Intercellular trafficking of adenovirus-delivered HSV VP22 from the retinal pigment epithelium to the photoreceptors--implications for gene therapy.
Adenovirus (Ad)-mediated gene transfer is a promising technology for therapy of a wide variety of genetic disorders of the retina. The tropism of Ad vectors limits their utility to cells that express the coxsackie-adenovirus receptor. Upon ocular delivery, Ad vectors primarily infect the retinal pigment epithelium (RPE) and the Müller cells of the retina. However, the most frequent blinding diseases such as retinitis pigmentosa and age-related macular degeneration are associated with the expression of mutant proteins in the photoreceptors. In this study we demonstrate that adenovirus-delivered heterologous proteins fused to the herpes simplex virus tegument protein VP22 can translocate from infected cells to uninfected cells in culture and in vivo. We tested three different ocular cell lines, specifically Y79, RPE-J, and Chang C. We show that there is a 3.25-fold increase in the number of Y79 cells that take up GFP mediated by the intercellular trafficking properties of VP22. Our data are based on FACS analysis of living cells and there was no need for cell fixation for the effect to be observed. When adenovirus expressing a VP22-GFP fusion was injected into the subretinal space of adult mice, the VP22-GFP fusion peptides translocated from the RPE to all of the other layers of the retina, including the outer nuclear layer, which contains the photoreceptor cell bodies. Our study has significant implications for a wide variety of diseases of the retina and other organ systems.
Cashman
Siobhan M
SM
Department of Ophthalmology, University of Utah, Salt Lake City, Utah 84112-5330, USA.
Sadowski
Sonia L
SL
Morris
David J
DJ
Frederick
Jeanne
J
Kumar-Singh
Rajendra
R
D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D009928:Organ Specificity; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D051381:Rats; D012160:Retina / Q000378:metabolism; D018139:Simplexvirus; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism*
D008164:Luminescent Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D049452:Green Fluorescent Proteins