Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication
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.
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Dissertation•
01 Jan 2012
22 citations
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
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Harvard University1, University of Pennsylvania2, University of Florida3, University of Texas Health Science Center at San Antonio4, Henry Ford Health System5, University of Miami6, Scripps Health7, Saint Louis University8, University of New Mexico9, Duke University10, Johns Hopkins University11, Indiana University12
TL;DR: Treatment with a once-daily, single-tablet regimen of ledipasvir and sofosbuvir resulted in high rates of sustained virologic response among patients with HCV genotype 1 infection who had not had a sustained virologyic response to prior interferon-based treatment.
Abstract: Background Effective treatment for hepatitis C virus (HCV) genotype 1 infection in patients who have not had a sustained virologic response to prior interferon-based therapy represents an unmet medical need. Methods We conducted a phase 3, randomized, open-label study involving patients infected with HCV genotype 1 who had not had a sustained virologic response after treatment with peginterferon and ribavirin, with or without a protease inhibitor. Patients were randomly assigned to receive the NS5A inhibitor ledipasvir and the nucleotide polymerase inhibitor sofosbuvir in a once-daily, fixed-dose combination tablet for 12 weeks, ledipasvir–sofosbuvir plus ribavirin for 12 weeks, ledipasvir–sofosbuvir for 24 weeks, or ledipasvir–sofosbuvir plus ribavirin for 24 weeks. The primary end point was a sustained virologic response at 12 weeks after the end of therapy. Results Among the 440 patients who underwent randomization and were treated, 20% had cirrhosis and 79% had HCV genotype 1a infection. The rates of ...
1,258 citations
TL;DR: The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.
Abstract: The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.
1,222 citations
TL;DR: It is demonstrated that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-α) and that a higher frequency of cured cells could support both sub genomic and full-length HCV replication.
Abstract: Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-α) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.
1,203 citations
TL;DR: It is reported here that a substantial number of shRNA vectors can trigger an interferon response and is thought to be too short to induce interferons expression.
Abstract: DNA vectors that express short hairpin RNAs (shRNAs) from RNA polymerase III (Pol III) promoters are a promising new tool to reduce gene expression in mammalian cells. shRNAs are processed to small interfering RNAs (siRNAs) of 21 nucleotides (nt) that guide the cleavage of the cognate mRNA by the RNA-induced silencing complex. Although siRNAs are thought to be too short to induce interferon expression, we report here that a substantial number of shRNA vectors can trigger an interferon response.
1,043 citations
TL;DR: This review explains about the history of gene therapy, all types of gene delivery systems for germline and somatic cells, and advantages, disadvantages, and practical use of each system are discussed.
Abstract: Gene therapy is the process of introducing foreign genomic materials into host cells to elicit a therapeutic benefit. Although initially the main focus of gene therapy was on special genetic disorders, now diverse diseases with different patterns of inheritance and acquired diseases are targets of gene therapy. There are 2 major categories of gene therapy, including germline gene therapy and somatic gene therapy. Although germline gene therapy may have great potential, because it is currently ethically forbidden, it cannot be used; however, to date human gene therapy has been limited to somatic cells. Although numerous viral and nonviral gene delivery systems have been developed in the last 3 decades, no delivery system has been designed that can be applied in gene therapy of all kinds of cell types in vitro and in vivo with no limitation and side effects. In this review we explain about the history of gene therapy, all types of gene delivery systems for germline (nuclei, egg cells, embryonic stem cells, pronuclear, microinjection, sperm cells) and somatic cells by viral [retroviral, adenoviral, adeno association, helper-dependent adenoviral systems, hybrid adenoviral systems, herpes simplex, pox virus, lentivirus, Epstein-Barr virus)] and nonviral systems (physical: Naked DNA, DNA bombardant, electroporation, hydrodynamic, ultrasound, magnetofection) and (chemical: Cationic lipids, different cationic polymers, lipid polymers). In addition to the above-mentioned, advantages, disadvantages, and practical use of each system are discussed.
668 citations