Abstract
Abstract: :
Purpose: The purpose of this study is to evaluate the potential of VP22, a structural protein of the herpes simplex virus tegument, for the delivery of nucleic acids into ocular cells. Methods:The C–terminal half of VP22 was expressed in Escherichia coli and purified in high yields. VP22 was mixed with 20 mer phosphorothioate oligonucleotides for the preparation of particles. The cellular uptake of VP22/oligonucleotide complexes was evaluated in living ocular cells by confocal microscopy using fluorescently labelled oligonucleotides. The activity of an antisense oligonucleotide corresponding to the 3’–untranslated region of human c–raf kinase gene was assessed on human ocular choroids melanoma cell. We tested the effects of the VP22/anti–raf complexes on the cell proliferation, including in parallel complexes assembled with a control mismatched oligonucleotide, and also free anti–c–raf oligonucleotide as an additional control. Results:VP22 has the unusual property of spontaneously recruiting oligonucleotides into spherical particles which enter human ocular choroid melanoma cells and bovine retina pigmentary epithelium cells efficiently. The particles remained stable in the cell cytoplasm unless they were illuminated with either a cold halogen light or a laser. Remarkably, light activation induced particle disruption and release of oligonucleotides to the cell cytosol and the nuclei. We demonstrated that an overnight incubation of such complexes with ocular cells lead to a high level of cellular uptake. No effect on the cell proliferation was observed for any of the test samples in the absence of illumination. However, a significant inhibition of the cell proliferation after illumination was observed for the complexes assembled with the anti–c–raf oligonucleotide. The release of the oligonucleotide from the complexes was required for the biological function of the oligonucleotide. Conclusions: This technology has the advantage of achieving efficient and controllable site–specific delivery. It can potentially be used for precise delivery of therapeutic nucleic acids to a desired location in the eye by simple and direct illumination of the target tissues with the lasers currently used in ophthalmology.
Keywords: melanoma • microscopy: confocal/tunneling • proliferation