Search

This study reports the use of erythrocyte ghosts (EG) as a biocompatible nonviral delivery system for extended circulation and prolonged expression of plasmid DNA in the blood. Murine interleukin-2-expressing plasmid DNA was efficiently loaded to EG by electroporation in hypotonic condition. The presence of plasmid DNA in EG was confirmed by fluorescence-labeled plasmid DNA. At 21 min after intravenous administration into mice, the level of plasmid DNA in the blood was 92 000-fold higher following EG-mediated delivery as compared to the injection of naked form. EG-mediated gene delivery revealed higher and more prolonged mRNA expression levels of plasmid DNA in the blood until 9 days after the single intravenous injection. Moreover, plasmid DNA-loaded EG showed gene expression targeted to the blood cells. At 3 days post-dose, substantial expression levels of plasmid DNA delivered in EG were observed only in the blood and not in the other organs. Of the blood cells, the subpopulation containing granulocytes showed higher expression of plasmid DNA than mononuclear cells. These results indicate the potential of EG as a safe, prolonged and blood-targeted delivery system of therapeutic genes.

To increase the potency of human papillomavirus (HPV) DNA vaccines, we constructed a series of HPV16 L1 vaccines genetically fused with a secretion signal and/or immune cell-recruiting RANTES. The DNA vaccines encoding secretory HPV L1 were constructed by inserting HPV L1 gene into a vector with an ER-targeting secretory signal sequence. The expression plasmid encoding secretory HPV L1 (pER/L1) was fused with cDNA of RANTES, generating pER/L1/R. For comparison, HPV L1 genes were cloned into pVAX1 vector with no signal sequence (pL1), and further linked to the N-terminus (pL1/R) or C-terminus of RANTES (pR/L1). The secretion of L1 proteins was observed in the pER/L1, pER/L1/R, and pR/L1-transfected cells, except the pL1/R-transfected group. Cytoplasmic localization of L1 protein was observed in the cells transfected with pL1/R, but not with pER/L1/R at 48 h after transfection. In mice, RANTES-fused vaccines more effectively elicited the levels of HPV16 L1-specific IgG and IgG2a antibodies than pL1. Of RANTES-fused vaccines, pER/L1/R encoding the secreted fusion protein induced the highest humoral and CD8(+) T-cell-stimulating responses. These results suggest that the immunogenicity of HPV L1 DNA vaccines could be enhanced by genetic fusion to a chemokine and secretory signal peptide sequences.

Some mutations in FOXL2 result in premature ovarian failure accompanied by blepharophimosis, ptosis, epicanthus inversus syndrome type I disease, and FOXL2-null mice exhibit developmental defects in granulosa cells. Recently, FOXL2 c.402C>G, a new somatic mutation that leads to a p.C134W change, was found in the majority of adult-type ovarian granulosa cell tumors (GCTs). In this study, we investigated the possible mechanisms by which the C134W mutation contributes to the development of GCTs. Wild-type (WT) and mutant FOXL2 displayed differential apoptotic activities. Specifically, WT FOXL2 induced significant granulosa cell death, but the mutant exhibited minimal cell death. The FOXL2-induced apoptotic response was greatly dependent on caspase 8, BID and BAK because the depletion of any of these three proteins inhibited FOXL2 from eliciting the full apoptotic response. Activation of caspase 8 and subsequent increased production of truncated BID, and oligomerization of BAK, and release of cytochrome c were all associated with the apoptosis induced by WT FOXL2 expression. In contrast, the mutant FOXL2 was unable to elicit the full array of apoptotic signaling responses. In addition, we found differential TNF-R1 (tumor necrosis factor-receptor 1) and Fas (CD95/APO-1) upregulation between the WT and the mutant, and the silencing of TNF-R1 or Fas and the blockage of the death signaling mediated by TNF-R1 or Fas using TNF-Fc or Fas-Fc, respectively, resulted in significant attenuations of FOXL2-induced apoptosis. Moreover, granulosa cells that expressed either WT FOXL2 or mutant exhibited distinct cell death sensitivities on activation of death receptors and deprivation of serum. Thus, the differential activities of FOXL2 and its mutant may partially account for the pathophysiology of GCT development.

Although polyethylenimine (PEI) has been widely used as a nonviral vector, there is little mechanistic understanding on PEI-mediated delivery. Here, we studied whether the expression of murine interleukin-2 (mIL-2) plasmids could be improved by complexation with PEI at various N/P ratios, and whether the cellular uptake, nuclear translocation, and retention of plasmids could be affected by the N/P ratios. Compared with the naked mIL-2, PEI/mIL-2 complexes showed at least two orders of magnitude higher expression at Raw264 cells in the N/P ratio-dependent manner. PEI-mediated cellular uptake and nuclear trafficking of plasmids, quantitated by competitive polymerase chain reaction, also depended on the N/P ratios showing the highest cell and nuclear levels of plasmids at 10/1. The higher cellular levels of plasmid DNA after PEI-mediated delivery were also observed in other cell lines. Unlike naked plasmids, PEI/mIL-2 complexes (N/P ratios >/=4/1) showed prolonged cellular and nuclear retention of mIL-2 plasmids. The nuclear translocation and higher cellular level of plasmids given in PEI complexes were similarly observed by fluorescence microscopy. Moreover, PEI/mIL-2 complexes revealed high stability against DNase I, partly explaining the prolonged subcellular retention. These results indicate that the expression of plasmid mIL-2 might be highly enhanced by complexation with PEI and that such increased expression could be attributed by the higher cellular uptake, nuclear translocation and prolonged retention.