Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors
Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different...
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| Veröffentlicht in: | Methods (San Diego, Calif.) Calif.), 2002-10, Vol.28 (2), p.158-167 |
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| creator | Zolotukhin, Sergei Potter, Mark Zolotukhin, Irene Sakai, Yoshihisa Loiler, Scott Fraites, Thomas J Chiodo, Vince A Phillipsberg, Tina Muzyczka, Nicholas Hauswirth, William W Flotte, Terance R Byrne, Barry J Snyder, Richard O |
| description | Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2
rep/AAV1
cap and AAV2
rep/AAV5
cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1×10
12 to 1×10
13
vector genomes/ml. |
| doi_str_mv | 10.1016/S1046-2023(02)00220-7 |
| format | Article |
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rep/AAV1
cap and AAV2
rep/AAV5
cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1×10
12 to 1×10
13
vector genomes/ml.</description><identifier>ISSN: 1046-2023</identifier><identifier>EISSN: 1095-9130</identifier><identifier>DOI: 10.1016/S1046-2023(02)00220-7</identifier><identifier>PMID: 12413414</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Adeno-associated virus ; Adenoviridae - classification ; Adenoviridae - genetics ; Adenoviridae - growth & development ; Adenoviridae Infections - virology ; Animals ; Anion Exchange Resins ; Cell Line ; Chromatography ; DNA, Recombinant - genetics ; Gene Expression Regulation, Viral ; Gene therapy ; Genetic Therapy - methods ; Genetic Vectors - genetics ; Genetic Vectors - isolation & purification ; Humans ; Male ; Plasmids - genetics ; Plasmids - isolation & purification ; Purification ; Rats ; Rats, Inbred F344 ; Sepharose ; Serotype ; Virion ; Virus Replication</subject><ispartof>Methods (San Diego, Calif.), 2002-10, Vol.28 (2), p.158-167</ispartof><rights>2002 Elsevier Science (USA)</rights><rights>Copyright 2002 Elsevier Science (USA)</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c479t-a063b1aa5d614b0ce614e14f6cae90f8a309b4fd8d7f74293162c8cda47c22e33</citedby><cites>FETCH-LOGICAL-c479t-a063b1aa5d614b0ce614e14f6cae90f8a309b4fd8d7f74293162c8cda47c22e33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1046202302002207$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65534</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12413414$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zolotukhin, Sergei</creatorcontrib><creatorcontrib>Potter, Mark</creatorcontrib><creatorcontrib>Zolotukhin, Irene</creatorcontrib><creatorcontrib>Sakai, Yoshihisa</creatorcontrib><creatorcontrib>Loiler, Scott</creatorcontrib><creatorcontrib>Fraites, Thomas J</creatorcontrib><creatorcontrib>Chiodo, Vince A</creatorcontrib><creatorcontrib>Phillipsberg, Tina</creatorcontrib><creatorcontrib>Muzyczka, Nicholas</creatorcontrib><creatorcontrib>Hauswirth, William W</creatorcontrib><creatorcontrib>Flotte, Terance R</creatorcontrib><creatorcontrib>Byrne, Barry J</creatorcontrib><creatorcontrib>Snyder, Richard O</creatorcontrib><title>Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors</title><title>Methods (San Diego, Calif.)</title><addtitle>Methods</addtitle><description>Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2
rep/AAV1
cap and AAV2
rep/AAV5
cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1×10
12 to 1×10
13
vector genomes/ml.</description><subject>Adeno-associated virus</subject><subject>Adenoviridae - classification</subject><subject>Adenoviridae - genetics</subject><subject>Adenoviridae - growth & development</subject><subject>Adenoviridae Infections - virology</subject><subject>Animals</subject><subject>Anion Exchange Resins</subject><subject>Cell Line</subject><subject>Chromatography</subject><subject>DNA, Recombinant - genetics</subject><subject>Gene Expression Regulation, Viral</subject><subject>Gene therapy</subject><subject>Genetic Therapy - methods</subject><subject>Genetic Vectors - genetics</subject><subject>Genetic Vectors - isolation & purification</subject><subject>Humans</subject><subject>Male</subject><subject>Plasmids - genetics</subject><subject>Plasmids - isolation & purification</subject><subject>Purification</subject><subject>Rats</subject><subject>Rats, Inbred F344</subject><subject>Sepharose</subject><subject>Serotype</subject><subject>Virion</subject><subject>Virus Replication</subject><issn>1046-2023</issn><issn>1095-9130</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkN9LwzAQx4Mobk7_BCVPorDqJU2b9Ulk-AsGCuqrIU2uEOmambSD_fd2W8FHn-44Pt877kPIOYMbBiy_fWcg8oQDT6-AXwNwDok8IGMGRZYULIXDbT8gI3IS4zcAMC5nx2TEuGCpYGJMvt6Ct51pnW-obixddcFVzujdwFc0YvDtZoWUTSmf7pCMBjR-WbpGNy3VFhuf6Bi9cbpFS9cu6Jqu0bQ-xFNyVOk64tlQJ-Tz8eFj_pwsXp9e5veLxAhZtImGPC2Z1pnNmSjBYF-QiSo3GguoZjqFohSVnVlZScGLlOXczIzVQhrOMU0n5HK_dxX8T4exVUsXDda1btB3UUmeC5mLrAezPWiCjzFgpVbBLXXYKAZqK1btxKqtNQVc7cQq2ecuhgNduUT7lxpM9sDdHsD-zbXDoKJx2Bi0rtfVKuvdPyd-ARvAh7Y</recordid><startdate>20021001</startdate><enddate>20021001</enddate><creator>Zolotukhin, Sergei</creator><creator>Potter, Mark</creator><creator>Zolotukhin, Irene</creator><creator>Sakai, Yoshihisa</creator><creator>Loiler, Scott</creator><creator>Fraites, Thomas J</creator><creator>Chiodo, Vince A</creator><creator>Phillipsberg, Tina</creator><creator>Muzyczka, Nicholas</creator><creator>Hauswirth, William W</creator><creator>Flotte, Terance R</creator><creator>Byrne, Barry J</creator><creator>Snyder, Richard O</creator><general>Elsevier Inc</general><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>7X8</scope></search><sort><creationdate>20021001</creationdate><title>Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors</title><author>Zolotukhin, Sergei ; 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Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2
rep/AAV1
cap and AAV2
rep/AAV5
cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1×10
12 to 1×10
13
vector genomes/ml.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>12413414</pmid><doi>10.1016/S1046-2023(02)00220-7</doi><tpages>10</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1046-2023 |
| ispartof | Methods (San Diego, Calif.), 2002-10, Vol.28 (2), p.158-167 |
| issn | 1046-2023 1095-9130 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_72647645 |
| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Adeno-associated virus Adenoviridae - classification Adenoviridae - genetics Adenoviridae - growth & development Adenoviridae Infections - virology Animals Anion Exchange Resins Cell Line Chromatography DNA, Recombinant - genetics Gene Expression Regulation, Viral Gene therapy Genetic Therapy - methods Genetic Vectors - genetics Genetic Vectors - isolation & purification Humans Male Plasmids - genetics Plasmids - isolation & purification Purification Rats Rats, Inbred F344 Sepharose Serotype Virion Virus Replication |
| title | Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors |
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