Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi)

Pan, Qiuwei; Ramakrishnaiah, Vedashree; Henry, Scot D.; Fouraschen, Suomi M. G.; Ruiter, Petra E. de; Kwekkeboom, Jaap; Tilanus, Hugo W.; Janssen, Harry L A; Laan, Luc J. W. van der

doi:10.1136/gutjnl-2011-300449

Cited by 152 publications

(101 citation statements)

References 53 publications

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“…As determined by direct Sanger sequencing, the amplified products from exosomes were identical to the HCV genotype 2a of Jc1. Consistent with our earlier report (18), both control and HCVpositive exosomes contained high levels of miRNAs, including miR-122.…”

Section: Exosomes Derived From Hcv-infected Hepatoma Cells Contain Virussupporting

confidence: 79%

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Ramakrishnaiah

Thumann

Fofana

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.

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Section: Exosomes Derived From Hcv-infected Hepatoma Cells Contain Virussupporting

confidence: 79%

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Ramakrishnaiah

Thumann

Fofana

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

440

399

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“…Factors involved in initial virion assembly will likely be necessary for both cell-free and cell-to-cell spread, while those involved in trafficking or late-stage maturation may have differing importance in these two processes and may indirectly provide insight into the nature of the viral particle involved in cell-to-cell spread. In this regard, the exosome pathway is of interest, as several reports have documented that HCV RNA is secreted from infected cells in exosomes, which has been proposed to transfer the infection when taken up by other cells (61)(62)(63)(64). While by nature this would appear to fall within the category of extracellular (i.e., cell-free) spread, a role for exosome pathway components in cell-to-cell spread cannot be ruled out.…”

Section: Discussionmentioning

confidence: 99%

Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

Barretto

Sáinz

Hussain

et al. 2014

J Virol

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Hepatitis C virus (HCV) infects 180 million people worldwide and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. It has been shown that HCV can spread to naive cells using two distinct entry mechanisms, "cellfree" entry of infectious extracellular virions that have been released by infected cells and direct "cell-to-cell" transmission. Here, we examined host cell requirements for HCV spread and found that the cholesterol uptake receptor NPC1L1, which we recently identified as being an antiviral target involved in HCV cell-free entry/spread, is also required for the cell-to-cell spread. In contrast, the very low density lipoprotein (VLDL) pathway, which is required for the secretion of cell-free infectious virus and thus has been identified as an antiviral target for blocking cell-free virus secretion/spread, is not required for cell-to-cell spread. Noting that HCV cell-free and cell-to-cell spread share some common factors but not others, we tested the therapeutic implications of these observations and demonstrate that inhibitors that target cell factors required for both forms of HCV spread exhibit synergy when used in combination with interferon (a representative inhibitor of intracellular HCV production), while inhibitors that block only cell-free spread do not. This provides insight into the mechanistic basis of synergy between interferon and HCV entry inhibitors and highlights the broader, previously unappreciated impact blocking HCV cell-to-cell spread can have on the efficacy of HCV combination therapies. IMPORTANCEHCV can spread to naive cells using distinct mechanisms: "cell-free" entry of extracellular virus and direct "cell-to-cell" transmission. Herein, we identify the host cell HCV entry factor NPC1L1 as also being required for HCV cell-to-cell spread, while showing that the VLDL pathway, which is required for the secretion of cell-free infectious virus, is not required for cell-to-cell spread. While both these host factors are considered viable antiviral targets, we demonstrate that only inhibitors that block factors required for both forms of HCV entry/spread (i.e., NPC1L1) exhibit synergy when used in combination with interferon, while inhibitors that block factors required only for cell-free spread (i.e., VLDL pathway components) do not. Thus, this study advances our understanding of HCV cell-to-cell spread, provides mechanistic insight into the basis of drug synergy, and highlights inhibition of HCV spread as a previously unappreciated consideration in HCV therapy design.

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“…There is additional evidence that siRNAs can be passed cell to cell in the liver partially via exosomes. 35 Alternatively, chemical modifications can stabilize or protect siRNAs. For example, attaching siRNAs to polyethylenimines tightly packages siRNAs until phagocytosed, and are released intracellularly following endosomal disruption.…”

Section: Demonstration Of Oncogenesis Of Pdx1mentioning

confidence: 99%

Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform

Liu

et al. 2014

Cancer Gene Ther

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RNA interference (RNAi) represents a powerful, new tool for scientific investigation as well as a promising new form of targeted gene therapy, with applications currently in clinical trials. Bifunctional short hairpin RNA (shRNA) are synthetic RNAi molecules, engineered to utilize multiple endogenous RNAi pathways to specifically silence target genes. Pancreatic and duodenal homeobox 1 (PDX1) is a key regulator of pancreatic development, β-cell differentiation, normal β-cell function and pancreatic cancer. Our aim is to review the process of identifying PDX1 as a specific, potential RNAi target in pancreatic cancer, as well as the underlying mechanisms and various forms of RNAi, with subsequent testing and development of PDX1-targeted bifunctional shRNA therapy.

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Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi)

Abstract: Both human and mouse hepatic cells exchange small silencing RNAs, partially mediated by shuttling of exosomes. Transmission of siRNA potentially extends the therapeutic reach of RNAi-based therapies against HCV as well as other liver diseases.

Cited by 152 publications

References 53 publications

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform

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