Limits...
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.


Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the SV40 sequence was used to spike crude (left) and HPLC-purified (middle) AAV8 samples and AAV2 (right) samples before HPLC purification at a vector concentration of 6.25×1013 copies/ml; qPCR analysis was performed, targeting spike-specific SV40 (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown (n=3). Asterisks indicate statistical significance between crude and purified samples, where *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4492554&req=5

f3: Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the SV40 sequence was used to spike crude (left) and HPLC-purified (middle) AAV8 samples and AAV2 (right) samples before HPLC purification at a vector concentration of 6.25×1013 copies/ml; qPCR analysis was performed, targeting spike-specific SV40 (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown (n=3). Asterisks indicate statistical significance between crude and purified samples, where *p<0.05.

Mentions: To further evaluate the influence of plasmid DNA conformation on AAV vector quantitation, three batches of AAV8 samples were subjected to qPCR targeting ITR, GFP, or CMV sequences and vector genome titers (VG/ml) were subsequently calculated on the basis of uncut circular or linear DNA calibration curves (Table 3). Although the amplification efficiency (E) in ITR and GFP qPCR was different between circular and linear plasmids (Fig. 2A and B), there was no significant difference in vector genome titers (p>0.05) between the quantitation based on circular and linear calibration curves for the same target sequences. On the basis of our data, circular plasmid DNA is adequate or may be a preferred standard for AAV8 vector quantitation, because when using linear DNA plasmid as a standard, the amplification efficiency in ITR and GFP qPCR (Fig. 3A and B) was out of the desired range (0.9–1.1) and may increase the risk of underestimating actual titers. There was no significant difference in vector genome titers among qPCRs targeting ITR, GFP, and CMV sequences (Table 3), indicating a nominal influence of qPCR target sequences on vector genome quantitation.


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)

Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the SV40 sequence was used to spike crude (left) and HPLC-purified (middle) AAV8 samples and AAV2 (right) samples before HPLC purification at a vector concentration of 6.25×1013 copies/ml; qPCR analysis was performed, targeting spike-specific SV40 (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown (n=3). Asterisks indicate statistical significance between crude and purified samples, where *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the SV40 sequence was used to spike crude (left) and HPLC-purified (middle) AAV8 samples and AAV2 (right) samples before HPLC purification at a vector concentration of 6.25×1013 copies/ml; qPCR analysis was performed, targeting spike-specific SV40 (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown (n=3). Asterisks indicate statistical significance between crude and purified samples, where *p<0.05.
Mentions: To further evaluate the influence of plasmid DNA conformation on AAV vector quantitation, three batches of AAV8 samples were subjected to qPCR targeting ITR, GFP, or CMV sequences and vector genome titers (VG/ml) were subsequently calculated on the basis of uncut circular or linear DNA calibration curves (Table 3). Although the amplification efficiency (E) in ITR and GFP qPCR was different between circular and linear plasmids (Fig. 2A and B), there was no significant difference in vector genome titers (p>0.05) between the quantitation based on circular and linear calibration curves for the same target sequences. On the basis of our data, circular plasmid DNA is adequate or may be a preferred standard for AAV8 vector quantitation, because when using linear DNA plasmid as a standard, the amplification efficiency in ITR and GFP qPCR (Fig. 3A and B) was out of the desired range (0.9–1.1) and may increase the risk of underestimating actual titers. There was no significant difference in vector genome titers among qPCRs targeting ITR, GFP, and CMV sequences (Table 3), indicating a nominal influence of qPCR target sequences on vector genome quantitation.

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.