Long-Term, Targeted Genetic Modification of the Aqueous Humor Outflow Tract Coupled with Noninvasive Imaging of Gene Expression In Vivo

To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been sh...

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Veröffentlicht in:Investigative ophthalmology & visual science 2004-09, Vol.45 (9), p.3091-3098
Hauptverfasser: Loewen, Nils, Fautsch, Michael P, Teo, Wu-Lin, Bahler, Cindy K, Johnson, Douglas H, Poeschla, Eric M
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container_end_page 3098
container_issue 9
container_start_page 3091
container_title Investigative ophthalmology & visual science
container_volume 45
creator Loewen, Nils
Fautsch, Michael P
Teo, Wu-Lin
Bahler, Cindy K
Johnson, Douglas H
Poeschla, Eric M
description To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been shown. More generally in the field of gene therapy, the authors hypothesized that distinctive properties of the intraocular aqueous circulation could facilitate solving problems of accessibility, targeting, and scale that have hindered realization of gene therapy in other settings. A domestic cat model was developed in which long-term in vivo studies were performed. After dose-response studies in primary human TM cells, 19 cats received anterior chamber (AC) injections of stepped doses (10(6)-10(8) transduction units) of lentiviral vectors encoding different marker transgenes (beta-galactosidase, Aequorea victoria green fluorescent protein [GFP], or Renilla reniformis GFP). Animals were monitored serially for transgene expression and IOP. High-grade, stable transgene expression in the TM was achieved and monitored noninvasively over time in living animals. Extensive expression resulted after a single transcorneal injection, persisted for at least 10 months (time of death in the present studies), and was targeted to the TM. The initial IOP did not differ significantly from the IOP at the end of the study (P = 0.4). Aequorea GFP was superior to Renilla GFP. Vectors were effective enough to cause GFP-specific overexpression cytotoxicity at the highest dose, which was solved by dose reduction. High-grade transgene expression in this large-animal model persisted stably for at least 10 months after a single transcorneal lentiviral vector injection, was highly targeted, and could be monitored serially and noninvasively in living animals. These studies provide a basis for developing realistic disease models and administering glaucoma gene therapy.
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Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been shown. More generally in the field of gene therapy, the authors hypothesized that distinctive properties of the intraocular aqueous circulation could facilitate solving problems of accessibility, targeting, and scale that have hindered realization of gene therapy in other settings. A domestic cat model was developed in which long-term in vivo studies were performed. After dose-response studies in primary human TM cells, 19 cats received anterior chamber (AC) injections of stepped doses (10(6)-10(8) transduction units) of lentiviral vectors encoding different marker transgenes (beta-galactosidase, Aequorea victoria green fluorescent protein [GFP], or Renilla reniformis GFP). Animals were monitored serially for transgene expression and IOP. High-grade, stable transgene expression in the TM was achieved and monitored noninvasively over time in living animals. Extensive expression resulted after a single transcorneal injection, persisted for at least 10 months (time of death in the present studies), and was targeted to the TM. The initial IOP did not differ significantly from the IOP at the end of the study (P = 0.4). Aequorea GFP was superior to Renilla GFP. Vectors were effective enough to cause GFP-specific overexpression cytotoxicity at the highest dose, which was solved by dose reduction. High-grade transgene expression in this large-animal model persisted stably for at least 10 months after a single transcorneal lentiviral vector injection, was highly targeted, and could be monitored serially and noninvasively in living animals. 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Transplantations, organ and tissue grafts. Graft diseases</topic><topic>Surgery of the eye and orbit</topic><topic>Time Factors</topic><topic>Trabecular Meshwork - cytology</topic><topic>Trabecular Meshwork - physiology</topic><topic>Transduction, Genetic</topic><topic>Transgenes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Loewen, Nils</creatorcontrib><creatorcontrib>Fautsch, Michael P</creatorcontrib><creatorcontrib>Teo, Wu-Lin</creatorcontrib><creatorcontrib>Bahler, Cindy K</creatorcontrib><creatorcontrib>Johnson, Douglas H</creatorcontrib><creatorcontrib>Poeschla, Eric M</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Investigative ophthalmology &amp; visual science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Loewen, Nils</au><au>Fautsch, Michael P</au><au>Teo, Wu-Lin</au><au>Bahler, Cindy K</au><au>Johnson, Douglas H</au><au>Poeschla, Eric M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Long-Term, Targeted Genetic Modification of the Aqueous Humor Outflow Tract Coupled with Noninvasive Imaging of Gene Expression In Vivo</atitle><jtitle>Investigative ophthalmology &amp; visual science</jtitle><addtitle>Invest Ophthalmol Vis Sci</addtitle><date>2004-09-01</date><risdate>2004</risdate><volume>45</volume><issue>9</issue><spage>3091</spage><epage>3098</epage><pages>3091-3098</pages><issn>0146-0404</issn><issn>1552-5783</issn><eissn>1552-5783</eissn><coden>IOVSDA</coden><abstract>To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). 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identifier ISSN: 0146-0404
ispartof Investigative ophthalmology & visual science, 2004-09, Vol.45 (9), p.3091-3098
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1552-5783
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source MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals
subjects Animals
Animals, Genetically Modified
Aqueous Humor - physiology
beta-Galactosidase - genetics
Biological and medical sciences
Cats
Cells, Cultured
Gene Expression
Gene Targeting
Genetic Vectors
Green Fluorescent Proteins
Humans
Indicators and Reagents - metabolism
Intraocular Pressure
Lentivirus - genetics
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Medical sciences
Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases
Surgery of the eye and orbit
Time Factors
Trabecular Meshwork - cytology
Trabecular Meshwork - physiology
Transduction, Genetic
Transgenes
title Long-Term, Targeted Genetic Modification of the Aqueous Humor Outflow Tract Coupled with Noninvasive Imaging of Gene Expression In Vivo
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