In Vitro Inhibition of Replication of Dengue Virus Serotypes 1-4 by siRNAs Bound to Non-Toxic Liposomes.

Carlos Andrés Rodriguez-Salazar,Maria Isabel Giraldo,Leonardo Padilla-Sanabria,Jhon Carlos Castaño-Osorio,Delia Piedad Recalde-Reyes,Juan Pablo Bedoya

doi:10.3390/v14020339

Carlos Andrés Rodriguez-Salazar, Maria Isabel Giraldo + Show 4 more

Open Access

https://doi.org/10.3390/v14020339

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Abstract

Dengue virus is a ssRNA+ flavivirus, which produces the dengue disease in humans. Currently, no specific treatment exists. siRNAs regulate gene expression and have been used systematically to silence viral genomes; however, they require controlled release. Liposomes show favorable results encapsulating siRNA for gene silencing. The objective herein was to design and evaluate in vitro siRNAs bound to liposomes that inhibit DENV replication. siRNAs were designed against DENV1–4 from conserved regions using siDirect2.0 and Web-BLOCK-iT™ RNAiDesigner; the initial in vitro evaluation was carried out through transfection into HepG2 cells. siRNA with silencing capacity was encapsulated in liposomes composed of D-Lin-MC3-DMA, DSPC, Chol. Cytotoxicity, hemolysis, pro-inflammatory cytokine release and antiviral activity were evaluated using plaque assay and RT-qPCR. A working concentration of siRNA was established at 40 nM. siRNA1, siRNA2, siRNA3.1, and siRNA4 were encapsulated in liposomes, and their siRNA delivery through liposomes led to a statistically significant decrease in viral titers, yielded no cytotoxicity or hemolysis and did not stimulate release of pro-inflammatory cytokines. Finally, liposomes were designed with siRNA against DENV, which proved to be safe in vitro.

Highlights

Dengue is a systemic viral infectious disease caused by the dengue virus (DENV), a flavivirus with a single-stranded positive polarity RNA genome of approximately 11 Kb, which is methylated on the 5 end, and the 3 end is not polyadenylated
A total of 72, 30, 53, and 91 regions conserved for DENV1, DENV2, DENV3, and DENV4, respectively, were used in siDirect to predict small interfering RNAs (siRNA)
Dengue virus is known for being the etiological agent responsible for dengue disease

Summary

Introduction

Dengue is a systemic viral infectious disease caused by the dengue virus (DENV), a flavivirus with a single-stranded positive polarity RNA genome of approximately 11 Kb, which is methylated on the 5 end, and the 3 end is not polyadenylated. It has a single open reading frame that encodes a single poly-protein, which is processed by cellular enzymes and viral proteases into 10 proteins; three structural (E: envelope, M: membrane-associated, and C: capsid) that form the viral particle and seven non-structural (NS1, 2A, 2B, 3, 4A, 4B, and NS5), which are necessary for processing and assembly of new viruses [1]. Four serotypes are well established (named DENV 1–4 including various genotypes), all affecting humans [2] This arbovirus is transmitted through the bite of female Aedes aegypti or Aedes albopictus mosquitoes. Viral replication produces an infection with a broad spectrum of clinical presentations, ranging from subclinical or asymptomatic forms to severe forms of the disease characterized by abnormalities in hemostasis and increased vascular permeability, which may lead to hemorrhagic complications, organ damage, and even cause the death of the individual [3,4]

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