title: Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences.
abstract: Viral vectors based on various naturally occurring adeno-associated virus (AAV) serotypes are among the most promising tools in human gene therapy. For the production of recombinant AAV (rAAV) vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for rAAV vector generation and depict the lowest common denominator of all AAV2-derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2 ITRs could be established because of their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we establish a molecular biological method that allows accurate and reproducible quantification of AAV2 genomes on the basis of an AAV2 ITR sequence-specific qPCR. Primers and labeled probe are located within the ITR sequence and have been designed to detect both wild-type AAV2 and AAV2-based vectors. This method is suitable for detecting single-stranded DNA derived from AAV2 vector particles and double-stranded DNA derived from vector plasmids. The limit of detection has been determined as 50 ITR sequence copies per reaction, by comparison with a plasmid standard. In conclusion, this method describes the first qPCR system facilitating the detection and quantification of AAV2 ITR sequences. Because this method can be used universally for all AAV2 genome-based vectors, it will significantly simplify rAAV2 vector titrations in the future.
authors.affiliation: Bavarian Health and Food Safety Authority, 85764 Oberschleissheim, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Aurnhammer
authors.forename: Maren
authors.initials: M
authors.lastname: Haase
authors.forename: Nadine
authors.initials: N
authors.lastname: Muether
authors.forename: Martin
authors.initials: M
authors.lastname: Hausl
authors.forename: Christina
authors.initials: C
authors.lastname: Rauschhuber
authors.forename: Ingrid
authors.initials: I
authors.lastname: Huber
authors.forename: Hans
authors.initials: H
authors.lastname: Nitschko
authors.forename: Ulrich
authors.initials: U
authors.lastname: Busch
authors.forename: Andreas
authors.initials: A
authors.lastname: Sing
authors.forename: Anja
authors.initials: A
authors.lastname: Ehrhardt
authors.forename: Armin
authors.initials: A
authors.lastname: Baiker
mesh_terms: D002213:Capsid / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020079:Terminal Repeat Sequences / Q000235:genetics*