TY - JOUR
T1 - Development of a rapid, robust, and universal PicoGreen-based method to titer adeno-associated vectors
AU - Piedra, Jose
AU - Ontiveros, Maria
AU - Miravet, Susana
AU - Penalva, Cristina
AU - Monfar, Mercè
AU - Chillon, Miguel
PY - 2015/1/1
Y1 - 2015/1/1
N2 - © 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.
AB - © 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.
U2 - 10.1089/hgtb.2014.120
DO - 10.1089/hgtb.2014.120
M3 - Article
VL - 26
SP - 35
EP - 42
JO - Human gene therapy methods
JF - Human gene therapy methods
SN - 1946-6536
IS - 1
ER -