Adeno-associated virus capsid protein expression in Escherichia coli and chemically defined capsid assembly

Abstract

Research and clinical applications of recombinant adeno-associated virus (rAAV) significantly increased in recent years alongside regulatory approvals of rAAV gene therapy products. To date, all rAAV vectors as well as AAV empty capsids are produced in eukaryotic cells. We explored a new route to generate AAV capsids with the aim to analyze capsid assembly in a chemically defined setting and pave the way for new production methods and applications based on AAV virus-like particles (VLPs). We generated these empty capsids by bacterial expression and subsequent concomitant protein refolding and VLP formation. AAV serotype 2 structural protein VP3 was expressed in Escherichia coli. VLPs formed as demonstrated by dynamic light scattering, atomic force microscopy, and ELISA. Furthermore, VLPs internalized into human HeLa cells. To extend the application range of the VLPs, we tested peptide insertions, at the genetic level, in a surface loop (amino acid position 587) or at the C-terminus of VP3 and these variants also formed VLPs. VLPs developed without assembly-activating protein (AAP), but adding purified recombinant AAP to the refolding process increased capsid yield. Our findings offer a new route to understand AAV assembly biology and open a toolbox for AAV production strategies that might enable capsid display for vaccination and matching of capsids with cargoes at large scale and low cost.

Comparative evaluation of integrated purification pathways for bacterial modular polyomavirus major capsid protein VP1 to produce virus-like particles using high throughput process technologies

“…As SEC-HPLC still reveals a near uniform capsomere peak we conclude, that partially digested VP1-J8 still remains in pentameric form together with intact VP1-J8 monomers and therefore are impossible to remove. The formation of truncation products of viral protein during the expression in E. coli has also been reported for adeno associated viral protein VP3 and might therefore also be a result of E. coli proteases [28] . Further research needs to be undertaken to minimize the formation of these digestion products, and how to remove the bound chaperons, but using protease inhibitors throughout the whole process instead of only during cell disruption, run at reduced temperature and addition of ATP to remove chaperones will likely solve the issue.…”

“…China approved E. coli produced VLP vaccines Hecolin® and Cecolin® showing high efficiency and safety and providing proof of concept for E. coli produced VLP vaccines [ 26 , 27 ]. Several modular and non-modular VLPs based on a variety of structural viral protein such as hepatitis B core antigen (HBcAg), papilloma major capsid protein L1, bacteriophage Qβ, adeno-associated virus structural protein VP3 and polyomavirus major capsid protein VP1 have been produced in E. coli [ 24 , 25 , 28 , 29 ]. One of the most advanced approaches is the platform technology using modularized murine polyomavirus major capsid protein VP1 [10] .…”

Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies

“…31 The particle size distribution obtained from DLS is only semiquantitative compared to microscopy and of low resolution. 50 , 96 , 97 , 98 Overall precision of the method is low, with repeatability reported as poor as 45% CV for capsid titer and 85% CV for aggregate content using multi-angle DLS. 31 Operated in batch mode, DLS is a non-destructive method with high throughput and commonly used in conjunction with static light scattering (SLS).…”

Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers

“…Subsequently it was shown that virus capsid, envelope and, sometimes, core viral proteins can form VLP structures. VLPs can be experimentally generated in the laboratory using recombinant viral proteins that are expressed in a range of expression systems including prokaryotic cells [6], yeast [7], insect cell lines [8,9], plants [10] and mammalian cell lines [11,12]. While VLPs are commonly produced using proteins(s) from a single virus type, chimeric VLPs can also be created by the assembly of structural proteins from different viruses [6].…”