Virus-like particles (VLPs) are increasingly used for vaccine development and drug delivery. Assembly of VLPs from purified monomers in a chemically defined reaction is advantageous compared to in vivo assembly, because it avoids encapsidation of host-derived components and enables loading with added cargoes. This review provides an overview of ex cella VLP production methods focusing on capsid protein production, factors that impact the in vitro assembly, and approaches to characterize in vitro VLPs. The uses of in vitro produced VLPs as vaccines and for therapeutic delivery are also reported.

Strategies for site-specific modification of proteins have increased in number, complexity, and specificity over the last years. Such modifications hold the promise to broaden the use of existing biopharmaceuticals or to tailor novel proteins for therapeutic or diagnostic applications. The recent quest for next-generation antibody–drug conjugates (ADCs) sparked research into techniques with site selectivity. While purely chemical approaches often impede control of dosage or locus of derivatization, naturally occurring enzymes and proteins bear the ability of co- or post-translational protein modifications at particular residues, thus enabling unique coupling reactions or protein fusions. This review provides a general overview and focuses on chemo-enzymatic methods including enzymes such as formylglycine-generating enzyme, sortase, and transglutaminase. Applications for the conjugation of antibodies and antibody mimetics are reported.

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, whilst additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with quantitative polymerase chain reaction results demonstrated the technique's future potential for quantification of DNA impurities in AAV vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.

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.

Monomeric RGD peptides show unspecific fluid‐phase uptake in cells, whereas multimeric RGD peptides are thought to be internalized by integrin‐mediated endocytosis. However, a potential correlation between uptake mechanism and molecular mass has been neglected so far. A dual derivatization of peptide c(RGDw(7Br)K) was performed to investigate this. A fluorescent probe was installed by chemoselective Suzuki–Miyaura cross‐coupling of the 7‐bromotryptophan and a poly(ethylene glycol) (PEG) linker was attached to the lysine residue. Flow cytometry and live cell imaging confirmed unspecific uptake of the small, non‐PEGylated peptide, whereas the PEG₅₀₀₀ peptide conjugate unveiled a selective internalization by M21 cells overexpressing α_vβ₃ and no uptake in α_v‐deficient M21L cells.