Validation of a qPCR method for determination of viral genome titres of AAV2-based vector preparations

The viral genome titre is universally used for the dosing of adeno-associated virus (AAV)-based vectors used for gene therapy. To standardise this determination, the development of a common method would be valuable to facilitate comparison of viral doses used in the clinic and in the subsequent quality control of the products. A collaborative study was initiated by the Gene Therapy Working Group of the General European Official Medicines Control Laboratories Network in order to validate a qPCR-based method targeting the ITR2 sequence common to a broad variety of AAV vectors, independently from the serotype of the capsid or from the specific transgene. Five preparations of AAV vectors from various serotypes, including the AAV2/2 (RSS2) and AAV2/8 (RSS8) Reference Standard Stocks (American Type Culture Collection, USA) were used in the study. A plasmid carrying the ITR2 sequence was used to prepare standard curves. Its digestion outside the ITR regions facilitated melting of the hairpin ITR sequence during PCR, allowing better accessibility to the DNA polymerase. The results show that this qPCR method is satisfactory in terms of accuracy and precision. The reproducibility is also acceptable when compared with other similar studies, as it was shown previously that titres obtained by qPCR generally show higher inter-laboratory variability. The use of RSS2 or RSS8 as normalisation control in each assay demonstrated a promising help to identify potential sources of variation in a given laboratory or to smooth out inter-laboratory variations, thus improving reproducibility.