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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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Effects of VLPs IL-10 shRNA on morphology and viability of RAW 264.7 cells. (A) Two preparations of IL-10 shRNA (IL-10i-1 and IL-10i-2) were packaged into JCV VLPs by osmotic shock. The presence of IL-10 shRNA template in VLPs was confirmed by PCR. Lane 1: VLPs; Lane 2: JCV VLPs with IL-10i-1 shRNA template; Lane 3: VLPs; Lane 4: VLPs with IL-10i-2 shRNA template. (B) RAW 264.7 cells were pseudo-infected with VLPs packaged with IL-10 shRNA and the effects of cell apoptosis were determined by 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. (C) Cell viability was determined by trypan blue staining. VLPs IL-10 shRNA, VLPs only, or shRNA only did not induce cell death.
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Figure 2: Effects of VLPs IL-10 shRNA on morphology and viability of RAW 264.7 cells. (A) Two preparations of IL-10 shRNA (IL-10i-1 and IL-10i-2) were packaged into JCV VLPs by osmotic shock. The presence of IL-10 shRNA template in VLPs was confirmed by PCR. Lane 1: VLPs; Lane 2: JCV VLPs with IL-10i-1 shRNA template; Lane 3: VLPs; Lane 4: VLPs with IL-10i-2 shRNA template. (B) RAW 264.7 cells were pseudo-infected with VLPs packaged with IL-10 shRNA and the effects of cell apoptosis were determined by 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. (C) Cell viability was determined by trypan blue staining. VLPs IL-10 shRNA, VLPs only, or shRNA only did not induce cell death.

Mentions: Two preparations of IL-10 shRNA (IL-10i-1 and IL-10i-2) were packaged into JCV VLPs by osmotic shock and were confirmed by PCR (Fig. 2A). The morphology of RAW 264.7 cells with pseudoinfection of VLPs-IL-10 shRNA was shown in Fig. 2B. The cells with the pseudoinfection of VLPs were stained with DAPI (Fig. 2B) and the cell viability was detected by trypan blue staining (Fig. 2C). Figure 3 shows suppression of IL-10 expression in RAW 264.7 cells. Both IL-10i-1 and IL-10i-2 could reduce IL-10 expression about 85% and 89%, respectively, by real-time PCR compared to VLPs only (Fig. 3A) (p < 0.005). The expression of TNF-α was not affected by IL-10 shRNA treatment (Fig. 3B). The suppression of IL-10 by IL-10 shRNA was dose-dependent (Fig. 3C). Figure 3(D) shows the suppression of IL-10 by IL-10 shRNA after LPS stimulation in RAW 264.7 by real-time PCR. The irrelevant shRNA did not suppress the production of IL-10. The IL-10 shRNA packaged in JCV VLPs could sufficiently suppress IL-10 expression in RAW 264.7 cells. We also found that the expression of TNF-α was not affected by IL-10 shRNA after LPS treatment in RAW264.7 cells by ELISA (Fig. 3E). Figure 3F shows reduction of IL-10 by IL-10 shRNA after LPS treatment in RAW 264.7 cells at 36 and 48 hours by ELISA. In addition, the IL-10 suppressive effects could be sustained for 48 h by IL-10 shRNA. Thus, we found suppression of IL-10 by VLPs-IL-10 shRNA not only at mRNA levels but also at protein level.


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)

Effects of VLPs IL-10 shRNA on morphology and viability of RAW 264.7 cells. (A) Two preparations of IL-10 shRNA (IL-10i-1 and IL-10i-2) were packaged into JCV VLPs by osmotic shock. The presence of IL-10 shRNA template in VLPs was confirmed by PCR. Lane 1: VLPs; Lane 2: JCV VLPs with IL-10i-1 shRNA template; Lane 3: VLPs; Lane 4: VLPs with IL-10i-2 shRNA template. (B) RAW 264.7 cells were pseudo-infected with VLPs packaged with IL-10 shRNA and the effects of cell apoptosis were determined by 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. (C) Cell viability was determined by trypan blue staining. VLPs IL-10 shRNA, VLPs only, or shRNA only did not induce cell death.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902427&req=5

Figure 2: Effects of VLPs IL-10 shRNA on morphology and viability of RAW 264.7 cells. (A) Two preparations of IL-10 shRNA (IL-10i-1 and IL-10i-2) were packaged into JCV VLPs by osmotic shock. The presence of IL-10 shRNA template in VLPs was confirmed by PCR. Lane 1: VLPs; Lane 2: JCV VLPs with IL-10i-1 shRNA template; Lane 3: VLPs; Lane 4: VLPs with IL-10i-2 shRNA template. (B) RAW 264.7 cells were pseudo-infected with VLPs packaged with IL-10 shRNA and the effects of cell apoptosis were determined by 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. (C) Cell viability was determined by trypan blue staining. VLPs IL-10 shRNA, VLPs only, or shRNA only did not induce cell death.
Mentions: Two preparations of IL-10 shRNA (IL-10i-1 and IL-10i-2) were packaged into JCV VLPs by osmotic shock and were confirmed by PCR (Fig. 2A). The morphology of RAW 264.7 cells with pseudoinfection of VLPs-IL-10 shRNA was shown in Fig. 2B. The cells with the pseudoinfection of VLPs were stained with DAPI (Fig. 2B) and the cell viability was detected by trypan blue staining (Fig. 2C). Figure 3 shows suppression of IL-10 expression in RAW 264.7 cells. Both IL-10i-1 and IL-10i-2 could reduce IL-10 expression about 85% and 89%, respectively, by real-time PCR compared to VLPs only (Fig. 3A) (p < 0.005). The expression of TNF-α was not affected by IL-10 shRNA treatment (Fig. 3B). The suppression of IL-10 by IL-10 shRNA was dose-dependent (Fig. 3C). Figure 3(D) shows the suppression of IL-10 by IL-10 shRNA after LPS stimulation in RAW 264.7 by real-time PCR. The irrelevant shRNA did not suppress the production of IL-10. The IL-10 shRNA packaged in JCV VLPs could sufficiently suppress IL-10 expression in RAW 264.7 cells. We also found that the expression of TNF-α was not affected by IL-10 shRNA after LPS treatment in RAW264.7 cells by ELISA (Fig. 3E). Figure 3F shows reduction of IL-10 by IL-10 shRNA after LPS treatment in RAW 264.7 cells at 36 and 48 hours by ELISA. In addition, the IL-10 suppressive effects could be sustained for 48 h by IL-10 shRNA. Thus, we found suppression of IL-10 by VLPs-IL-10 shRNA not only at mRNA levels but also at protein level.

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