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

Emerging role of RNA interference in immune cells engineering and its therapeutic synergism in immunotherapy

“…To date, several studies have investigated optimizing multiplex shRNA systems using various promoters and modifying vector systems with circular miRNA to increase the therapeutic potency while minimizing adverse events (Choi et al, 2015;Lambeth & Smith, 2013;Mandal & Chaubey, 2018).…”

“…However, it is currently difficult to distinguish which RNAi implementation strategies are optimal for efficient gene silencing. Indeed, several investigations have implied that shRNA‐mediated RNAi is more competent and results in a more potent therapeutic effect than siRNA‐mediated RNAi (Mandal & Chaubey, 2018; Stovall et al, 2012). However, oversaturation of exportin‐5 and high levels of anti‐sense strands compete with endogenous RNAi molecules, leading to fatal events caused by off‐target toxicities (Boudreau et al, 2009).…”

“…Though this strategy could enhance the inherent risks of RNAi, including off‐target toxicity, immunogenicity and oversaturation of the endogenous pathway, expression of multiple RNAi effectors could provide new therapeutic modalities (Grimm & Kay, 2007; Ter Brake et al, 2008). To date, several studies have investigated optimizing multiplex shRNA systems using various promoters and modifying vector systems with circular miRNA to increase the therapeutic potency while minimizing adverse events (Choi et al, 2015; Lambeth & Smith, 2013; Mandal & Chaubey, 2018).…”