title: Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors.
abstract: Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen®. This method allows detection from 3×10(10) viral genome/ml up to 2.4×10(13) viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.
authors: 1 Vector Production Unit, Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona , Bellaterra 08193, Barcelona, Spain .
authors: Jose
authors: J
authors: Piedra
authors: Maria
authors: M
authors: Ontiveros
authors: Susana
authors: S
authors: Miravet
authors: Cristina
authors: C
authors: Penalva
authors: Mercè
authors: M
authors: Monfar
authors: Miguel
authors: M
authors: Chillon
mesh_terms: D000229:Dependovirus / Q000737:chemistry* / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry; D017186:Titrimetry / Q000379:methods
chemical_list: D009930:Organic Chemicals; C099476:PicoGreen