6PWA

AAV8 human HEK293-produced, full capsid

6PWA の概要

• エントリーDOI: 10.2210/pdb6pwa/pdb
• EMDBエントリー: 20502
• 分子名称: Capsid protein (1 entity in total)
• 機能のキーワード: adeno-associated virus, aav, gene therapy vector, post translational modification, capsid, virus
• 由来する生物種: Adeno-associated virus - 8
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 58528.37

構造登録者

Paulk, N.K.,Poweleit, N. (登録日: 2019-07-22, 公開日: 2020-06-03, 最終更新日: 2024-03-20)

主引用文献

Rumachik, N.G.,Malaker, S.A.,Poweleit, N.,Maynard, L.H.,Adams, C.M.,Leib, R.D.,Cirolia, G.,Thomas, D.,Stamnes, S.,Holt, K.,Sinn, P.,May, A.P.,Paulk, N.K.
Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors.
Mol Ther Methods Clin Dev, 18:98-118, 2020

PubMed 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 () 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.

PubMed: 32995354
DOI: 10.1016/j.omtm.2020.05.018

実験手法

ELECTRON MICROSCOPY (3.3 Å)