qPCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2 (AAV2) ITR-Sequences

Abstract
Background: Viral vectors based on adeno-associated virus serotype 2 (AAV2) constitute promising tools in human gene therapy. The inverted terminal repeats (ITRs) within the viral genome are the only cis-acting viral elements required for functional AAV2 vector generation and constitute the lowest common denominator of all AAV2-based vectors. However, so far, no PCR-based method for the detection and quantification of AAV2-ITRs could be established due to their extensive secondary hairpin structure. Current PCR-based methods are therefore predominantly targeting vector-encoded transgenes or regulatory elements. Methods: We established an AAV2-ITR sequence-specific quantitative PCR (qPCR) method. Primers and BBQ-labeled probe are located within a particular subregion of the ITR sequence and have been designed to detect wild type AAV2 and AAV2-based vectors. Results: This method is suitable for the evidence of both, single-stranded (genomic) DNA derived from AAV2 vector particles and double-stranded DNA derived from producer plasmids. The linear dynamic range could be shown for 102 to 107 copies and the detection limit determined as 50 copies. A practical approach for the analysis of putative cross reactivities against closely related AAV serotypes utilizing synthetic oligo nucleotides revealed some cross reactivity against orthologous sequences of AAV1, 3, 6 and 7 but not against AAV4, 5, 8 and 9. For AAV vector production adenovirus type 5 (Ad5) is often used in terms of its helper virus properties. Therefore, we further investigated the specificity of our qPCR method using Ad5-DNA and could prove the method to result in no detectable cross reactivity with Ad5. Conclusion: This method comprises the first qPCR system facilitating the detection and quantification of AAV2 -ITR sequences. Since this method can be applied for all AAV2-based vectors in a “one for all”-based manner, it will significantly simplify AAV2 vector genome titrations in the future.