Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/36008 http://dx.doi.org/10.1167/iovs.12-10933 |
Resumo: | PURPOSE. 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.12-10933 |
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Lavinsky, Daniel [UNIFESP]Chalberg, Thomas W.Mandel, YossiHuie, PhilipDalal, RoopaMarmor, MichaelPalanker, DanielStanford UnivUniversidade Federal de São Paulo (UNIFESP)Avalanche Biotechnol Inc2016-01-24T14:31:18Z2016-01-24T14:31:18Z2013-03-01Investigative Ophthalmology & Visual Science. Rockville: Assoc Research Vision Ophthalmology Inc, v. 54, n. 3, p. 1873-1880, 2013.0146-0404http://repositorio.unifesp.br/handle/11600/36008http://dx.doi.org/10.1167/iovs.12-1093310.1167/iovs.12-10933WOS:000316942400040PURPOSE. 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.12-10933Guillingham Pan-American FellowshipPan-American Ophthalmological FoundationRetina Research FoundationStanford Univ, Dept Ophthalmol, Stanford, CA 94305 USAStanford Univ, Hansen Expt Phys Lab, Stanford, CA 94305 USAFed Univ São Paulo UNIFESP, São Paulo, BrazilAvalanche Biotechnol Inc, San Francisco, CA USAFed Univ São Paulo UNIFESP, São Paulo, BrazilWeb of Science1873-1880engAssoc Research Vision Ophthalmology IncInvestigative Ophthalmology & Visual ScienceModulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatmentinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP11600/360082016-01-24 12:31:18.353metadata only accessoai:repositorio.unifesp.br:11600/36008Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652016-01-24T14:31:18Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment |
title |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment |
spellingShingle |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment Lavinsky, Daniel [UNIFESP] |
title_short |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment |
title_full |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment |
title_fullStr |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment |
title_full_unstemmed |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment |
title_sort |
Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment |
author |
Lavinsky, Daniel [UNIFESP] |
author_facet |
Lavinsky, Daniel [UNIFESP] Chalberg, Thomas W. Mandel, Yossi Huie, Philip Dalal, Roopa Marmor, Michael Palanker, Daniel |
author_role |
author |
author2 |
Chalberg, Thomas W. Mandel, Yossi Huie, Philip Dalal, Roopa Marmor, Michael Palanker, Daniel |
author2_role |
author author author author author author |
dc.contributor.institution.none.fl_str_mv |
Stanford Univ Universidade Federal de São Paulo (UNIFESP) Avalanche Biotechnol Inc |
dc.contributor.author.fl_str_mv |
Lavinsky, Daniel [UNIFESP] Chalberg, Thomas W. Mandel, Yossi Huie, Philip Dalal, Roopa Marmor, Michael Palanker, Daniel |
description |
PURPOSE. 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.12-10933 |
publishDate |
2013 |
dc.date.issued.fl_str_mv |
2013-03-01 |
dc.date.accessioned.fl_str_mv |
2016-01-24T14:31:18Z |
dc.date.available.fl_str_mv |
2016-01-24T14:31:18Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Investigative Ophthalmology & Visual Science. Rockville: Assoc Research Vision Ophthalmology Inc, v. 54, n. 3, p. 1873-1880, 2013. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/36008 http://dx.doi.org/10.1167/iovs.12-10933 |
dc.identifier.issn.none.fl_str_mv |
0146-0404 |
dc.identifier.doi.none.fl_str_mv |
10.1167/iovs.12-10933 |
dc.identifier.wos.none.fl_str_mv |
WOS:000316942400040 |
identifier_str_mv |
Investigative Ophthalmology & Visual Science. Rockville: Assoc Research Vision Ophthalmology Inc, v. 54, n. 3, p. 1873-1880, 2013. 0146-0404 10.1167/iovs.12-10933 WOS:000316942400040 |
url |
http://repositorio.unifesp.br/handle/11600/36008 http://dx.doi.org/10.1167/iovs.12-10933 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.none.fl_str_mv |
Investigative Ophthalmology & Visual Science |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
1873-1880 |
dc.publisher.none.fl_str_mv |
Assoc Research Vision Ophthalmology Inc |
publisher.none.fl_str_mv |
Assoc Research Vision Ophthalmology Inc |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
|
_version_ |
1802764115855278080 |