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

Autor:	Scott S; Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.; Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Westmead, NSW 2145, Australia., Westhaus A; Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia., Nazareth D; Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia., Cabanes-Creus M; Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia., Navarro RG; Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia., Chandra D; Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia., Zhu E; 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., Venkateswaran A; Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Westmead, NSW 2145, Australia., Alexander IE; 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.; Discipline of Child and Adolescent Health, The University of Sydney, Sydney Medical School, Faculty of Medicine and Health, Westmead, NSW 2145, Australia., Bauer DC; Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Westmead, NSW 2145, Australia.; Department of Biomedical Sciences, Faculty of Medicine and Health Science, Macquarie University, Macquarie Park, NSW 2113, Australia.; Applied BioSciences, Faculty of Science and Engineering, Macquarie University, Macquarie Park, NSW 2113, Australia., Wilson LOW; Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Westmead, NSW 2145, Australia.; Applied BioSciences, Faculty of Science and Engineering, Macquarie University, Macquarie Park, NSW 2113, Australia., Lisowski L; Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.; Laboratory of Molecular Oncology and Innovative Therapies, Military Institute of Medicine - National Research Institute, 04-141 Warsaw, Poland.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Methods & clinical development [Mol Ther Methods Clin Dev] 2024 Oct 04; Vol. 32 (4), pp. 101351. Date of Electronic Publication: 2024 Oct 04 (Print Publication: 2024).
DOI:	10.1016/j.omtm.2024.101351
Abstrakt:	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. Competing Interests: L.L. is a cofounder of LogicBio Therapeutics, S.S. and L.L. are cofounders of Sendatu Therapeutics, and L.L. and I.E.A. are co-founders of Exigen Biotherapeutics, companies that utilize technologies similar to those broadly discussed in this paper. (© 2024 The Author(s).)
Databáze:	MEDLINE
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