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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: Whereas HIV-1 could escape from a single shRNA, it is shown that HIV- 1 escape can be prevented when four shRNAs are simultaneously expressed in a cell.

220 citations

Journal ArticleDOI
TL;DR: RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.
Abstract: RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.

194 citations

Journal ArticleDOI
TL;DR: Recent advances have been made in the shRNA vector design to mimic cellular miRNA processing and to express multiplex siRNAs in a tightly regulated and reversible manner to overcome toxicities, and in this review, some of these advances are described.

180 citations

Journal ArticleDOI
TL;DR: The results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.

177 citations

Journal ArticleDOI
Stephanie Pebernard1, Richard Iggo1
TL;DR: To avoid interferon induction by U6 vectors, it is recommended to preserve the wild-type sequence around the transcription start site, in particular a C/G sequence at positions -1/+1, and a simple cloning strategy using the Gateway recombination system that facilitates this task is described.

170 citations