Targeted Delivery of Small Interfering RNA to Human Dendritic Cells To Suppress Dengue Virus Infection and Associated Proinflammatory Cytokine Production

ABSTRACT Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-d-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34+ hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvED) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ−/− mouse model engrafted with human CD34+ hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

Download Full-text

420 Small interfering RNA(siRNA)-mediated inhibition of hepatitis C virus replication using bicistronic vector in vivo (cell line Huh-7) and in vitro

Journal of Hepatology ◽

10.1016/s0168-8278(05)81832-x ◽

2005 ◽

Vol 42 ◽

pp. 154 ◽

Cited By ~ 1

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Line ◽

Virus Replication ◽

Small Interfering Rna ◽

Bicistronic Vector ◽

Interfering Rna

Download Full-text

Inhibitory effect of small interfering RNA on dengue virus replication in mosquito cells

Virology Journal ◽

10.1186/1743-422x-7-270 ◽

2010 ◽

Vol 7 (1) ◽

pp. 270 ◽

Cited By ~ 24

Author(s):

Xinwei Wu ◽

Hua Hong ◽

Jinya Yue ◽

Yejian Wu ◽

Xiangzhong Li ◽

...

Keyword(s):

Dengue Virus ◽

Virus Replication ◽

Small Interfering Rna ◽

Inhibitory Effect ◽

Mosquito Cells ◽

Interfering Rna

Download Full-text

Inhibition of foot-and-mouth disease virus replication in vitro and in vivo by small interfering RNA

Virology Journal ◽

10.1186/1743-422x-5-86 ◽

2008 ◽

Vol 5 (1) ◽

pp. 86 ◽

Cited By ~ 14

Author(s):

Wang Pengyan ◽

Ren Yan ◽

Guo Zhiru ◽

Chen Chuangfu

Keyword(s):

Disease Virus ◽

Virus Replication ◽

Small Interfering Rna ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Interfering Rna

Download Full-text

In Vivo Injection of Synthetic Small Interfering RNA to Lethally Irradiated Mouse as a New Model to Study Apoptotic Pathways.

Blood ◽

10.1182/blood.v112.11.1336.1336 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1336-1336 ◽

Cited By ~ 1

Author(s):

Michel Drouet ◽

Philippe Garrigou ◽

Jean-François Mayol ◽

Christophe Delaunay ◽

Andre Peinnequin ◽

...

Keyword(s):

Small Interfering Rna ◽

Fas Ligand ◽

Ex Vivo ◽

Short Term ◽

Apoptotic Pathway ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Apoptosis Process ◽

Interfering Rna

Abstract The Fas/Fas-ligand system is a well known component of the extrinsic apoptotic pathway. Using a short term culture assay we have established that CD34+ hematopoietic stem and progenitor cells express Fas antigen within 10 hours following irradiation. Using the terminaldeoxynucleotidyl transferase test, we have also established that this expression was linked with apoptosis since only the Fas/Fas-ligand positive cells exhibited a high level of DNA fragmentation (Drouet et al, Stem cell 1999). However Fas is also involved in the CD34+ cells differentiation process as described in ex vivo expansion studies. Caspases are other important actors of radiation induced (RI) apoptosis process and our team has recently identified caspases one and six as key actors in RI apoptosis in CD34+ cells. The goal of the present study was to evaluate short term synthetic small interfering RNA (siRNA) as new tools to in vivo modulate apoptosis in order to allow pathophysiological studies at the hematopoietic niche level. Briefly, B6D2F1 mice were globally irradiated (9 Gy gamma, LD90% 30 days) and then injected at 2 hours following irradiation with siRNA (0.5 nmol/mice, Dharmacon). To ensure a proper delivery to the niche cell components, siRNA were intra-tibially injected under a volume of 30μl. The duration of gene inhibition is about 10 days long. Control mice were injected with non relevant mock siRNA (n=80). Treated animals were injected with Fas-siRNA (n=20) or Fas + a pool of siRNA against caspases 1+ 3 + 6 + 8)(n=20). All mice were given ciprofloxacin during a week and no early lethality was observed. The lethality curves show that animals treated with Fas-siRNA exhibited an accelerated death rate when compared with siRNA mice. These results are compatible with a janus model for Fas expression depending on the time following irradiation: initial proapoptotic role, then requirement for cell expansion. Globally this study suggests the feasibility of using synthetic siRNA in vivo to screen the role of apoptosis actors.

Download Full-text

Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc–siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5′-exonuclease

Nucleic Acids Research ◽

10.1093/nar/gkaa125 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4028-4040

Author(s):

Pawan Kumar ◽

Rohan Degaonkar ◽

Dale C Guenther ◽

Mikhail Abramov ◽

Guy Schepers ◽

...

Keyword(s):

Targeted Delivery ◽

Small Interfering Rna ◽

Antisense Strand ◽

Seed Region ◽

Chemically Modified ◽

Interfering Rna ◽

Insight Into ◽

Rnai Activity

Abstract In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2′-deoxy-2′-fluoro and 2′-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5′ end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5′ end was more stable in the presence of 5′-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5′-exonuclease degradation afforded by the ANA modification.

Download Full-text

Anti-β2 Glycoprotein I Antibodies Cause Inflammation and Recruit Dendritic Cells in Platelet Clearance

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616059 ◽

2001 ◽

Vol 86 (11) ◽

pp. 1257-1263 ◽

Cited By ~ 6

Author(s):

Attilio Bondanza ◽

Angelo Manfredi ◽

Valérie Zimmermann ◽

Matteo Iannacone ◽

Angela Tincani ◽

...

Keyword(s):

Dendritic Cells ◽

Inflammatory Responses ◽

Glycoprotein I ◽

Activated Platelets ◽

Tnf Α ◽

Per Se ◽

Antigen Presenting ◽

Antiinflammatory Cytokine

SummaryScavenger phagocytes are mostly responsible for the in vivo clearance of activated or senescent platelets. In contrast to other particulate substrates, the phagocytosis of platelets does not incite pro-inflammatory responses in vivo. This study assessed the contribution of macrophages and dendritic cells (DCs) to the clearance of activated platelets. Furthermore, we verified whether antibodies against the β2 Glycoprotein I (β2GPI), which bind to activated platelets, influence the phenomenon. DCs did not per se internalise activated platelets. In contrast, macrophages efficiently phagocytosed platelets. In agreement with the uneventful nature of the clearance of platelets in vivo, phagocytosing macrophages did not release IL-1β, TNF-α or IL-10. β2GPI bound to activated platelets and was required for their recognition by anti-ββ2GPI antibodies. DCs internalised platelets opsonised by anti-ββ2GPI antibodies. The phagocytosis of opsonised platelets determined the release of TNF-α and IL-1β by DCs and macrophages. Phagocytosing macrophages, but not DCs, secreted the antiinflammatory cytokine IL-1β0. We conclude that anti-ββ2GPI antibodies cause inflammation during platelet clearance and shuttle platelet antigens to antigen presenting DCs.

Download Full-text