Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column.
One of the most promising gene transfer vectors in human clinical trials is AAV2. The quality of the vector preparations is a key element in obtaining reliable and reproducible data in preclinical studies. However, established protocols either result in impure, low infectious virus (CsCl2 gradient centrifugation) or demand a high level of manual and technical skills (CsCl2 gradient centrifugation, iodixanol/heparin or HPLC purification). In this study, we present an easy-to-do single-step column purification (SSCP) of AAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation. Various vector preparations generated by our method reproducibly showed high titers, infectivity, and purity. In vivo, our single-step column-purified AAV2 vectors mediate significantly higher transduction efficiency compared with conventional protocols. Investigators still unsatisfied with previously published techniques or new to the field of AAV production may find in our method an interesting alternative.
Auricchio
A
A
Wistar Institute, Philadelphia, PA 19104, USA.
Hildinger
M
M
O'Connor
E
E
Gao
G P
GP
Wilson
J M
JM
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D000229:Dependovirus / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000302:isolation & purification*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007763:Lac Operon / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000821:virology; D014161:Transduction, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D006493:Heparin; D001616:beta-Galactosidase