Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks

Abstract

Despite the clinical application of adeno-associated virus (AAV) gene therapy, the titration of viral stocks has not yet been standardized. This complicates the comparison of viral stocks between laboratories. Functional titering of AAV is time-consuming, requires the manipulation of hazardous material, and often has a high degree of variability. We established an optimized real-time quantitative polymerase chain reaction (RQ-PCR) titration assay to determine viral titers and compared it with a functional green fluorescent protein (GFP)-based titration method. With a combination of improved lysis procedures and RQ-PCR protocols we could decrease the intraexperimental coefficient of variation (CV) from 0.24 ± 0.03 to 0.042 ± 0.004 and the interexperimental CV from 0.34 ± 0.06 to 0.093 ± 0.028 following functional and RQ-PCR-based titration, respectively. This low variability conforms to even the strictest quality standards required, for example, in clinical laboratories. The highly standardized titration by RQ-PCR described here will be especially advantageous for groups working on AAV-based gene therapy in a good manufacturing practice setting.