Journal article
Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors
Molecular therapy. Methods & clinical development, Vol.18, pp.98-118
09/11/2020
DOI: 10.1016/j.omtm.2020.05.018
PMCID: PMC7488757
PMID: 32995354
Abstract
Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05–0.0001), in various mouse tissues in vivo (p < 0.03–0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.
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Paulk and colleagues performed an unbiased comparative characterization of rAAV manufactured by transient transfection of HEK293 or baculoviral infection of insect cells. They discovered that rAAV capsids have post-translational modifications, including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms. Additionally, rAAV genomes are differentially methylated during production between platforms.
Details
- Title: Subtitle
- Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors
- Creators
- Neil G. Rumachik - Stanford UniversityStacy A. Malaker - Stanford UniversityNicole Poweleit - University of California, San FranciscoLucy H. Maynard - Chan Zuckerberg Initiative (United States)Christopher M. Adams - Stanford UniversityRyan D. Leib - Stanford UniversityGiana Cirolia - Chan Zuckerberg Initiative (United States)Dennis Thomas - Cold Spring Harbor LaboratorySusan Stamnes - Roy J. and Lucille A. Carver College of MedicineKathleen Holt - Roy J. and Lucille A. Carver College of MedicinePatrick Sinn - University of IowaAndrew P. May - Chan Zuckerberg Initiative (United States)Nicole K. Paulk - University of California, San Francisco
- Resource Type
- Journal article
- Publication Details
- Molecular therapy. Methods & clinical development, Vol.18, pp.98-118
- DOI
- 10.1016/j.omtm.2020.05.018
- PMID
- 32995354
- PMCID
- PMC7488757
- NLM abbreviation
- Mol Ther Methods Clin Dev
- ISSN
- 2329-0501
- eISSN
- 2329-0501
- Publisher
- Elsevier Inc
- Grant note
- DOI: 10.13039/100000002, name: National Institutes of Health
- Language
- English
- Date published
- 09/11/2020
- Academic Unit
- Microbiology and Immunology; Pulmonary Medicine; Stead Family Department of Pediatrics
- Record Identifier
- 9984297330602771
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