Development of a rapid, robust, and universal PicoGreen-based method to titer adeno-associated vectors

Jose Piedra, Maria Ontiveros, Susana Miravet, Cristina Penalva, Mercè Monfar, Miguel Chillon

Research output: Contribution to journalArticleResearchpeer-review

27 Citations (Scopus)

Abstract

© Mary Ann Liebert, Inc. 2015. 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 3×1010viral genome/ml up to 2.4×1013viral 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.
Original languageEnglish
Pages (from-to)35-42
JournalHuman gene therapy methods
Volume26
Issue number1
DOIs
Publication statusPublished - 1 Jan 2015

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