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Hum Gene Ther. 2020 Jan 30. doi: 10.1089/hum.2019.264. [Epub ahead of print]

High throughput <i>in vitro</i>, <i>ex vivo</i> and i<i>n vivo</i> screen of AAV vectors based on physical and functional transduction.

Author information

1
The University of Sydney Faculty of Medicine and Health, 522555, Translational Vectorology Group, Children's Medical Research Institute, Sydney, New South Wales, Australia.
2
University College London, 4919, Great Ormond Street Institute of Child Health, London, London, United Kingdom of Great Britain and Northern Ireland; awesthaus@cmri.org.au.
3
The University of Sydney Faculty of Medicine and Health, 522555, Translational Vectorology Group, Children's Medical Research Institute, Sydney, New South Wales, Australia; mcabanescreus@cmri.org.au.
4
The University of Sydney Faculty of Medicine and Health, 522555, Translational Vectorology Group, Children's Medical Research Institute, Sydney, New South Wales, Australia; arkadius.rybicki@gmail.com.
5
The University of Sydney Faculty of Medicine and Health, 522555, Translational Vectorology Group, Children's Medical Research Institute, Sydney, New South Wales, Australia; gbaltazar@cmri.org.au.
6
The University of Sydney Faculty of Medicine and Health, 522555, Translational Vectorology Group, Children's Medical Research Institute, Sydney, New South Wales, Australia; inavarro@cmri.org.au.
7
The University of Sydney Faculty of Medicine and Health, 522555, Gene Therapy Research Unit, Children's Medical Research Institute , Sydney, New South Wales, Australia; czhu@cmri.org.au.
8
The University of Sydney Faculty of Medicine and Health, 522555, Translational Vectorology Group, Children's Medical Research Institute, Sydney, New South Wales, Australia; MDrouyer@cmri.org.au.
9
The University of Sydney Faculty of Medicine and Health, 522555, Vector and Genome Engineering Facility, Children's Medical Research Institute, Sydney, New South Wales, Australia; mknight@cmri.org.au.
10
The University of Sydney Faculty of Medicine and Health, 522555, Vector and Genome Engineering Facility, Children's Medical Research Institute, Sydney, New South Wales, Australia; ralbu@cmri.org.au.
11
The University of Sydney Faculty of Medicine and Health, 522555, Vector and Genome Engineering Facility, Children's Medical Research Institute, Sydney, New South Wales, Australia; boaz.ng@monash.edu.
12
The University of Sydney Faculty of Medicine and Health, 522555, Vector and Genome Engineering Facility, Children's Medical Research Institute, Sydney, New South Wales, Australia; pkalajdzic@cmri.org.au.
13
General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, 326803, The Biological Threats Identification and Countermeasure Centre, Puławy, Poland; mkwiatek@wihe.pulawy.pl.
14
Children's Hospital at Westmead, 8538, Children's Cancer Research Unit, Kids Research, Westmead, New South Wales, Australia; Kenneth.Hsu@health.nsw.gov.au.
15
University College London, 4919, Great Ormond Street Institute of Child Health, London, London, United Kingdom of Great Britain and Northern Ireland; g.santilli@ucl.ac.uk.
16
Children's Hospital at Westmead, 8538, Molecular Neurobiology Research Lab, Kids Research, Westmead, New South Wales, Australia.
17
The University of Sydney Faculty of Medicine and Health, 522555, Discipline of Child and Adolescent Health, Sydney, New South Wales, Australia.
18
Children's Hospital at Westmead, 8538, Kids Neuroscience Centre, Kids Research, Westmead, New South Wales, Australia; wendy.gold@sydney.edu.au.
19
Children's Hospital at Westmead, 8538, Children's Cancer Research Unit, Kids Research, Westmead, New South Wales, Australia; belinda.kramer@health.nsw.gov.au.
20
The University of Sydney Faculty of Medicine and Health, 522555, Stem Cell & Organoid Facility and Stem Cell Medicine Group, Children's Medical Research Institute, Sydney, New South Wales, Australia; agonzalez-cordero@cmri.org.au.
21
University College London, 4919, Great Ormond Street Institute of Child Health, London, London, United Kingdom of Great Britain and Northern Ireland; A.Thrasher@ucl.ac.uk.
22
The University of Sydney Faculty of Medicine and Health, 522555, Gene Therapy Research Unit, Children's Medical Research Institute, Sydney, New South Wales, Australia.
23
The University of Sydney Faculty of Medicine and Health, 522555, Discipline of Child and Adolescent Health,, Sydney, New South Wales, Australia; ian.alexander@health.nsw.gov.au.
24
The University of Sydney Faculty of Medicine and Health, 522555, Vector and Genome Engineering Facility, Children's Medical Research Institute, Sydney, New South Wales, Australia.
25
General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, 326803, The Biological Threats Identification and Countermeasure Centre, Puławy, Poland; llisowski@cmri.org.au.

Abstract

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) remain the most important features 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 complicate 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 <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i> 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 iPSCs in vitro, as well as <i>in vivo</i> transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we identified also novel previously unknown tropisms for some AAV variants.

PMID:
32000541
DOI:
10.1089/hum.2019.264

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