for its potential against the hepatitis V virus (HCV). However, to achieve a sustained virological response, a consistent presence of siRNA is needed and it can be achieved by constitutively expressing shRNAs. In this context, the lentiviral vector has emerged as an attractive tool for shRNA delivery against HCV. Methods: We monitored HCV inhibition after single and multiple rounds of siRNA treatments against La autoantigen and HCV-NS5B in Huh-7.5 cells infected with the FL-J6/JFH chimeric HCV strain. A bicistronic self-inactivating third-generation lentiviral vector expressing shRNA under U6 and H1 promoters was constructed. To ascertain the long-term HCV inhibition, cells were transduced with lentiviral vectors and HCV inhibition was monitored by RT-PCR and Western blotting at regular intervals. Results: We observed transient antiviral activity after a single round of siRNA treatment, and consecutive rounds of treatments with siRNA demonstrated a sustained HCV inhibition. Delivery of duplex shRNA expressing lentiviral vectors provided constant expression of shRNA leading to synergistic and sustained HCV inhibition. Conclusion: A lentiviral vector-based delivery system is a “single-shot” therapeutic strategy. It can express duplex shRNA for long-term synergistic inhibition of HCV and qualify as a promising therapeutic approach for sustained inhibition of HCV replication.
Citations
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Journal ArticleDOI
TL;DR: RNAi effectors (e.g., siRNA, shRNA, and miRNA) can efficiently trigger the silencing of specific genes, and its genomic alteration functions allowed to pursue clinical trials in distinct areas, including infectious diseases, neurodegenerative disorders, and cancer as discussed by the authors.
Abstract: RNAi effectors (e.g., siRNA, shRNA, and miRNA) can efficiently trigger the silencing of specific genes, and its genomic alteration functions allowed to pursue clinical trials in distinct areas, including infectious diseases, neurodegenerative disorders, and cancer. Moreover, regarding cancer immunotherapy, RNAi therapeutics showed potential immunomodulatory ability by downregulating suppressive receptors such as PD-1 and CTLA-4, which restrict immune cell function and present challenges in cancer immunotherapy. Therefore, compared with extracellular targeting by antibodies, RNAi-mediated, cell-intrinsic disruption of inhibitory pathways in immune cells can promote an increased antitumor immune response. Along with nonviral vectors, DNA-based RNAi strategies might be a more promising method for immunomodulation to silence multiple inhibitory pathways in T cells than immune checkpoint blockade antibodies. Thus, in this review, we discuss diverse RNAi implementation strategies, with recent viral and non-viral mediated RNAi synergism to immunotherapy that augments the antitumor immunity. Finally, we provide the current progress of RNAi in clinical pipeline.

9 citations

References
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Journal ArticleDOI
TL;DR: It is found that the 3' end of positive polarity HCV RNA is sensitive to cytosolic RNases whereas the 3', untranslated region with the cellular La protein prevented premature degradation of the HCV RNAs.
Abstract: We have analysed hepatitis C virus (HCV) RNAs in an in vitro RNA degradation assay. We found that the 3′ end of positive polarity HCV RNA is sensitive to cytosolic RNases whereas the 3′ end of negative polarity HCV RNA is relatively stable. Interaction of the HCV 3′ untranslated region with the cellular La protein prevented premature degradation of the HCV RNA. One may speculate that HCV RNAs interact with La protein in infected cells to prevent premature degradation of the viral RNAs.

67 citations

Journal ArticleDOI
TL;DR: Both nucleoside and non-nucleoside inhibitors of HCV polymerase have been identified through the innovative use of new screening tools and rational drug design, and initiation of clinical trials in the near future promises to yield exciting new information on the ability of these compounds to achieve sustained responses in suppressing HCV replication.

64 citations

Journal ArticleDOI
TL;DR: A novel drug is described, intended as a “single-shot ” therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system.

52 citations

Journal ArticleDOI
TL;DR: It is demonstrated that exogenous anti‐HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity.

50 citations

Journal ArticleDOI
TL;DR: In conclusion, even after 4 weeks of treatment of replicon cells with HCV‐siRNA, HCv‐RNA andHCV‐NS5A protein expression could not be completely eliminated.
Abstract: Summary. RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.

38 citations