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 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, 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.
Zolotukhin
Sergei
S
Powell Gene Therapy Center, 1600 SW Archer Road, College of Medicine, University of Florida, 32610-0266, Gainesville, FL 32610-0266, USA.
Potter
Mark
M
Zolotukhin
Irene
I
Sakai
Yoshihisa
Y
Loiler
Scott
S
Fraites
Thomas J
TJ
Chiodo
Vince A
VA
Phillipsberg
Tina
T
Muzyczka
Nicholas
N
Hauswirth
William W
WW
Flotte
Terance R
TR
Byrne
Barry J
BJ
Snyder
Richard O
RO
D000256:Adenoviridae / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D000257:Adenoviridae Infections / Q000821:virology; D000818:Animals; D000837:Anion Exchange Resins; D002460:Cell Line; D002845:Chromatography; D004274:DNA, Recombinant / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008297:Male; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D011916:Rats, Inbred F344; D012685:Sepharose; D014771:Virion; D014779:Virus Replication
D000837:Anion Exchange Resins; D004274:DNA, Recombinant; D012685:Sepharose