Molecular Therapy
Volume 6, Issue 2, August 2002, Pages 272-278
Journal home page for Molecular Therapy

Method
Development and Optimization of a Real-Time Quantitative PCR-Based Method for the Titration of AAV-2 Vector Stocks

https://doi.org/10.1006/mthe.2002.0659Get rights and content
Under a Creative Commons license
open archive

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 RQPCR-based titration, respectively. This low variability conforms to even the strictest quality standards required, for example, in clinical laboratories. The highly standardized titration by RQPCR described here will be especially advantageous for groups working on AAV-based gene therapy in a good manufacturing practice setting.

Key Words

adeno-associated virus-2 (AAV-2)
titration
TaqMan PCR
RQ-PCR

Cited by (0)