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Daniel Lavinsky, Thomas W. Chalberg, Yossi Mandel, Philip Huie, Roopa Dalal, Michael Marmor, Daniel Palanker; Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment. Invest. Ophthalmol. Vis. Sci. 2013;54(3):1873-1880. doi: 10.1167/iovs.12-10933.
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© 2016 Association for Research in Vision and Ophthalmology.
To develop a method for modulation of transgene expression in retinal pigment epithelium (RPE) using scanning laser that spares neurosensory retina.
Fifteen pigmented rabbits received subretinal injection of recombinant adeno-associated virus (rAAV-2) encoding green fluorescent protein (GFP). GFP expression was measured using confocal scanning laser ophthalmoscopy (cSLO) fluorescence imaging and immunohistochemistry. To reduce the total expression in RPE by half, 50% of the transfected RPE cells were selectively destroyed by microsecond exposures to scanning laser with 50% pattern density. The selectivity of RPE destruction and its migration and proliferation were monitored using fluorescein angiography, spectral-domain optical coherence tomography (SD-OCT), and light, transmission, and scanning electron microscopy. 5-Bromo-2′-dioxyuridine (BrdU) assay was performed to evaluate proliferation of RPE cells.
RPE cells were selectively destroyed by the line scanning laser with 15 μs exposures, without damage to the photoreceptors or Bruch's membrane. RPE cells started migrating after the first day, and in 1 week there was complete restoration of RPE monolayer. Selective laser treatment decreased the GFP fluorescence by 54% as compared to control areas; this was further decreased by an additional 48% following a second treatment 1 month later. BrdU assay demonstrated proliferation in approximately half of the RPE cells in treatment areas.
Microsecond exposures produced by scanning laser destroyed RPE cells selectively, without damage to neural retina. Continuity of RPE layer is restored within days by migration and proliferation, but transgene not integrated into the nucleus is not replicated. Therefore, gene expression can be modulated in a precise manner by controlling the laser pattern density and further adjusted using repeated applications.
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