Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium
Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low tran...
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| Veröffentlicht in: | Molecular therapy 2009-12, Vol.17 (12), p.2067-2077 |
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| creator | Li, Wuping Zhang, Liqun Johnson, Jarrod S Zhijian, Wu Grieger, Joshua C Ping-Jie, Xiao Drouin, Lauren M Agbandje-McKenna, Mavis Pickles, Raymond J Samulski, R Jude |
| description | Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease. |
| doi_str_mv | 10.1038/mt.2009.155 |
| format | Article |
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However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.</description><identifier>ISSN: 1525-0016</identifier><identifier>EISSN: 1525-0024</identifier><identifier>DOI: 10.1038/mt.2009.155</identifier><identifier>PMID: 19603002</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Bacterial infections ; Blotting, Western ; Cells, Cultured ; Chlorides - metabolism ; Cilia - metabolism ; Cystic Fibrosis - genetics ; Cystic Fibrosis - therapy ; Cystic Fibrosis Transmembrane Conductance Regulator - genetics ; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism ; Dependovirus - genetics ; Efficiency ; Epithelium - metabolism ; Gene expression ; Genetic engineering ; Genetic Therapy ; Genetic Vectors - therapeutic use ; HeLa Cells ; Humans ; Infections ; Lung diseases ; Original ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Respiratory System - cytology ; Respiratory System - metabolism ; Transduction, Genetic ; Transfection</subject><ispartof>Molecular therapy, 2009-12, Vol.17 (12), p.2067-2077</ispartof><rights>2009 The American Society of Gene & Cell Therapy</rights><rights>Copyright Nature Publishing Group Dec 2009</rights><rights>Copyright 2009, The American Society of Gene & Cell Therapy 2009 The American Society of Gene & Cell Therapy</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c544t-637289f3f011e585dacc36e1760d69ec384bb581c023f37832de4fe43806eb403</citedby><cites>FETCH-LOGICAL-c544t-637289f3f011e585dacc36e1760d69ec384bb581c023f37832de4fe43806eb403</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801879/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/1792591070?pq-origsite=primo$$EHTML$$P50$$Gproquest$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793,64385,64387,64389,72469</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19603002$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Wuping</creatorcontrib><creatorcontrib>Zhang, Liqun</creatorcontrib><creatorcontrib>Johnson, Jarrod S</creatorcontrib><creatorcontrib>Zhijian, Wu</creatorcontrib><creatorcontrib>Grieger, Joshua C</creatorcontrib><creatorcontrib>Ping-Jie, Xiao</creatorcontrib><creatorcontrib>Drouin, Lauren M</creatorcontrib><creatorcontrib>Agbandje-McKenna, Mavis</creatorcontrib><creatorcontrib>Pickles, Raymond J</creatorcontrib><creatorcontrib>Samulski, R Jude</creatorcontrib><title>Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium</title><title>Molecular therapy</title><addtitle>Mol Ther</addtitle><description>Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.</description><subject>Bacterial infections</subject><subject>Blotting, Western</subject><subject>Cells, Cultured</subject><subject>Chlorides - metabolism</subject><subject>Cilia - metabolism</subject><subject>Cystic Fibrosis - genetics</subject><subject>Cystic Fibrosis - therapy</subject><subject>Cystic Fibrosis Transmembrane Conductance Regulator - genetics</subject><subject>Cystic Fibrosis Transmembrane Conductance Regulator - metabolism</subject><subject>Dependovirus - genetics</subject><subject>Efficiency</subject><subject>Epithelium - metabolism</subject><subject>Gene expression</subject><subject>Genetic engineering</subject><subject>Genetic Therapy</subject><subject>Genetic Vectors - therapeutic use</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Infections</subject><subject>Lung diseases</subject><subject>Original</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Respiratory System - cytology</subject><subject>Respiratory System - metabolism</subject><subject>Transduction, Genetic</subject><subject>Transfection</subject><issn>1525-0016</issn><issn>1525-0024</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNpt0c1r2zAYBnAzNtau3Wn3TdDDDiOZZFm2dBmENP2AskHpehWy_HpVsaVMkl3y30-uQ_bBThLo50ev_GTZO4KXBFP-uY_LHGOxJIy9yI4Jy9kC47x4ediT8ih7E8Jj2hEmytfZERElpgkdZ8MlWPAqGmeRa9FXN0KHVqt7dK-8UTYGVO_QufGgIzRoM7pueLat8-i63_rkG7S-uLtF59CZEfwORYeuhl5ZtDadUdNnK-Of1A5ttiY-JDX0p9mrVnUB3u7Xk-z7xeZufbW4-XZ5vV7dLDQrirgoaZVz0dI2DQ6Ms0ZpTUsgVYmbUoCmvKhrxonGOW1pxWneQNFCQTkuoS4wPcm-zLnboe6h0WCjV53cetMrv5NOGfn3iTUP8ocbZc4x4ZVIAR_3Ad79HCBE2ZugoeuUBTcEWVEqMKXldNXZP_LRDd6m10lSiZwJgqtJfZqV9i4ED-1hFoLl1Kbso5zalKnNpN__Of5vu68vgQ8zsCoOHg6gj1PGHMFmAek3jwa8DNqA1dA8dyobZ_579S-hgrcs</recordid><startdate>20091201</startdate><enddate>20091201</enddate><creator>Li, Wuping</creator><creator>Zhang, Liqun</creator><creator>Johnson, Jarrod