Inhibiting avian influenza virus shedding using a novel RNAi antiviral vector technology: proof of concept in an avian cell model

Abstract

Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.

The Influenza Virus Polymerase Complex: An Update on Its Structure, Functions, and Significance for Antiviral Drug Design

“…5ʹ‐Triphosphate modification of siRNA (for NP) provided a dual effect by combining gene silencing with RIG‐I activation . Alternatively, to circumvent the poor delivery of siRNA, a lentiviral or Escherichia coli vector was explored to express NP‐ and PB1‐targeting, or NP‐ and PA‐targeting RNA interfering sequences, respectively.…”

An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

“…9 CFU / 100 µL As shown in Figure2A, SVC1 bacteria remained localized following administration, suggesting…”

“…For over three decades, there has been increasing interest in the use of engineered commensal or probiotic bacteria as an in vivo nucleic acid and protein delivery modality as they represent a powerful and unique approach for the delivery of therapeutic moieties that can overcome many of the limitations of viral or chemical delivery systems. As part of the normal human flora, Escherichia coli has garnered particular interest [6][7][8][9][10][11][12][13] .…”

“…RNAimediated silencing is guided by small interfering RNAs (siRNAs) [46][47][48] . Substrate siRNAs can be generated from the enzymatic processing of short hairpin RNAs (shRNAs), which, importantly, can be expressed by bacteria 9,28 . E. coli strains can be engineered to express shRNAs against a target gene of interest.…”

“…While professional phagocytic cells (e.g., neutrophils) readily take up bacteria via phagocytosis 14 , other cell types, which are clear front runners as therapeutic targets (e.g., epithelial cells), do not have intrinsic phagocytic activity; thus, the bacteria must be engineered to enable cellular uptake. In the Escherichia coli -based system described here, the E. coli strain SVC1 has been genetically modified to meet these needs 9 . First, the SVC1 bacteria carry a heterologous gene encoding the Yersinia pseudotuberculosis invasin (inv) protein to allow uptake by the targeted eukaryotic cells.…”