National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)
K01-DK107607
United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
U01-HL145795
United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
P01-HL51670
United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)
P30-DK54759
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
P30-CA086862
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
U01-CA207702
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
F32-GM126663
United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
T32-AI060537
United States
Citation
Journal: Mol Ther Methods Clin Dev / Year: 2020 Title: Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors. Authors: Neil G Rumachik / Stacy A Malaker / Nicole Poweleit / Lucy H Maynard / Christopher M Adams / Ryan D Leib / Giana Cirolia / Dennis Thomas / Susan Stamnes / Kathleen Holt / Patrick Sinn / ...Authors: Neil G Rumachik / Stacy A Malaker / Nicole Poweleit / Lucy H Maynard / Christopher M Adams / Ryan D Leib / Giana Cirolia / Dennis Thomas / Susan Stamnes / Kathleen Holt / Patrick Sinn / Andrew P May / Nicole K Paulk / 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 ...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 () 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 and , 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- vectors in various cell types (p < 0.05-0.0001), in various mouse tissues (p < 0.03-0.0001), and in human liver (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.
History
Deposition
Aug 18, 2019
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Header (metadata) release
Oct 2, 2019
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Map release
Jun 3, 2020
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Update
Mar 20, 2024
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Current status
Mar 20, 2024
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 1647 / Average exposure time: 6.0 sec. / Average electron dose: 66.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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