© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

摘要

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.

詳細資料

標題
Accurate Quantification of AAV Vector Genomes by Quantitative PCR
作者
Cristina Martinez-Fernandez de la Camara 1   VIAFID ORCID 標誌  ; McClements, Michelle E 2   VIAFID ORCID 標誌  ; MacLaren, Robert E 1   VIAFID ORCID 標誌 

 Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, John Radcliffe Hospital, Level 5&6, West Wing, Headley Way, Oxford OX3 9DU, UK; michelle.mcclements@eye.ox.ac.uk (M.E.M.); robert.maclaren@eye.ox.ac.uk (R.E.M.); Oxford Eye Hospital, Oxford University Hospitals NHS Trust, John Radcliffe Hospital, West Wing, Headley Way, Oxford OX3 9DU, UK 
 Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, John Radcliffe Hospital, Level 5&6, West Wing, Headley Way, Oxford OX3 9DU, UK; michelle.mcclements@eye.ox.ac.uk (M.E.M.); robert.maclaren@eye.ox.ac.uk (R.E.M.) 
第一頁
601
出版年份
2021
出版日期
2021
出版者
MDPI AG
e-ISSN
20734425
來源類型
學術期刊
出版物語言
English
ProQuest 文件識別碼
2531383497
著作權
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.