Accurate Quantification of AAV Vector Genomes by Quantitative PCR
The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this...
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doaj-8154513df659410a81bed0c84f3459952021-04-19T23:04:09ZengMDPI AGGenes2073-44252021-04-011260160110.3390/genes12040601Accurate Quantification of AAV Vector Genomes by Quantitative PCRCristina Martinez-Fernandez de la Camara0Michelle E. McClements1Robert E. MacLaren2Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, John Radcliffe Hospital, Level 5 & 6, West Wing, Headley Way, Oxford OX3 9DU, UKNuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, John Radcliffe Hospital, Level 5 & 6, West Wing, Headley Way, Oxford OX3 9DU, UKNuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, John Radcliffe Hospital, Level 5 & 6, West Wing, Headley Way, Oxford OX3 9DU, UKThe ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario.https://www.mdpi.com/2073-4425/12/4/601adeno-associated virusgene therapyquantitative PCR |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Cristina Martinez-Fernandez de la Camara Michelle E. McClements Robert E. MacLaren |
spellingShingle |
Cristina Martinez-Fernandez de la Camara Michelle E. McClements Robert E. MacLaren Accurate Quantification of AAV Vector Genomes by Quantitative PCR Genes adeno-associated virus gene therapy quantitative PCR |
author_facet |
Cristina Martinez-Fernandez de la Camara Michelle E. McClements Robert E. MacLaren |
author_sort |
Cristina Martinez-Fernandez de la Camara |
title |
Accurate Quantification of AAV Vector Genomes by Quantitative PCR |
title_short |
Accurate Quantification of AAV Vector Genomes by Quantitative PCR |
title_full |
Accurate Quantification of AAV Vector Genomes by Quantitative PCR |
title_fullStr |
Accurate Quantification of AAV Vector Genomes by Quantitative PCR |
title_full_unstemmed |
Accurate Quantification of AAV Vector Genomes by Quantitative PCR |
title_sort |
accurate quantification of aav vector genomes by quantitative pcr |
publisher |
MDPI AG |
series |
Genes |
issn |
2073-4425 |
publishDate |
2021-04-01 |
description |
The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario. |
topic |
adeno-associated virus gene therapy quantitative PCR |
url |
https://www.mdpi.com/2073-4425/12/4/601 |
work_keys_str_mv |
AT cristinamartinezfernandezdelacamara accuratequantificationofaavvectorgenomesbyquantitativepcr AT michelleemcclements accuratequantificationofaavvectorgenomesbyquantitativepcr AT robertemaclaren accuratequantificationofaavvectorgenomesbyquantitativepcr |
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