Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment

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dc.contributor.authorLavinsky, Daniel [UNIFESP]
dc.contributor.authorChalberg, Thomas W.
dc.contributor.authorMandel, Yossi
dc.contributor.authorHuie, Philip
dc.contributor.authorDalal, Roopa
dc.contributor.authorMarmor, Michael
dc.contributor.authorPalanker, Daniel
dc.contributor.institutionStanford Univ
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionAvalanche Biotechnol Inc
dc.date.accessioned2016-01-24T14:31:18Z
dc.date.available2016-01-24T14:31:18Z
dc.date.issued2013-03-01
dc.description.abstractPURPOSE. To develop a method for modulation of transgene expression in retinal pigment epithelium (RPE) using scanning laser that spares neurosensory retina.METHODS. 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-20-dioxyuridine (BrdU) assay was performed to evaluate proliferation of RPE cells.RESULTS. RPE cells were selectively destroyed by the line scanning laser with 15 mu s 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.CONCLUSIONS. 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. (Invest Ophthalmol Vis Sci. 2013;54:1873-1880) DOI:10.1167/iovs.12-10933en
dc.description.affiliationStanford Univ, Dept Ophthalmol, Stanford, CA 94305 USA
dc.description.affiliationStanford Univ, Hansen Expt Phys Lab, Stanford, CA 94305 USA
dc.description.affiliationFed Univ São Paulo UNIFESP, São Paulo, Brazil
dc.description.affiliationAvalanche Biotechnol Inc, San Francisco, CA USA
dc.description.affiliationUnifespFed Univ São Paulo UNIFESP, São Paulo, Brazil
dc.description.sourceWeb of Science
dc.description.sponsorshipGuillingham Pan-American Fellowship
dc.description.sponsorshipPan-American Ophthalmological Foundation
dc.description.sponsorshipRetina Research Foundation
dc.format.extent1873-1880
dc.identifierhttp://dx.doi.org/10.1167/iovs.12-10933
dc.identifier.citationInvestigative Ophthalmology & Visual Science. Rockville: Assoc Research Vision Ophthalmology Inc, v. 54, n. 3, p. 1873-1880, 2013.
dc.identifier.doi10.1167/iovs.12-10933
dc.identifier.issn0146-0404
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/36008
dc.identifier.wosWOS:000316942400040
dc.language.isoeng
dc.publisherAssoc Research Vision Ophthalmology Inc
dc.relation.ispartofInvestigative Ophthalmology & Visual Science
dc.rightsinfo:eu-repo/semantics/openAccess
dc.titleModulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatmenten
dc.typeinfo:eu-repo/semantics/article
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