A High Throughput In Vivo Model for Testing Delivery and Antiviral Effects of siRNAs in Vertebrates

Abstract

Despite the promise of small interfering RNAs (siRNAs) in antiviral therapy, few in vivostudies of them as inhibitors of viral replication and disease have been published, a lack that is most probably due to problems with obtaining successful delivery. Here we introduce a novel in vivomodel composed of small juvenile rainbow trout and a fish pathogenic virus to analyze the delivery and antiviral effects of formulated siRNAs. Intraperitoneally (IP) injected siRNAs formulated in polycationic liposomes, and to a lesser degree naked siRNAs, primarily entered free IP cells, including macrophage-like cells. Uptake in these cells correlated with antiviral activity, seen as reduced mortality of virus-challenged fish. However, protection at the disease level was not dependent upon which of three tested siRNAs was used, and protection correlated with up-regulation of an interferon (IFN)-related gene in the liver, indicating a systemic IFN response. The results emphasize the compromise in using transfection reagents for improved uptake of siRNAs, where these reagents also increase the risk of the siRNAs ending up in a cellular compartment in which stimulation of non-specific anti-viral defence mechanisms will be initiated.

A RNAi-based therapeutic proof of concept targets salmonid whirling disease in vivo

“…For successful silencing of intracellular parasite genes, siRNAs must: (i) be systemically delivered from the area of uptake to body tissues harbouring and affected by the parasite, (ii) be taken up by the RNA-induced silencing complex and (iii) degrade specific messenger RNA targets of the parasite [10]. In whirling disease, this would suggest siRNA delivery to the rainbow trout cartilage because it is the specific tissue affected by M .…”

“…RNAi has been demonstrated in cells of mammals and other vertebrates including fish [10,11]. Since 2012, there has been a resurgence in clinical trials using RNA interference (RNAi)-based therapeutics for the treatment of diseases by specifically silencing the expression of disease-relevant genes [12].…”

In vivo screening of modified siRNAs for non-specific antiviral effect in a small fish model: number and localization in the strands are important

“…Duplexes were formulated in the liposome-formulated polycationic transfection agent 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP; Roche Diagnostics, Basel, Switzerland), injected into the intraperitoneum (IP) of 1 g large rainbow trout followed by challenge with VHSV (27). Briefly, 1 μg siRNA was mixed with 2 μg DOTAP in 0.9% NaCl (physiological saline, Nycomed, Denmark).…”

“…In this study, we test a range of modified siRNAs for their ability to induce a non-specific antiviral response in a small vertebrate model. We exploit our previous finding that the activation of a functional innate immune mechanisms by delivered siRNAs can delay and reduce the mortality of small juvenile rainbow trout challenged with the fish pathogenic rhabdovirus Viral Haemorrhagic Septicaemia Virus (VHSV) (27). Our data indicate that the induced immune effect of modified siRNAs in vivo is determined by several factors such as nucleotide sequence of the siRNA, modification chemistries, number and localization of modifications and probably, as our results seem to indicate, the thermal stability of RNA bindings.…”

RNA interference technology used for the study of aquatic virus infections

“…Schyth et al [84] have investigated siRNA suppression of intracellular pathogens using the envelope glycoprotein of the Rhabdovirus family member viral hemorrhagic septicemia virus (VHSV). Preliminary in vitro studies involved the transfection of human kidney 293T cells with synthesized envelope protein siRNA along with a eukaryotic expression vector carrying the VHSV envelope glycoprotein gene.…”