Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences

Abstract

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...