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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.


Related in: MedlinePlus

Systematic analysis of buffer effects on qPCR quantitation of plasmid DNA. (A) qPCR results of 108 to 101 copies of vector plasmid DNA performed in (1) H2O (cross-hatched columns), (2) DT buffer only (open columns), (3) buffer/inactivated DT (hatched columns), and (4) buffer/inactivated DT/PK/heat at 95°C (solid columns); (B) calibration curves generated from qPCR of plasmid DNA in various buffers, showing data linear range, R2 and amplification E values (n=3).
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f4: Systematic analysis of buffer effects on qPCR quantitation of plasmid DNA. (A) qPCR results of 108 to 101 copies of vector plasmid DNA performed in (1) H2O (cross-hatched columns), (2) DT buffer only (open columns), (3) buffer/inactivated DT (hatched columns), and (4) buffer/inactivated DT/PK/heat at 95°C (solid columns); (B) calibration curves generated from qPCR of plasmid DNA in various buffers, showing data linear range, R2 and amplification E values (n=3).

Mentions: To investigate the effects of buffer, enzyme, and capsid proteins presented when using method A on qPCR results, we carried out qPCR of 108 to 101 copies of vector plasmid DNA in (1) H2O (Fig. 4A, cross-hatched columns), (2) DT buffer only (open columns), (3) buffer/inactivated DT (hatched columns) and (4) buffer/inactivated DT/PK/heat at 95°C (solid columns). Figure 4 shows that qPCR of plasmid DNA in buffer/inactivated DT/PK (solid columns) gave the closest results to that of plasmid DNA in H2O (Fig. 4A, cross-hatched columns), for example, 1.88±0.3×10 and 1.22±0.22×106 copies of plasmid DNA detected from 108 and 107 copies of plasmid DNA, respectively; whereas both buffer alone (Fig. 4A, open columns) and buffer/inactivated DT (Fig. 4A, hatched columns) showed significantly lower detection of plasmid DNA than that of qPCR in H2O, demonstrating significant buffer effects on qPCR quantitation. When using the qPCR results of qPCR plasmid DNA under different conditions to generate calibration standard curves, qPCR in H2O (Fig. 4B, solid line) showed both R2 and amplification efficiency (E) within the desired range (R2>0.99, E=0.9–1.1) and six data points within a linear range. However, the R2 and E values of plasmid DNA in buffer (R2=0.86, E=1.6), DT (R2=0.82, E=1.8), and DT/PK (R2=0.95, E=1.4) were all out of the desired range, with the qPCR in buffer/DT/PK showing a linear range and R2 and E values closest to qPCR in H2O, demonstrating the importance of using comparable buffer–protein mix to generate a standard curve for titer calculation.


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)

Systematic analysis of buffer effects on qPCR quantitation of plasmid DNA. (A) qPCR results of 108 to 101 copies of vector plasmid DNA performed in (1) H2O (cross-hatched columns), (2) DT buffer only (open columns), (3) buffer/inactivated DT (hatched columns), and (4) buffer/inactivated DT/PK/heat at 95°C (solid columns); (B) calibration curves generated from qPCR of plasmid DNA in various buffers, showing data linear range, R2 and amplification E values (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Systematic analysis of buffer effects on qPCR quantitation of plasmid DNA. (A) qPCR results of 108 to 101 copies of vector plasmid DNA performed in (1) H2O (cross-hatched columns), (2) DT buffer only (open columns), (3) buffer/inactivated DT (hatched columns), and (4) buffer/inactivated DT/PK/heat at 95°C (solid columns); (B) calibration curves generated from qPCR of plasmid DNA in various buffers, showing data linear range, R2 and amplification E values (n=3).
Mentions: To investigate the effects of buffer, enzyme, and capsid proteins presented when using method A on qPCR results, we carried out qPCR of 108 to 101 copies of vector plasmid DNA in (1) H2O (Fig. 4A, cross-hatched columns), (2) DT buffer only (open columns), (3) buffer/inactivated DT (hatched columns) and (4) buffer/inactivated DT/PK/heat at 95°C (solid columns). Figure 4 shows that qPCR of plasmid DNA in buffer/inactivated DT/PK (solid columns) gave the closest results to that of plasmid DNA in H2O (Fig. 4A, cross-hatched columns), for example, 1.88±0.3×10 and 1.22±0.22×106 copies of plasmid DNA detected from 108 and 107 copies of plasmid DNA, respectively; whereas both buffer alone (Fig. 4A, open columns) and buffer/inactivated DT (Fig. 4A, hatched columns) showed significantly lower detection of plasmid DNA than that of qPCR in H2O, demonstrating significant buffer effects on qPCR quantitation. When using the qPCR results of qPCR plasmid DNA under different conditions to generate calibration standard curves, qPCR in H2O (Fig. 4B, solid line) showed both R2 and amplification efficiency (E) within the desired range (R2>0.99, E=0.9–1.1) and six data points within a linear range. However, the R2 and E values of plasmid DNA in buffer (R2=0.86, E=1.6), DT (R2=0.82, E=1.8), and DT/PK (R2=0.95, E=1.4) were all out of the desired range, with the qPCR in buffer/DT/PK showing a linear range and R2 and E values closest to qPCR in H2O, demonstrating the importance of using comparable buffer–protein mix to generate a standard curve for titer calculation.

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.


Related in: MedlinePlus