In vitro adenovirus mediated gene transfer to the human cornea
Background/aims: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. Methods: Adenoviral vectors (AdV) encod...
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| Veröffentlicht in: | British journal of ophthalmology 2005-06, Vol.89 (6), p.658-661 |
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| creator | Jessup, C F Brereton, H M Coster, D J Williams, K A |
| description | Background/aims: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. Methods: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72–96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. Results: Transduction of human corneas with high doses (5×107–3×108 pfu) of AdV caused eGFP expression in 12–100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2×109 pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm2 (compared to 2114 (716) nuclei/mm2 for all other groups). Transgenic protein production peaked at 2.4 (0.9) μg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) μg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm2. Conclusion: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro. |
| doi_str_mv | 10.1136/bjo.2004.061754 |
| format | Article |
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The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. Methods: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72–96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. Results: Transduction of human corneas with high doses (5×107–3×108 pfu) of AdV caused eGFP expression in 12–100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2×109 pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm2 (compared to 2114 (716) nuclei/mm2 for all other groups). Transgenic protein production peaked at 2.4 (0.9) μg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) μg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm2. Conclusion: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.</description><identifier>ISSN: 0007-1161</identifier><identifier>EISSN: 1468-2079</identifier><identifier>DOI: 10.1136/bjo.2004.061754</identifier><identifier>PMID: 15923495</identifier><identifier>CODEN: BJOPAL</identifier><language>eng</language><publisher>BMA House, Tavistock Square, London, WC1H 9JR: BMJ Publishing Group Ltd</publisher><subject>Adenoviridae - genetics ; Adenovirus ; adenovirus based vector ; AdGFP ; AdV ; Aged ; Aged, 80 and over ; Biological and medical sciences ; Clinical Science - Scientific Reports ; Cold Temperature ; cornea ; E3 deleted adenovirus serotype 5 encoding GFP ; Efficiency ; eGFP ; Endothelium ; Endothelium, Corneal - metabolism ; Endothelium, Corneal - virology ; enhanced green fluorescent protein ; Experimental and animal immunopathology. Animal models ; FCS ; fetal calf serum ; Flow cytometry ; Gene Expression ; Gene therapy ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy - methods ; Genetic Vectors - administration & dosage ; Green Fluorescent Proteins - biosynthesis ; Green Fluorescent Proteins - genetics ; Humans ; Immunoglobulin Variable Region - biosynthesis ; Immunoglobulin Variable Region - genetics ; Immunopathology ; Medical sciences ; Microscopy, Fluorescence ; Middle Aged ; MOI ; multiplicity of infection ; Organ Culture Techniques ; PBS ; pfu ; phosphate buffered saline ; plaque forming units ; Proteins ; replication deficient E1 ; reporter gene ; Rodents ; scFv ; single chain antibody fragment ; Transduction, Genetic ; Transgenes ; Vectors (Biology)</subject><ispartof>British journal of ophthalmology, 2005-06, Vol.89 (6), p.658-661</ispartof><rights>Copyright 2005 British Journal of Ophthalmology</rights><rights>2005 INIST-CNRS</rights><rights>Copyright: 2005 Copyright 2005 British Journal of Ophthalmology</rights><rights>Copyright © Copyright 2005 British Journal of Ophthalmology 2005</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-b553t-15039fcfbdbc27871205f395b467712a4f389571462a04a5d042bd4397b1da7a3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1772682/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1772682/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27903,27904,53770,53772</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=16847713$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15923495$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jessup, C F</creatorcontrib><creatorcontrib>Brereton, H M</creatorcontrib><creatorcontrib>Coster, D J</creatorcontrib><creatorcontrib>Williams, K A</creatorcontrib><title>In vitro adenovirus mediated gene transfer to the human cornea</title><title>British journal of