AAVolve: Concatenated long-read deep sequencing enables whole capsid tracking during shuffled AAV library selection

Suzanne Scott; Adrian Westhaus; Deborah Nazareth; Marti Cabanes-Creus; Renina Gale Navarro; Deborah Chandra; Erhua Zhu; Aravind Venkateswaran; Ian E Alexander; Denis C Bauer; Laurence O W Wilson; Leszek Lisowski

doi:10.1016/j.omtm.2024.101351

AAVolve: Concatenated long-read deep sequencing enables whole capsid tracking during shuffled AAV library selection

Mol Ther Methods Clin Dev. 2024 Oct 4;32(4):101351. doi: 10.1016/j.omtm.2024.101351. eCollection 2024 Dec 12.

Authors

Suzanne Scott^{1

2}, Adrian Westhaus¹, Deborah Nazareth¹, Marti Cabanes-Creus¹, Renina Gale Navarro¹, Deborah Chandra¹, Erhua Zhu³, Aravind Venkateswaran², Ian E Alexander^{3

4}, Denis C Bauer^{2

5

6}, Laurence O W Wilson^{2

6}, Leszek Lisowski^{1

7}

Affiliations

¹ Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.
² Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Westmead, NSW 2145, Australia.
³ Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Faculty of Medicine and Health, The University of Sydney, and Sydney Children's Hospitals Network, Westmead, NSW 2145, Australia.
⁴ Discipline of Child and Adolescent Health, The University of Sydney, Sydney Medical School, Faculty of Medicine and Health, Westmead, NSW 2145, Australia.
⁵ Department of Biomedical Sciences, Faculty of Medicine and Health Science, Macquarie University, Macquarie Park, NSW 2113, Australia.
⁶ Applied BioSciences, Faculty of Science and Engineering, Macquarie University, Macquarie Park, NSW 2113, Australia.
⁷ Laboratory of Molecular Oncology and Innovative Therapies, Military Institute of Medicine - National Research Institute, 04-141 Warsaw, Poland.

Abstract

Gene therapies using recombinant adeno-associated virus (AAV) vectors have demonstrated considerable clinical success in the treatment of genetic disorders. Improved vectors with favorable tropism profiles, decreased immunogenicity, and enhanced manufacturability are poised to further improve the state of gene therapies. Such vectors can be identified through directed evolution, a process of subjecting a diverse capsid library to a selection pressure to identify individual variants with a desired trait. Currently, libraries that involve changes distributed throughout the AAV capsid coding region, such as DNA family shuffled libraries, are largely characterized using low-throughput Sanger sequencing of individual clones. However, improvements in long-read sequencing technologies have increased their applicability to capsid libraries and evaluation of the selection process. Here, we explore the application of Oxford Nanopore Technologies refined by a concatemeric consensus method for initial library characterization and monitoring selection of a shuffled AAV capsid library. Furthermore, we present AAVolve, a bioinformatic pipeline for processing long-read data from AAV-directed evolution experiments. Our approach allows high-throughput characterization of AAV capsids in a streamlined manner, facilitating deeper insights into library composition through multiple rounds of selection, and generalization through training of machine learning models.

Keywords: AAV vector; DNA family shuffling; Oxford Nanopore Technologies; capsid engineering; directed evolution; gene therapy; rolling circle amplification to concatemeric consensus.