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

Werling NJ, Satkunanathan S, Thorpe R, Zhao Y - Hum Gene Ther Methods (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Advanced Therapies, NIBSC/Medicines and Healthcare Products Regulatory Agency , Potters Bar, Hertfordshire, EN6 3QG United Kingdom .

ABSTRACT
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.

No MeSH data available.


Comparison of amplification background signals obtained from PBS control samples after treatment by method A or method B and calculated on the basis of circular [(A), (C), and (E)] or linear [(B), (D), and (F)] plasmid DNA, using ITR primers (A and B), GFP primers (C and D), or CMV primers (E and F). Dashed lines indicate the set Cq cutoff value and a valid detection value, and potentially the cross-contamination of AAV DNA sequence during sample preparation when the copy number is above the dashed lines (n=3). ns, not significantly different.
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f5: Comparison of amplification background signals obtained from PBS control samples after treatment by method A or method B and calculated on the basis of circular [(A), (C), and (E)] or linear [(B), (D), and (F)] plasmid DNA, using ITR primers (A and B), GFP primers (C and D), or CMV primers (E and F). Dashed lines indicate the set Cq cutoff value and a valid detection value, and potentially the cross-contamination of AAV DNA sequence during sample preparation when the copy number is above the dashed lines (n=3). ns, not significantly different.

Mentions: A column purification step has been added, after enzymatic DT/PK treatment, to AAV sample preparation as described by other laboratories. For the convenience of method comparison, we termed the enzymatic DT/PK treatment as method A and the combined treatment with method A and column purification as method B. Typically, method A would generate unpurified samples containing vector DNA, digested vector capsid, and cellular proteins and inactivated enzymes; in contrast, method B resulted in the preparation of purified vector DNA free from enzymes, capsid, and cellular proteins. Control PBS samples free of AAV vectors were first subjected to the whole process of sample preparation, using methods A or B, to monitor potential method influence and cross-contamination risk during sample preparation. Figure 5 shows that 66% of processed PBS control samples showed copy numbers greater than the set Cq cutoff value (dashed lines), indicating a valid detection value and potentially the cross-contamination of AAV DNA sequence during sample preparation. In particular, control samples prepared by column-based method B showed significantly higher amplification background compared with enzymatic treatment alone (method A) (p<0.05), reflecting an increased likelihood of cross-contamination from multistep processing.


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

Werling NJ, Satkunanathan S, Thorpe R, Zhao Y - Hum Gene Ther Methods (2015)

Comparison of amplification background signals obtained from PBS control samples after treatment by method A or method B and calculated on the basis of circular [(A), (C), and (E)] or linear [(B), (D), and (F)] plasmid DNA, using ITR primers (A and B), GFP primers (C and D), or CMV primers (E and F). Dashed lines indicate the set Cq cutoff value and a valid detection value, and potentially the cross-contamination of AAV DNA sequence during sample preparation when the copy number is above the dashed lines (n=3). ns, not significantly different.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4492554&req=5

f5: Comparison of amplification background signals obtained from PBS control samples after treatment by method A or method B and calculated on the basis of circular [(A), (C), and (E)] or linear [(B), (D), and (F)] plasmid DNA, using ITR primers (A and B), GFP primers (C and D), or CMV primers (E and F). Dashed lines indicate the set Cq cutoff value and a valid detection value, and potentially the cross-contamination of AAV DNA sequence during sample preparation when the copy number is above the dashed lines (n=3). ns, not significantly different.
Mentions: A column purification step has been added, after enzymatic DT/PK treatment, to AAV sample preparation as described by other laboratories. For the convenience of method comparison, we termed the enzymatic DT/PK treatment as method A and the combined treatment with method A and column purification as method B. Typically, method A would generate unpurified samples containing vector DNA, digested vector capsid, and cellular proteins and inactivated enzymes; in contrast, method B resulted in the preparation of purified vector DNA free from enzymes, capsid, and cellular proteins. Control PBS samples free of AAV vectors were first subjected to the whole process of sample preparation, using methods A or B, to monitor potential method influence and cross-contamination risk during sample preparation. Figure 5 shows that 66% of processed PBS control samples showed copy numbers greater than the set Cq cutoff value (dashed lines), indicating a valid detection value and potentially the cross-contamination of AAV DNA sequence during sample preparation. In particular, control samples prepared by column-based method B showed significantly higher amplification background compared with enzymatic treatment alone (method A) (p<0.05), reflecting an increased likelihood of cross-contamination from multistep processing.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Advanced Therapies, NIBSC/Medicines and Healthcare Products Regulatory Agency , Potters Bar, Hertfordshire, EN6 3QG United Kingdom .

ABSTRACT
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.

No MeSH data available.