S</creator><creator>Zhijian, Wu</creator><creator>Grieger, Joshua C</creator><creator>Ping-Jie, Xiao</creator><creator>Drouin, Lauren M</creator><creator>Agbandje-McKenna, Mavis</creator><creator>Pickles, Raymond J</creator><creator>Samulski, R Jude</creator><general>Elsevier Inc</general><general>Elsevier Limited</general><general>Nature Publishing Group</general><scope>6I.</scope><scope>AAFTH</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>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20091201</creationdate><title>Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium</title><author>Li, Wuping ; Zhang, Liqun ; Johnson, Jarrod S ; Zhijian, Wu ; Grieger, Joshua C ; Ping-Jie, Xiao ; Drouin, Lauren M ; Agbandje-McKenna, Mavis ; Pickles, Raymond J ; Samulski, R Jude</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c544t-637289f3f011e585dacc36e1760d69ec384bb581c023f37832de4fe43806eb403</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Bacterial infections</topic><topic>Blotting, Western</topic><topic>Cells, Cultured</topic><topic>Chlorides - metabolism</topic><topic>Cilia - metabolism</topic><topic>Cystic Fibrosis - genetics</topic><topic>Cystic Fibrosis - therapy</topic><topic>Cystic Fibrosis Transmembrane Conductance Regulator - genetics</topic><topic>Cystic Fibrosis Transmembrane Conductance Regulator - metabolism</topic><topic>Dependovirus - genetics</topic><topic>Efficiency</topic><topic>Epithelium - metabolism</topic><topic>Gene expression</topic><topic>Genetic engineering</topic><topic>Genetic Therapy</topic><topic>Genetic Vectors - therapeutic use</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>Infections</topic><topic>Lung diseases</topic><topic>Original</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Respiratory System - cytology</topic><topic>Respiratory System - metabolism</topic><topic>Transduction, Genetic</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Wuping</creatorcontrib><creatorcontrib>Zhang, Liqun</creatorcontrib><creatorcontrib>Johnson, Jarrod S</creatorcontrib><creatorcontrib>Zhijian, Wu</creatorcontrib><creatorcontrib>Grieger, Joshua C</creatorcontrib><creatorcontrib>Ping-Jie, Xiao</creatorcontrib><creatorcontrib>Drouin, Lauren M</creatorcontrib><creatorcontrib>Agbandje-McKenna, Mavis</creatorcontrib><creatorcontrib>Pickles, Raymond J</creatorcontrib><creatorcontrib>Samulski, R Jude</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</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>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</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 Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science 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>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular therapy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Wuping</au><au>Zhang, Liqun</au><au>Johnson, Jarrod S</au><au>Zhijian, Wu</au><au>Grieger, Joshua C</au><au>Ping-Jie, Xiao</au><au>Drouin, Lauren M</au><au>Agbandje-McKenna, Mavis</au><au>Pickles, Raymond J</au><au>Samulski, R Jude</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium</atitle><jtitle>Molecular therapy</jtitle><addtitle>Mol Ther</addtitle><date>2009-12-01</date><risdate>2009</risdate><volume>17</volume><issue>12</issue><spage>2067</spage><epage>2077</epage><pages>2067-2077</pages><issn>1525-0016</issn><eissn>1525-0024</eissn><abstract>Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>19603002</pmid><doi>10.1038/mt.2009.155</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bacterial infections Blotting, Western Cells, Cultured Chlorides - metabolism Cilia - metabolism Cystic Fibrosis - genetics Cystic Fibrosis - therapy Cystic Fibrosis Transmembrane Conductance Regulator - genetics Cystic Fibrosis Transmembrane Conductance Regulator - metabolism Dependovirus - genetics Efficiency Epithelium - metabolism Gene expression Genetic engineering Genetic Therapy Genetic Vectors - therapeutic use HeLa Cells Humans Infections Lung diseases Original Recombinant Proteins - genetics Recombinant Proteins - metabolism Respiratory System - cytology Respiratory System - metabolism Transduction, Genetic Transfection |
| title | Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium |
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