ophthalmology</title><addtitle>Br J Ophthalmol</addtitle><description>Background/aims: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. Methods: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72–96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. Results: Transduction of human corneas with high doses (5×107–3×108 pfu) of AdV caused eGFP expression in 12–100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2×109 pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm2 (compared to 2114 (716) nuclei/mm2 for all other groups). Transgenic protein production peaked at 2.4 (0.9) μg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) μg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm2. Conclusion: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.</description><subject>Adenoviridae - genetics</subject><subject>Adenovirus</subject><subject>adenovirus based vector</subject><subject>AdGFP</subject><subject>AdV</subject><subject>Aged</subject><subject>Aged, 80 and over</subject><subject>Biological and medical sciences</subject><subject>Clinical Science - Scientific Reports</subject><subject>Cold Temperature</subject><subject>cornea</subject><subject>E3 deleted adenovirus serotype 5 encoding GFP</subject><subject>Efficiency</subject><subject>eGFP</subject><subject>Endothelium</subject><subject>Endothelium, Corneal - metabolism</subject><subject>Endothelium, Corneal - virology</subject><subject>enhanced green fluorescent protein</subject><subject>Experimental and animal immunopathology. Animal models</subject><subject>FCS</subject><subject>fetal calf serum</subject><subject>Flow cytometry</subject><subject>Gene Expression</subject><subject>Gene therapy</subject><subject>Gene Transfer Techniques</subject><subject>Genes, Reporter</subject><subject>Genetic Therapy - methods</subject><subject>Genetic Vectors - administration & dosage</subject><subject>Green Fluorescent Proteins - biosynthesis</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Humans</subject><subject>Immunoglobulin Variable Region - biosynthesis</subject><subject>Immunoglobulin Variable Region - genetics</subject><subject>Immunopathology</subject><subject>Medical sciences</subject><subject>Microscopy, Fluorescence</subject><subject>Middle Aged</subject><subject>MOI</subject><subject>multiplicity of infection</subject><subject>Organ Culture Techniques</subject><subject>PBS</subject><subject>pfu</subject><subject>phosphate buffered saline</subject><subject>plaque forming units</subject><subject>Proteins</subject><subject>replication deficient E1</subject><subject>reporter gene</subject><subject>Rodents</subject><subject>scFv</subject><subject>single chain antibody fragment</subject><subject>Transduction, Genetic</subject><subject>Transgenes</subject><subject>Vectors (Biology)</subject><issn>0007-1161</issn><issn>1468-2079</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><recordid>eNqFkU1v1DAQhi0EotvCmRuyhNpDpWz97fhSia6grVrBBbhaTuJ0syR2sZNV-ffMKqsWuPRkjfx4_My8CL2jZEkpV2fVJi4ZIWJJFNVSvEALKlRZMKLNS7QghOiCUkUP0GHOGygZYK_RAZWGcWHkAp1fB7ztxhSxa3yI2y5NGQ--6dzoG3zng8djciG3PuEx4nHt8XoaXMB1TMG7N-hV6_rs3-7PI_T986dvq6vi9uvl9erjbVFJyceCSsJNW7dVU9VMl5oyIltuZCWUhsKJlpdGanBnjggnGyJY1QhudEUbpx0_Qudz3_upArvaB7Dq7X3qBpd-2-g6--9N6Nb2Lm4t1ZqpkkGDk32DFH9NPo926HLt-94FH6dsFVgpwuSzIDXAackB_PAfuIlTCrCF3aeloYopA9TZTNUp5px8--hMid1FaCFCu4vQzhHCi_d_j_rE7zMD4HgPuFy7voV46i4_caoUsNSdYDFzXR79w-O9Sz9hWq6l_fJjZS9upL66UDe2BP505qth86zlH77-vtQ</recordid><startdate>20050601</startdate><enddate>20050601</enddate><creator>Jessup, C F</creator><creator>Brereton, H M</creator><creator>Coster, D J</creator><creator>Williams, K A</creator><general>BMJ Publishing Group Ltd</general><general>BMJ</general><general>BMJ Publishing Group LTD</general><general>Copyright 2005 British Journal of Ophthalmology</general><scope>BSCLL</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>BENPR</scope><scope>BTHHO</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20050601</creationdate><title>In vitro adenovirus mediated gene transfer to the human cornea</title><author>Jessup, C F ; Brereton, H M ; Coster, D J ; Williams, K A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-b553t-15039fcfbdbc27871205f395b467712a4f389571462a04a5d042bd4397b1da7a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Adenoviridae - genetics</topic><topic>Adenovirus</topic><topic>adenovirus based vector</topic><topic>AdGFP</topic><topic>AdV</topic><topic>Aged</topic><topic>Aged, 80 and over</topic><topic>Biological and medical sciences</topic><topic>Clinical Science - Scientific Reports</topic><topic>Cold Temperature</topic><topic>cornea</topic><topic>E3 deleted