Journal
METHODS
Volume 28, Issue 2, Pages 158-167Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S1046-2023(02)00220-7
Keywords
adeno-associated virus; gene therapy; chromatography; purification; serotype
Funding
- NHLBI NIH HHS [HL51811, HL59412] Funding Source: Medline
- NIDDK NIH HHS [DK58327] Funding Source: Medline
- NINDS NIH HHS [NS36302] Funding Source: Medline
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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 (scrota pes 1, 3, 4, 5 and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, e 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, AAV2rep/AAV1cap and AAV2rep/AAV5cap 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 X 10(12) to 1 X 10(13) vector genomes/ml. (C) 2002 Elsevier Science (USA). All rights reserved.
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