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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 calibration curves generated from circular (dotted lines) and ScaI-linearized (solid lines) pAAV2-hrGFP plasmid DNA standards with (A)ITR primers, (B) hrGFP primers, and (C)CMV primers. Data shown represent the mean quantification cycle (Cq)±standard deviation (n=3).
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f2: Comparison of calibration curves generated from circular (dotted lines) and ScaI-linearized (solid lines) pAAV2-hrGFP plasmid DNA standards with (A)ITR primers, (B) hrGFP primers, and (C)CMV primers. Data shown represent the mean quantification cycle (Cq)±standard deviation (n=3).

Mentions: Suppression of real-time PCR by uncut circular plasmid DNA has been previously reported by several groups,25,26 indicating a low amplification efficiency of a circular DNA compared with that of a linearized DNA plasmid under identical qPCR conditions. For a direct comparison, two AAV standard calibration curves were generated for each of the three qPCR target sequences ITR (Fig. 2A), GFP (Fig. 2B), and CMV (Fig. 2C), using uncut circular (dotted line) or ScaI-linearized (solid line) DNA plasmids. Figure 2 shows that the amplification efficiency (E), which is primarily governed by the annealing efficiency of primers to a target sequence and represents the quantitation accuracy, was within the designated range of 0.9–1.1 for all circular plasmid calibration curves (dotted lines; Fig. 2A–C) regardless of the PCR target sequences; however, E was greater than 1.1 for linear ITR (solid line; Fig. 2A) and GFP (solid line; Fig. 2B) plasmids, indicating a potentially lower amplification efficiency for circular DNA standards than for linear ITR and GFP plasmids. These results is in contrast to the early findings that circular plasmids suppressed qPCR amplification.15,16,25,26 Aurnhammer and colleagues16 have developed a universal qPCR method based on an AAV2 ITR-specific sequence, which has become widely used for AAV dose determination and allows a direct comparison of AAV titration results between laboratories. The observed difference in E between linear and circular ITR standard should be taken into account when using ITR qPCR quantitation. In contrast, the amplification efficiency of CMV target sequences was comparable between linear and circular plasmids (Fig. 2C), indicating that the influence of DNA conformation on qPCR quantitation may be target sequence dependent and may be optimized by qPCR primer design.


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 calibration curves generated from circular (dotted lines) and ScaI-linearized (solid lines) pAAV2-hrGFP plasmid DNA standards with (A)ITR primers, (B) hrGFP primers, and (C)CMV primers. Data shown represent the mean quantification cycle (Cq)±standard deviation (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Comparison of calibration curves generated from circular (dotted lines) and ScaI-linearized (solid lines) pAAV2-hrGFP plasmid DNA standards with (A)ITR primers, (B) hrGFP primers, and (C)CMV primers. Data shown represent the mean quantification cycle (Cq)±standard deviation (n=3).
Mentions: Suppression of real-time PCR by uncut circular plasmid DNA has been previously reported by several groups,25,26 indicating a low amplification efficiency of a circular DNA compared with that of a linearized DNA plasmid under identical qPCR conditions. For a direct comparison, two AAV standard calibration curves were generated for each of the three qPCR target sequences ITR (Fig. 2A), GFP (Fig. 2B), and CMV (Fig. 2C), using uncut circular (dotted line) or ScaI-linearized (solid line) DNA plasmids. Figure 2 shows that the amplification efficiency (E), which is primarily governed by the annealing efficiency of primers to a target sequence and represents the quantitation accuracy, was within the designated range of 0.9–1.1 for all circular plasmid calibration curves (dotted lines; Fig. 2A–C) regardless of the PCR target sequences; however, E was greater than 1.1 for linear ITR (solid line; Fig. 2A) and GFP (solid line; Fig. 2B) plasmids, indicating a potentially lower amplification efficiency for circular DNA standards than for linear ITR and GFP plasmids. These results is in contrast to the early findings that circular plasmids suppressed qPCR amplification.15,16,25,26 Aurnhammer and colleagues16 have developed a universal qPCR method based on an AAV2 ITR-specific sequence, which has become widely used for AAV dose determination and allows a direct comparison of AAV titration results between laboratories. The observed difference in E between linear and circular ITR standard should be taken into account when using ITR qPCR quantitation. In contrast, the amplification efficiency of CMV target sequences was comparable between linear and circular plasmids (Fig. 2C), indicating that the influence of DNA conformation on qPCR quantitation may be target sequence dependent and may be optimized by qPCR primer design.

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.