In vivo Intra–ocular Delivery of Oligonucleotides Using Photosensitive Vectosomes

Abstract

Abstract: : Purpose: To evaluate the potential of VP22/ODN complexes (vectosomes) to deliver and enable the control release of oligonucleotides to intraocular tissues. Methods: The C–terminal half of VP22 is expressed in Escherichia coli and purified in high yields. VP22 is mixed with fluorescent phosphorothioate oligonucleotides in PBS. VP22 has the unusual property of spontaneously recruiting oligonucleotides into spherical particles forming complex particles (vectosomes). The vectosomes preparations are administered to Lewis rats by intravenous or intravitreal injections. 24 hours after the injection, controlled delivery of the complexed ODNs is carried out by transcleral illumination of the eye with a cold white light. A more localized and targeted delivery is also attempted using a laser compatible with the excitation wave–length of the fluorochrome used to label the oligonucleotides (532nm). Subsequent studies of the intraocular distribution of the oligonucleotides were carried out. After illumination, the rats are sacrificed, the eyes enucleated, snap frozen in OCT compound, cryosectioned and analysed by fluorescence microscopy. Results: The day following the intravitreous injection of vectosomes, they are detected in ganglion cells, inner and outer nuclear layers and retinal pigment epithelial (RPE) cells. A few vectosomes are also seen in the choroid. They are also observed in the ciliary body iris epithel and the cornea endothel. Without illumination, the pattern of vectosomes fluorescence is punctuate and confined to the cell cytoplasm. Illumination of the treated eyes, triggers a disruption of the vectosomes accompanied by a redistribution of the released fluorescent oligonucleotides from the cell cytoplasm to the cell nuclei. When administered intravenously, the vectosomes localize mainly within the choroid. No significant intraocular inflammatory reaction is observed even after repeated injection of vectosomes. Conclusions:Efficient delivery of therapeutic ODNs to intraocular structures can be achieved using VP22 protein. The paramount advantage of this strategy is the ability to specifically control the timing and site of ODN delivery by simple illumination of the target tissue.

Keywords: retinal pigment epithelium • gene transfer/gene therapy • choroid