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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.


qPCR analysis of artificial crude (left) and pure (middle and right) ATCC reference materials (RSS) prepared by method A (left and middle) or method B (right), quantified on the basis of calibration curves of circular (left and middle) or linearized (right) plasmid DNA in H2O (hatched and cross-hatched columns) or DT/PK mixture (gray columns). The RSS given titer (3.28×1010 VG/ml) is shown in the solid column. Data for each column represent mean data of nine PCRs of the same sample prepared in triplicate and then qPCR in triplicate (n=9). All study results are significantly different from the RSS given titers of 3.28×1010 VG/ml, p<0.05.
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f7: qPCR analysis of artificial crude (left) and pure (middle and right) ATCC reference materials (RSS) prepared by method A (left and middle) or method B (right), quantified on the basis of calibration curves of circular (left and middle) or linearized (right) plasmid DNA in H2O (hatched and cross-hatched columns) or DT/PK mixture (gray columns). The RSS given titer (3.28×1010 VG/ml) is shown in the solid column. Data for each column represent mean data of nine PCRs of the same sample prepared in triplicate and then qPCR in triplicate (n=9). All study results are significantly different from the RSS given titers of 3.28×1010 VG/ml, p<0.05.

Mentions: Figure 7 shows qPCR of the validated ATCC AAV2 Reference Materials (rAAV2-RSS, ATCC VR-1616) with a given titer of 3.28×1010 VG/ml (Fig. 7, solid column), to study the effect of sample preparation, DNA conformation, and qPCR buffer conditions on AAV titer quantitation. It is noteworthy that all our data were significantly different from the given RSS (Reference Standard Stock) titers, regardless of preparation methods, DNA conformation, or standard curves used, demonstrating the intrinsic variation of qPCR quantitation. Three sets/panels of data are presented in Fig. 7, that is, (1) RSS being added to cell lysate to generate artificial crude samples and prepared by method A (Fig. 7, left), (2) pure RSS prepared by method A (Fig. 7, middle), and pure RSS prepared by method B (Fig. 7, right). We also used qPCR of plasmid DNA in H2O (Fig. 7, hatched columns) and in DT/PK buffer mix (Fig. 7, gray columns) to generate standard curves for titer calculation, showing that RSS recovered titers were closer to the RSS given titer when using sample-comparable buffer mix (Fig. 7, gray columns) rather than using H2O (Fig. 7, hatched columns) to generate standard curves for titer calculation. Comparable amounts of vector DNA was detected in both DT- and DT/PK-treated samples (Fig. 7, left and middle, DT and DT/PK), confirming that the qPCR 95°C heating step resulted in the release of vector DNA from AAV2 capsids. The titers of RSS prepared by method B and qPCR conducted by various laboratories (Fig. 7, right, cross-hatched columns) were all significantly higher than the given RSS titer (p<0.05) and that of the RSS samples prepared by method A (Fig. 7, left and middle). The results presented in Fig. 7 were all from qPCR targeting ITR sequences and further confirmed our data (Fig. 6) that the ITR titers of high-titer samples were significantly higher when using method B rather than method A.


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)

qPCR analysis of artificial crude (left) and pure (middle and right) ATCC reference materials (RSS) prepared by method A (left and middle) or method B (right), quantified on the basis of calibration curves of circular (left and middle) or linearized (right) plasmid DNA in H2O (hatched and cross-hatched columns) or DT/PK mixture (gray columns). The RSS given titer (3.28×1010 VG/ml) is shown in the solid column. Data for each column represent mean data of nine PCRs of the same sample prepared in triplicate and then qPCR in triplicate (n=9). All study results are significantly different from the RSS given titers of 3.28×1010 VG/ml, p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: qPCR analysis of artificial crude (left) and pure (middle and right) ATCC reference materials (RSS) prepared by method A (left and middle) or method B (right), quantified on the basis of calibration curves of circular (left and middle) or linearized (right) plasmid DNA in H2O (hatched and cross-hatched columns) or DT/PK mixture (gray columns). The RSS given titer (3.28×1010 VG/ml) is shown in the solid column. Data for each column represent mean data of nine PCRs of the same sample prepared in triplicate and then qPCR in triplicate (n=9). All study results are significantly different from the RSS given titers of 3.28×1010 VG/ml, p<0.05.
Mentions: Figure 7 shows qPCR of the validated ATCC AAV2 Reference Materials (rAAV2-RSS, ATCC VR-1616) with a given titer of 3.28×1010 VG/ml (Fig. 7, solid column), to study the effect of sample preparation, DNA conformation, and qPCR buffer conditions on AAV titer quantitation. It is noteworthy that all our data were significantly different from the given RSS (Reference Standard Stock) titers, regardless of preparation methods, DNA conformation, or standard curves used, demonstrating the intrinsic variation of qPCR quantitation. Three sets/panels of data are presented in Fig. 7, that is, (1) RSS being added to cell lysate to generate artificial crude samples and prepared by method A (Fig. 7, left), (2) pure RSS prepared by method A (Fig. 7, middle), and pure RSS prepared by method B (Fig. 7, right). We also used qPCR of plasmid DNA in H2O (Fig. 7, hatched columns) and in DT/PK buffer mix (Fig. 7, gray columns) to generate standard curves for titer calculation, showing that RSS recovered titers were closer to the RSS given titer when using sample-comparable buffer mix (Fig. 7, gray columns) rather than using H2O (Fig. 7, hatched columns) to generate standard curves for titer calculation. Comparable amounts of vector DNA was detected in both DT- and DT/PK-treated samples (Fig. 7, left and middle, DT and DT/PK), confirming that the qPCR 95°C heating step resulted in the release of vector DNA from AAV2 capsids. The titers of RSS prepared by method B and qPCR conducted by various laboratories (Fig. 7, right, cross-hatched columns) were all significantly higher than the given RSS titer (p<0.05) and that of the RSS samples prepared by method A (Fig. 7, left and middle). The results presented in Fig. 7 were all from qPCR targeting ITR sequences and further confirmed our data (Fig. 6) that the ITR titers of high-titer samples were significantly higher when using method B rather than method A.

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.