Abstract
BackgroundRNA 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.MethodsJCV 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.ResultsIn 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.ConclusionsWe 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.
Highlights
RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally
The JC virion contains three capsid pro- In this study, we show that JC virus (JCV) virus-like particles (VLPs) can be used as a teins (VP1, VP2 and VP3) and a viral mini chromosome. gene delivery vector for IL-10 RNAi and for the possibil
The JCV VLPs showed strong hemagglutination activity and the activity was completely inhibited by JCV-positive human serum
Summary
Purification of JCV virus-like particles (VLPs) 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. Suppression of IL-10 expression by IL-10 shRNA in RAW 264.7 cells 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). 10 shRNA packaged in JCV VLPs could sufficiently suppress IL-10 expression in RAW 264.7 cells. We 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). Effects of IL-10 shRNA on cytokines production of IL-10 and TNF-α in BALB/c mice After LPS treatment in mice, IL-10 was increased and the peak concentration of IL-10 was at 6 h decreased at Figure 2 Effects of VLPs IL-10 shRNA on morphology and viability of RAW 264.7 cells. The mice treated with LPS-VLPs-IL-10 shRNA had longer lifespan than those treated with LPS-VLPs irrelevant shRNA or LPS. (Fig. 5)
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