Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products

被引：45

作者：

Werling, Natalie Jayne ^{[1
]}

Satkunanathan, Stifani ^{[1
]}

Thorpe, Robin ^{[1
]}

Zhao, Yuan ^{[1
]}

机构：

[1] NIBSC Med & Healthcare Prod Regulatory Agcy, Div Adv Therapies, Potters Bar EN6 3QG, Herts, England

来源：

HUMAN GENE THERAPY METHODS | 2015年 / 26卷 / 03期

关键词：

ION-EXCHANGE CHROMATOGRAPHY; VIRUS VECTORS; INFECTIOUS TITER; QUANTIFICATION; PURIFICATION; EXPRESSION; TITRATION; HELPER; TRANSDUCTION; GENERATION;

D O I：

10.1089/hgtb.2015.013

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Adeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge. With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target sequences and calibration DNA conformation on the qPCR quantitation of AAV products. Our results emphasize the importance of designing qPCR primers and conducting sample preparation and demonstrate the need for extensive characterization, vigorous control, and use of reference materials in clinical dose determination.

引用

页码：82 / 92

页数：11

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