In vivo application of adenoviral vectors purified by a Taqman Real Time PCR-supported chromatographic protocol.
Recombinant adenoviral vectors encoding human HDL-cholesterol receptor SR-BI (Ad/hSR-BI) or beta-galactosidase (Ad/lacZ), respectively, were purified using a Source15 Q anion-exchange (AEX) column and quantified using two parallel Taqman Real Time PCR systems with different target sequences. Adenovirus concentrations were ascertained by 260 nm measurements, purity by 260/280 nm ratio and SDS-PAGE. Subsequently, adenoviruses were validated by experimental intravenous application into New Zealand White rabbits. Transgene expression was verified by functional assays determining plasma clearance rate of 3H-HDL-cholesterol, and was not affected by 21-months storage at -80 degrees C. No alterations of liver enzymes and C-reactive protein (CRP) upon Source15 Q adenovirus treatment could be detected, demonstrating biological safety of our protocol.
Tancevski
Ivan
I
Department of Internal Medicine, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria.
Wehinger
Andreas
A
Patsch
Josef R
JR
Ritsch
Andreas
A
D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D002852:Chromatography, Ion Exchange; D015925:Cryopreservation; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051122:Scavenger Receptors, Class B / Q000235:genetics; D014161:Transduction, Genetic
D051122:Scavenger Receptors, Class B