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High-Throughput In Vitro , Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction

  • Westhaus, Adrian1, 2
  • Cabanes-Creus, Marti1
  • Rybicki, Arkadiusz1
  • Baltazar, Grober1
  • Navarro, Renina Gale1
  • Zhu, Erhua3
  • Drouyer, Matthieu1
  • Knight, Maddison4
  • Albu, Razvan F.4
  • Ng, Boaz H.4
  • Kalajdzic, Predrag4
  • Kwiatek, Magdalena5
  • Hsu, Kenneth6
  • Santilli, Giorgia2
  • Gold, Wendy7, 8, 9
  • Kramer, Belinda6
  • Gonzalez-Cordero, Anai10
  • Thrasher, Adrian J.2
  • Alexander, Ian E.3, 8
  • Lisowski, Leszek1, 4, 5
  • 1 Translational Vectorology Research Unit, Children's Medical Research Institute, The University of Sydney, Westmead, Australia
  • 2 Great Ormond Street Institute of Child Health, University College London, London, United Kingdom
  • 3 Gene Therapy Research Unit, Children's Medical Research Institute and Sydney Children's Hospitals Network, The University of Sydney, Westmead, Australia
  • 4 Vector and Genome Engineering Facility, Children's Medical Research Institute, , The University of Sydney, Westmead, Australia
  • 5 Military Institute of Hygiene and Epidemiology, The Biological Threats Identification and Countermeasure Centre, Puławy, Poland
  • 6 Children's Cancer Research Unit, The Children's Hospital at Westmead, Westmead, Australia
  • 7 Molecular Neurobiology Research Lab, The Children's Hospital at Westmead, Westmead, Australia
  • 8 Discipline of Child and Adolescent Health, Faculty of Medicine and Health, The University of Sydney, Sydney, Australia.
  • 9 Kids Neuroscience Centre, Kids Research, The Children's Hospital at Westmead, Westmead, Australia
  • 10 Stem Cell & Organoid Facility and Stem Cell Medicine Group, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia
Published Article
Human Gene Therapy
Mary Ann Liebert
Publication Date
May 01, 2020
DOI: 10.1089/hum.2019.264
PMID: 32000541
PMCID: PMC7232709
PubMed Central


Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro , ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

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