Quantitative and Digital Droplet-Based AAV Genome Titration

Methods Mol Biol. 2019;1950:51-83. doi: 10.1007/978-1-4939-9139-6_4.

Abstract

The adeno-associated viral vector (AAV) platform has developed into a primary modality for efficient in vivo, and in more limited settings, in vitro or ex vivo gene transfer. Its applications range from a tool for experimental purposes to preclinical and clinical gene therapy. The ability to accurately and reproducibly quantify vector concentration is critical for any of these applications. While several quantification assays are available, here we outline a detailed protocol for the quantification of DNase-I protected vector genomes reliant on the polymerase chain reaction (PCR) as a measure of the active component of the vector, namely its transgene cargo. With the emergence of droplet digital PCR (ddPCR), we provide side-by-side protocols for traditional TaqMan™ real-time, quantitative PCR (qPCR) and ddPCR, as well as comparative data generated with both methods. Lastly, we discuss the importance of the use of surfactant (here, Pluronic® F-68) in the execution of the assay to limit DNA and AAV adherence to various carriers during the titration, particularly at low concentrations. We believe these protocols can lead to reduced variability and increased comparability between AAV studies.

Keywords: AAV; Adeno-associated virus; Droplet digital PCR; Genome; Quantitative PCR; Real-time PCR; Titration; Vector; ddPCR; qPCR.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Dependovirus / genetics*
  • Genetic Vectors / genetics*
  • Genome, Viral*
  • Humans
  • Real-Time Polymerase Chain Reaction* / methods
  • Real-Time Polymerase Chain Reaction* / standards
  • Sensitivity and Specificity
  • Viral Load*
  • Workflow