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Daniel M. Lipinski, Peter Charbel Issa, Mandeep S. Singh, Antonio Trabalza, Stuart M. Elison, Nicholas D. Mazarakis, Robert E. MacLaren; Gene Transfer Into Corneal, Trabecular Meshwork And Retinal Cells Using Pseudotyped Lentiviral Vectors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1889.
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The use of adeno-associated virus for therapeutic gene delivery into dividing cells and for the delivery of large genes is limited. Lentivirus vectors provide a feasible alternative as they typically integrate into the host genome and have a larger coding capacity. Furthermore, lentivirus vectors may be pseudotyped through substitution of heterologous surface glycoproteins in order to alter cellular tropism. Herein, the ocular tropism of a novel lentivirus pseudotype, derived from Venezuelan equine encephalitis virus (VEEV), was explored to determine its utility for gene delivery in the eye.
HIV-1 lentiviral vectors expressing enhanced green fluorescent protein (eGFP) were pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or VEEV-G (strain 3908) and concentrated 2000-fold. 1μl of vector (max titre 2.63x10^9 TU293T/ml) was administered via subretinal, intravitreal or intracameral injections in 5-week-old C57BL/6 mice (n=5 per group). In vivo fluorescence imaging of the fundus or anterior chamber was performed by confocal scanning laser ophthalmoscopy (cSLO) on days 1, 2, 7, 14 and 21 post-injection to assess transgene expression. Eyes were removed post mortem for immunohistochemistry (IHC) to determine cellular tropism.
cSLO imaging one day post subretinal injection of VSV-G and VEEV-G revealed retinal pigment epithelium (RPE) transduction, which was confirmed by IHC. RPE65 expression was reduced in regions of RPE transduction compared to neighbouring areas. cSLO imaging following intracameral VSV-G injection revealed transduction of cells in the central and far peripheral cornea. Histological localization of eGFP showed transduction of endothelial cells in the central cornea and of trabecular meshwork cells in the iridocorneal angle. Intracameral VEEV-G injection resulted in greater transduction of stromal keratocytes. Intravitreal VSV-G and VEEV-G delivery resulted in RPE transduction only at the site of injection.
Efficient gene delivery to the RPE, cornea and trabecular meshwork implicates lentiviral vectors as potentially useful tools for the treatment of ocular disorders such as glaucoma and corneal dystrophies. As integrating vectors they may be particularly useful for the transduction of corneal endothelium, and for the expression of neuroprotective factors in dividing cells.
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