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Engineered AAV8 capsid acquires heparin and AVB sepharose binding capacity but has altered in vivo transduction efficiency

Gene Therapy volume 30, pages 236–244 (2023)Cite this article

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Abstract

Naturally occurring adeno-associated virus (AAV) serotypes that bind to ligands such as AVB sepharose or heparin can be purified by affinity chromatography, which is a more efficient and scalable method than gradient ultracentrifugation. Wild-type AAV8 does not bind effectively to either of these molecules, which constitutes a barrier to using this vector when a high throughput design is required. Previously, AAV8 was engineered to contain a SPAKFA amino acid sequence to facilitate purification using AVB sepharose resin; however, in vivo studies were not conducted to examine whether these capsid mutations altered the transduction profile. To address this gap in knowledge, a mutant AAV8 capsid was engineered to bind to AVB sepharose and heparan sulfate (AAV8-AVB-HS), which efficiently bound to both affinity columns, resulting in elution yields of >80% of the total vector loaded compared to <5% for wild-type AAV8. However, in vivo comparison by intramuscular, intravenous, and intraperitoneal vector administration demonstrated a significant decrease in AAV8-AVB-HS transduction efficiency without alteration of the transduction profile. Therefore, although it is possible to engineer AAV capsids to bind various affinity ligands, the consequences associated with mutating surface exposed residues have the potential to negatively impact other vector characteristics including in vivo potency and production yield. This study demonstrates the importance of evaluating all aspects of vector performance when engineering AAV capsids.

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Fig. 1: Affinity column binding profiles and in vitro characterization of AAV8 and AAV8-AVB-HS vectors.
Fig. 2: AAV8 and AAV8-AVB-HS transduction profile following intranasal administration.
Fig. 3: AAV8 and AAV8-AVB-HS transduction profile following intramuscular administration.
Fig. 4: AAV8 and AAV8-AVB-HS transduction profile following two routes of systemic administration.
Fig. 5: Structural model of AAV8 capsid highlighting mutated binding domains.

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Acknowledgements

We thank all those involved in the care of the animals for these studies at all institutions.

Funding

Operating funds were from the Canadian Institute of Health Research (SKW—grant # 352532), the Canadian Lung Association (SKW—grant # 052919), and the National Institute of Health (JPB—HL131634). LPvL was funded by an Ontario Graduate Scholarship. AAS was funded by a Vanier Canada Graduate Scholarship, Brock Doctoral Scholarship, and the Ethel Rose Charney Scholarship in the Human/Animal Bond.

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Authors and Affiliations

  1. Department of Pathobiology, University of Guelph, Guelph, ON, Canada

    Laura P. van Lieshout, Ashley A. Stegelmeier & Sarah K. Wootton

  2. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Tara N. Rindler

  3. Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Tara N. Rindler

  4. Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    John J. Lawder

  5. Molecular Pathobiology, National Microbiology Laboratory NML, Public Health Agency of Canada, Winnipeg, MB, Canada

    Debra L. Sorensen, Kathy L. Frost & Stephanie A. Booth

  6. Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB, Canada

    Stephanie A. Booth

  7. Division of Pulmonary, Critical Care and Sleep Medicine, National Jewish Health, Denver, CO, USA

    James P. Bridges

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  1. Laura P. van Lieshout
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Correspondence to Sarah K. Wootton.

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The authors declare that they have no conflict of interest.

Ethical approval

All animal experiments were approved by the institutional Animal Care Committees at the University of Guelph, the Cincinnati Children’s Hospital Medical Center, and the National Microbiology Laboratory, in accordance with the Canadian Council on Animal Care guidelines (AUPP 3827). Six to eight-week-old female BALB/c mice were purchased from Charles River Laboratories (St. Constant, Quebec, Canada) and allowed to acclimatize 1 week prior to experimentation. All mice were healthy and criteria was established prior to ordering mice. Mice were of the same age, sex, and shipment. Thus, no further randomization was conducted aside from randomly selecting one cage per experimental group. The investigator was not blinded during the experiment as this study was analyzing expression as quantified objectively by a machine, and not subjective parameters such as survival endpoint in a disease model.

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van Lieshout, L.P., Stegelmeier, A.A., Rindler, T.N. et al. Engineered AAV8 capsid acquires heparin and AVB sepharose binding capacity but has altered in vivo transduction efficiency. Gene Ther 30, 236–244 (2023). https://doi.org/10.1038/s41434-020-00198-7

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