Analysis of the production efficiency and titration of various recombinant adeno-associated viruses.
Recombinant adeno-associated virus type 2 (rAAV2) viral vector, a non-pathogenic human parvovirus, has recently emerged as a gene transfer vehicle for cancer gene therapy. To utilize rAAV2 properly and safely while carrying out preclinical and clinical studies, it is crucial to exactly titer the virus. We therefore compared biological infectious rAAV2 titers with physical titers of rAAV2 vectors encoding various transgenes with different sized viral genomes. Biological rAAV2 infectivity was assayed by measuring the number of virus particles able to transduce Hela cells using several detection methods, including X-gal staining and immunocytostaining. Physical titers of rAAV2 were determined using a commercially available rAAV2 particle-specific enzyme-linked immunosorbent assay. We found that total rAAV2 particle production was consistent within the limited size variations of the rAAV2 genome, regardless of the difference in transgenes. In contrast, the infectious titer of rAAV2 differed greatly, even for the same viruses, due to variation in the sensitivity of the relevant assays. Thus, the results suggest that both infectious virus titer and total virus particle should be precisely measured for rAAV2 vector utilized in each study.
Nam
Young Ran
YR
Department of Microbiology, University of Ulsan College of Medicine, Seoul, Korea.
Kim
Sung Jin
SJ
Lee
Boyoung
B
Chang
Jin Woo
JW
Joo
Chul-Hyen
CH
Kim
Yoo Kyum
YK
Lee
Heuiran
H
D004279:DNA, Viral / Q000737:chemistry*; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity*; D005822:Genetic Vectors / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D011995:Recombination, Genetic; D017186:Titrimetry; D014779:Virus Replication; D001616:beta-Galactosidase / Q000378:metabolism
D004279:DNA, Viral; D001616:beta-Galactosidase