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 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.

Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors

“…Genomic titers were determined by quantitative real-time PCR (TaqMan, Applied Biosystems, Darmstadt, Germany) using CMV-specific or rep-specific primers and probe for vectors and viruses, respectively, as described previously. 49 Replicative titers of AAV virus particles were determined by dot blot analysis. 50 Detection of AAV genomes was performed with a In vitro neutralization assay with wt AAV and single mutants…”

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…Genomic titers (vg ml À1 ) were determined by quantitative real-time PCR as described earlier. 57 Comparative analysis of in vivo biodistribution of targeted and non-targeted AAV clones upon co-injection Individual NMRI mice were intravenously co-injected with a mixture of each 5 Â 10 10 vg of the targeted virus (rep-cap genome) and non-targeted vector (luciferase genome) (n ¼ 4 mice per co-injection group). One week after co-injection, organs were harvested and snap frozen in liquid nitrogen.…”

Augmentation of AAV-mediated cardiac gene transfer after systemic administration in adult rats

“…Extracted DNA (15-50 ng) was applied for QR-PCR under the conditions previously described. 47 Products were evaluated with TaqMan data analysis software (Applied Biosystems, Darmstadt, Germany).…”