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A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision.

Abstract：

:Recombinant adeno-associated virus (rAAV) is one of the main vectors used in gene therapy. An accurate genome titer is not only critical for clinical dosing, but also a prerequisite for many analytical assays for AAV product characterization. AAV genome titer is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR (ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. However, currently ddPCR is not as widely available as qPCR and operates at a lower throughput and a higher cost. In this paper, we introduce an improved qPCR method with two major optimizations: (1) using an AAV reference material as qPCR standard instead of plasmid DNA and (2) implementing a "digestion-free" method by adding 5% Tween 20 to standard and sample preparations. The new method has been extensively tested with AAV of different serotypes, purification status, and transgenes encapsidated and was found to be highly accurate, precise, and robust. This significantly improved and simplified assay can be easily adopted by researchers in the gene therapy field and further automated for high-throughput applications.

journal_name

Mol Ther Methods Clin Dev

journal_title

Molecular therapy. Methods & clinical development

authors

Wang Y,Menon N,Shen S,Feschenko M,Bergelson S

doi

10.1016/j.omtm.2020.09.017

subject

Has Abstract

pub_date

2020-10-01 00:00:00

pages

341-346

issn

2329-0501

pii

S2329-0501(20)30202-3

journal_volume

19

pub_type

杂志文章

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