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In vitro and in vivo targeted delivery of IL-10 interfering RNA by JC virus-like particles.

Chou MI, Hsieh YF, Wang M, Chang JT, Chang D, Zouali M, Tsay GJ - J. Biomed. Sci. (2010)

Bottom Line: In RAW 264.7 cells, IL-10 shRNA was found to reduce IL-10 expression by 85 to 89%, as compared with VLPs alone.IL-10 shRNA did not cross-react with TNF-alpha mRNA or influence the expression of TNF-alpha.In BALB/c mice IL-10 shRNA could reduce 95% of IL-10 secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan.

ABSTRACT

Background: RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Delivering sequences of RNAi in vivo remains a problem. The aim of this study was to use JC virus (JCV) virus-like particles (VLPs) as a vector for delivering RNAi in silencing the cytokine gene of IL-10.

Methods: JCV VLPs were generated by recombinant JCV VP1 protein in yeast expression system. DNA fragment containing IL-10 shRNA was packaged into VLPs by osmotic shock.

Results: In RAW 264.7 cells, IL-10 shRNA was found to reduce IL-10 expression by 85 to 89%, as compared with VLPs alone. IL-10 shRNA did not cross-react with TNF-alpha mRNA or influence the expression of TNF-alpha. In BALB/c mice IL-10 shRNA could reduce 95% of IL-10 secretion. Surprisingly, it also down regulated TNF-alpha expression.

Conclusions: We show for the first time that JCV VLPs empty capsids are competent vectors to deliver RNAi and are nontoxic to cells, suggesting that JCV VLPs is an efficient agent to deliver RNAi in both murine macrophage cells and BALB/c mice. This system provides an efficient means for delivering the RNAi for gene therapy purposes.

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Purification of JCV virus-like particles (VLPs). Recombinant JCV VLPs protein in yeast cells was purified and identified by SDS-PAGE (A) and Western blot (B). Lane M: molecular weight marker; Lane VLPs: JCV virus-like particles. The rabbit antibody reactive to the JC virus-like particle is indicated by an arrow. (C) RAW 264.7 cells were pseudo-infected with VLPs packaged with pEGFP-N3 (II, III) and were analyzed by light microscopy (II) and fluorescence microscopy (III). Light microscopy of RAW 264.7 cells with pseudo-infected with VLPs as a control (I).
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Figure 1: Purification of JCV virus-like particles (VLPs). Recombinant JCV VLPs protein in yeast cells was purified and identified by SDS-PAGE (A) and Western blot (B). Lane M: molecular weight marker; Lane VLPs: JCV virus-like particles. The rabbit antibody reactive to the JC virus-like particle is indicated by an arrow. (C) RAW 264.7 cells were pseudo-infected with VLPs packaged with pEGFP-N3 (II, III) and were analyzed by light microscopy (II) and fluorescence microscopy (III). Light microscopy of RAW 264.7 cells with pseudo-infected with VLPs as a control (I).

Mentions: Recombinant JCV VLPs protein in yeast cells was purified and identified by SDS-PAGE (Fig. 1A) and Western blot (Fig. 1B) using the rabbit antibody to JCV VLPs. The JCV VLPs showed strong hemagglutination activity and the activity was completely inhibited by JCV-positive human serum. As the control, RAW 264.7 cells were pseudoinfected with VLPs (Fig. 1C (I)), VLPs packaged with pEGFP-N3 were analyzed by light microscopy (Fig. 1C (II)) and fluorescence microscopy (Fig. 1C (III)). All RAW 264.7 cells transfected with VLPs expressed green fluorescence (Fig. 1C (III)).


In vitro and in vivo targeted delivery of IL-10 interfering RNA by JC virus-like particles.

Chou MI, Hsieh YF, Wang M, Chang JT, Chang D, Zouali M, Tsay GJ - J. Biomed. Sci. (2010)

Purification of JCV virus-like particles (VLPs). Recombinant JCV VLPs protein in yeast cells was purified and identified by SDS-PAGE (A) and Western blot (B). Lane M: molecular weight marker; Lane VLPs: JCV virus-like particles. The rabbit antibody reactive to the JC virus-like particle is indicated by an arrow. (C) RAW 264.7 cells were pseudo-infected with VLPs packaged with pEGFP-N3 (II, III) and were analyzed by light microscopy (II) and fluorescence microscopy (III). Light microscopy of RAW 264.7 cells with pseudo-infected with VLPs as a control (I).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902427&req=5

Figure 1: Purification of JCV virus-like particles (VLPs). Recombinant JCV VLPs protein in yeast cells was purified and identified by SDS-PAGE (A) and Western blot (B). Lane M: molecular weight marker; Lane VLPs: JCV virus-like particles. The rabbit antibody reactive to the JC virus-like particle is indicated by an arrow. (C) RAW 264.7 cells were pseudo-infected with VLPs packaged with pEGFP-N3 (II, III) and were analyzed by light microscopy (II) and fluorescence microscopy (III). Light microscopy of RAW 264.7 cells with pseudo-infected with VLPs as a control (I).
Mentions: Recombinant JCV VLPs protein in yeast cells was purified and identified by SDS-PAGE (Fig. 1A) and Western blot (Fig. 1B) using the rabbit antibody to JCV VLPs. The JCV VLPs showed strong hemagglutination activity and the activity was completely inhibited by JCV-positive human serum. As the control, RAW 264.7 cells were pseudoinfected with VLPs (Fig. 1C (I)), VLPs packaged with pEGFP-N3 were analyzed by light microscopy (Fig. 1C (II)) and fluorescence microscopy (Fig. 1C (III)). All RAW 264.7 cells transfected with VLPs expressed green fluorescence (Fig. 1C (III)).

Bottom Line: In RAW 264.7 cells, IL-10 shRNA was found to reduce IL-10 expression by 85 to 89%, as compared with VLPs alone.IL-10 shRNA did not cross-react with TNF-alpha mRNA or influence the expression of TNF-alpha.In BALB/c mice IL-10 shRNA could reduce 95% of IL-10 secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan.

ABSTRACT

Background: RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Delivering sequences of RNAi in vivo remains a problem. The aim of this study was to use JC virus (JCV) virus-like particles (VLPs) as a vector for delivering RNAi in silencing the cytokine gene of IL-10.

Methods: JCV VLPs were generated by recombinant JCV VP1 protein in yeast expression system. DNA fragment containing IL-10 shRNA was packaged into VLPs by osmotic shock.

Results: In RAW 264.7 cells, IL-10 shRNA was found to reduce IL-10 expression by 85 to 89%, as compared with VLPs alone. IL-10 shRNA did not cross-react with TNF-alpha mRNA or influence the expression of TNF-alpha. In BALB/c mice IL-10 shRNA could reduce 95% of IL-10 secretion. Surprisingly, it also down regulated TNF-alpha expression.

Conclusions: We show for the first time that JCV VLPs empty capsids are competent vectors to deliver RNAi and are nontoxic to cells, suggesting that JCV VLPs is an efficient agent to deliver RNAi in both murine macrophage cells and BALB/c mice. This system provides an efficient means for delivering the RNAi for gene therapy purposes.

Show MeSH
Related in: MedlinePlus