adenovirus serotype 5 encoding GFP</topic><topic>Efficiency</topic><topic>eGFP</topic><topic>Endothelium</topic><topic>Endothelium, Corneal - metabolism</topic><topic>Endothelium, Corneal - virology</topic><topic>enhanced green fluorescent protein</topic><topic>Experimental and animal immunopathology. Animal models</topic><topic>FCS</topic><topic>fetal calf serum</topic><topic>Flow cytometry</topic><topic>Gene Expression</topic><topic>Gene therapy</topic><topic>Gene Transfer Techniques</topic><topic>Genes, Reporter</topic><topic>Genetic Therapy - methods</topic><topic>Genetic Vectors - administration & dosage</topic><topic>Green Fluorescent Proteins - biosynthesis</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Humans</topic><topic>Immunoglobulin Variable Region - biosynthesis</topic><topic>Immunoglobulin Variable Region - genetics</topic><topic>Immunopathology</topic><topic>Medical sciences</topic><topic>Microscopy, Fluorescence</topic><topic>Middle Aged</topic><topic>MOI</topic><topic>multiplicity of infection</topic><topic>Organ Culture Techniques</topic><topic>PBS</topic><topic>pfu</topic><topic>phosphate buffered saline</topic><topic>plaque forming units</topic><topic>Proteins</topic><topic>replication deficient E1</topic><topic>reporter gene</topic><topic>Rodents</topic><topic>scFv</topic><topic>single chain antibody fragment</topic><topic>Transduction, Genetic</topic><topic>Transgenes</topic><topic>Vectors (Biology)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jessup, C F</creatorcontrib><creatorcontrib>Brereton, H M</creatorcontrib><creatorcontrib>Coster, D J</creatorcontrib><creatorcontrib>Williams, K A</creatorcontrib><collection>Istex</collection><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>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central</collection><collection>BMJ Journals</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>British journal of ophthalmology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jessup, C F</au><au>Brereton, H M</au><au>Coster, D J</au><au>Williams, K A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>In vitro adenovirus mediated gene transfer to the human cornea</atitle><jtitle>British journal of ophthalmology</jtitle><addtitle>Br J Ophthalmol</addtitle><date>2005-06-01</date><risdate>2005</risdate><volume>89</volume><issue>6</issue><spage>658</spage><epage>661</epage><pages>658-661</pages><issn>0007-1161</issn><eissn>1468-2079</eissn><coden>BJOPAL</coden><abstract>Background/aims: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. Methods: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72–96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. Results: Transduction of human corneas with high doses (5×107–3×108 pfu) of AdV caused eGFP expression in 12–100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2×109 pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm2 (compared to 2114 (716) nuclei/mm2 for all other groups). Transgenic protein production peaked at 2.4 (0.9) μg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) μg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm2. Conclusion: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.</abstract><cop>BMA House, Tavistock Square, London, WC1H 9JR</cop><pub>BMJ Publishing Group Ltd</pub><pmid>15923495</pmid><doi>10.1136/bjo.2004.061754</doi><tpages>4</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Adenoviridae - genetics Adenovirus adenovirus based vector AdGFP AdV Aged Aged, 80 and over Biological and medical sciences Clinical Science - Scientific Reports Cold Temperature cornea E3 deleted adenovirus serotype 5 encoding GFP Efficiency eGFP Endothelium Endothelium, Corneal - metabolism Endothelium, Corneal - virology enhanced green fluorescent protein Experimental and animal immunopathology. Animal models FCS fetal calf serum Flow cytometry Gene Expression Gene therapy Gene Transfer Techniques Genes, Reporter Genetic Therapy - methods Genetic Vectors - administration & dosage Green Fluorescent Proteins - biosynthesis Green Fluorescent Proteins - genetics Humans Immunoglobulin Variable Region - biosynthesis Immunoglobulin Variable Region - genetics Immunopathology Medical sciences Microscopy, Fluorescence Middle Aged MOI multiplicity of infection Organ Culture Techniques PBS pfu phosphate buffered saline plaque forming units Proteins replication deficient E1 reporter gene Rodents scFv single chain antibody fragment Transduction, Genetic Transgenes Vectors (Biology) |
| title | In vitro adenovirus mediated gene transfer to the human cornea |
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