Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors.
Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications.
Mohiuddin
Imran
I
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32610-0266, USA.
Loiler
Scott
S
Zolotukhin
Irina
I
Byrne
Barry J
BJ
Flotte
Terence R
TR
Snyder
Richard O
RO
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D000229:Dependovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification*; D016679:Genome, Viral; D006378:Helper Viruses / Q000235:genetics / Q000502:physiology; D006801:Humans; D051381:Rats; D012703:Serotyping; D018139:Simplexvirus / Q000235:genetics / Q000502:physiology*