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Quantitative and Digital Droplet-Based AAV Genome Titration

  • Julio Sanmiguel
  • Guangping GaoEmail author
  • Luk H. VandenbergheEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1950)

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.

Key words

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

Notes

Acknowledgments

We wish to extend our appreciation to Qin Su and Ru Xiao, directors of Vector Production at the Horae Gene Therapy Center, UMass Medical School and the Gene Transfer Vector Core (vector.meei.harvard.edu) at the Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, respectively, for providing valuable vectors for the comparisons presented here. This work was supported by 1P01AI100263-05, 1R01NS076991-05, R01 HL097088, and 4P01HL131471-01 (GPG) and Giving/Grousbeck (LHV).

Conflict of Interest

LHV is an inventor on gene therapy technologies licensed to various biopharmaceutical companies, a founder of Akouos and Gensight, a consultant to multiple entities with gene and genome editing therapeutic interest, and receives sponsored research from Selecta and Solid Biosciences and Lonza Houston.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Grousbeck Gene Therapy CenterSchepens Eye Research Institute and Massachusetts Eye and Ear InfirmaryBostonUSA
  2. 2.Harvard Stem Cell InstituteHarvard UniversityCambridgeUSA
  3. 3.Horae Gene Therapy CenterUniversity of Massachusetts Medical SchoolWorcesterUSA
  4. 4.Li Weibo Institute for Rare Diseases ResearchUniversity of Massachusetts Medical SchoolWorcesterUSA
  5. 5.Department of Microbiology and Physiological SystemsUniversity of Massachusetts Medical SchoolWorcesterUSA
  6. 6.Department of OphthalmologyHarvard Medical SchoolBostonUSA
  7. 7.The Broad Institute of Harvard and MITCambridgeUSA

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