title: A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard.
abstract: Recombinant adeno-associated virus (rAAV) vectors are promising tools in gene therapy, but accurate quantification of the vector dose remains a critical issue for their successful application. We therefore aimed at the precise determination of the titer of self-complementary AAV (scAAV) vectors to improve the reliability of RNA interference (RNAi)-mediated knockdown approaches. Vector titers were initially determined by quantitative polymerase chain reaction (qPCR) using four primer sets targeting different regions within the AAV vector genome (VG) and either coiled or linearized plasmid standards. Despite very low variability between replicates in each assay, these quantification experiments revealed up to 20-fold variation in vector titers. Therefore, we developed a novel approach for the reproducible determination of titers of scAAV vectors based on the use of purified genomic vector DNA as a standard (scAAVStd). Consistent results were obtained in qPCR assays using the four primer sets mentioned above. RNAi-mediated silencing of human cyclophilin B (hCycB) by short hairpin RNA-expressing scAAV vectors was investigated in HeLa cells using two independent vector preparations. We found that the required vector titers for efficient knockdown differed by a factor of 3.5 between both preparations. Hence, we also investigated the number of internalized scAAV vectors, termed transduction units (TUs). TUs were determined by qPCR applying the scAAVStd. Very similar values for 80% hCycB knockdown were obtained for the two AAV vector preparations. Thus, only the determination of TUs, rather than vector concentration, allows for reproducible results in functional analyses using AAV vectors.
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin , Berlin 13355, Germany .
authors.forename: Anke
authors.initials: A
authors.lastname: Wagner
authors.forename: Viola
authors.initials: V
authors.lastname: Röhrs
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Kedzierski
authors.forename: Henry
authors.initials: H
authors.lastname: Fechner
authors.forename: Jens
authors.initials: J
authors.lastname: Kurreck
mesh_terms: D021983:Cyclophilins / Q000235:genetics; D004279:DNA, Viral / Q000592:standards*; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards
chemical_list: D004279:DNA, Viral; C081786:cyclophilin B; D021983:Cyclophilins