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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.


Validation of calibration curves generated from circular pAAV2-hrGFP plasmid DNA standards with (A)ITR, (B)GFP, (C)CMV, and (D)SV40 primers. Efficiency (E) confirms that each calibration curve is within the acceptable range of 0.9–1.1. The correlation coefficient (r2) confirms that each curve has correlation greater than 0.99. Data shown represent the mean quantification cycle (Cq)±standard deviation.
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f1: Validation of calibration curves generated from circular pAAV2-hrGFP plasmid DNA standards with (A)ITR, (B)GFP, (C)CMV, and (D)SV40 primers. Efficiency (E) confirms that each calibration curve is within the acceptable range of 0.9–1.1. The correlation coefficient (r2) confirms that each curve has correlation greater than 0.99. Data shown represent the mean quantification cycle (Cq)±standard deviation.

Mentions: The quantitative PCR assay was validated using a serial dilution of circular plasmid DNA to generate calibration curves for each primer set, that is, (1) plasmid pAAV2-hrGFP for ITR (Fig. 1A), GFP (Fig. 1B), and CMV (Fig. 1C) primers and (2) plasmid pRRLSIN.cPPT.PGK-GFP.WPRE for SV40 primers (Fig. 1D). Control samples of PBS and a blank well were also included in all qPCR analyses to monitor potential cross-contamination. The symbol Cq is used throughout the text as recommended by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments; www.rdml.org/miqe.php) guideline.24 The Cq values for all controls were greater than the cutoff Cq, that is, greater than the mean Cq of the 102 standard; therefore, the data for control samples are not shown. Only data sets with a control sample Cq greater than the cutoff Cq were considered valid and included in subsequent analyses. The amplification efficiency of qPCR was calculated using the equation: PCR efficiency (E)=10–1/slope – 1.24 Efficiency within the range of 0.9–1.1 was considered valid, where a value of 1 indicates 100% amplification efficiency (Table 2). The linear dynamic range for each experiment was also determined and confirmed to be 1×108 to 1×102 copies for each target sequence. All r2 values were greater than 0.99 for each experiment (Fig. 1A–D), demonstrating the fidelity of sample dilutions and assay performance and the robustness of the results presented in this study.


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)

Validation of calibration curves generated from circular pAAV2-hrGFP plasmid DNA standards with (A)ITR, (B)GFP, (C)CMV, and (D)SV40 primers. Efficiency (E) confirms that each calibration curve is within the acceptable range of 0.9–1.1. The correlation coefficient (r2) confirms that each curve has correlation greater than 0.99. Data shown represent the mean quantification cycle (Cq)±standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492554&req=5

f1: Validation of calibration curves generated from circular pAAV2-hrGFP plasmid DNA standards with (A)ITR, (B)GFP, (C)CMV, and (D)SV40 primers. Efficiency (E) confirms that each calibration curve is within the acceptable range of 0.9–1.1. The correlation coefficient (r2) confirms that each curve has correlation greater than 0.99. Data shown represent the mean quantification cycle (Cq)±standard deviation.
Mentions: The quantitative PCR assay was validated using a serial dilution of circular plasmid DNA to generate calibration curves for each primer set, that is, (1) plasmid pAAV2-hrGFP for ITR (Fig. 1A), GFP (Fig. 1B), and CMV (Fig. 1C) primers and (2) plasmid pRRLSIN.cPPT.PGK-GFP.WPRE for SV40 primers (Fig. 1D). Control samples of PBS and a blank well were also included in all qPCR analyses to monitor potential cross-contamination. The symbol Cq is used throughout the text as recommended by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments; www.rdml.org/miqe.php) guideline.24 The Cq values for all controls were greater than the cutoff Cq, that is, greater than the mean Cq of the 102 standard; therefore, the data for control samples are not shown. Only data sets with a control sample Cq greater than the cutoff Cq were considered valid and included in subsequent analyses. The amplification efficiency of qPCR was calculated using the equation: PCR efficiency (E)=10–1/slope – 1.24 Efficiency within the range of 0.9–1.1 was considered valid, where a value of 1 indicates 100% amplification efficiency (Table 2). The linear dynamic range for each experiment was also determined and confirmed to be 1×108 to 1×102 copies for each target sequence. All r2 values were greater than 0.99 for each experiment (Fig. 1A–D), demonstrating the fidelity of sample dilutions and assay performance and the robustness of the results presented in this study.

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.