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.

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

Current plasmid propagation in E. coli compromises large inverted repeats, such as inverted terminal repeats (ITRs) of adeno-associated virus (AAV). Direct long-read sequencing analyses upon varying strains and culture conditions revealed ITR instability caused by a slipped misalignment mechanism, although other mechanism probably contribute. ITRs stabilized in absence of SbcC, which is part of the SbcCD nuclease complex, a human Mre11–Rad50 homolog, or at elevated growth temperatures (e.g. 42°C), with a combination being optimal. Resulting full ITR transgene plasmids improved rAAV yield and purity in HEK-293 productions. The findings advance plasmid biology, cloneable sequences and therapeutic AAV manufacturing.

Current plasmid propagation compromises large inverted repeats, like inverted terminal repeats (ITRs) of adeno-associated virus (AAV). Direct long-read sequencing analyses upon varying culture conditions and strains revealed ITR instability mechanisms, which diminished in absence of SbcC or at elevated growth temperatures (e.g. 42 °C), with a combination being optimal. Resulting full ITRs improved rAAV yield and purity. The findings advance plasmid biology, cloneable sequences, and therapeutic AAV manufacturing.

BACKGROUND: The DES gene encodes the IF (intermediate filament) protein desmin, which connects different multiprotein complexes, such as the cardiac desmosomes, and is highly important for the structural integrity of cardiomyocytes. Pathogenic DES mutations cause filament assembly defects leading to cardiomyopathies. However, most DES variants listed in genetic disease databases are currently classified as variants of unknown significance. Here, we characterized 21 different DES variants of unknown significance and 18 additional proline variants, localized in a highly conserved stretch at the C terminus of the desmin coil-2 subdomain. METHODS: We inserted desmin variants via site-directed mutagenesis and investigated the filament assembly in transfected cell lines and cardiomyocytes derived from induced pluripotent stem cells by confocal microscopy. In addition, we purified recombinant wild-type and mutant desmin and analyzed the filament formation by atomic force microscopy. Coexpression with wild-type desmin delivered by adeno-associated virus was used to model the heterozygous status of cardiomyopathy patients. RESULTS: Twelve DES variants of unknown significance formed cytoplasmic aggregates, which were likewise verified by atomic force microscopy. Of note, these 12 variants disturb the filament assembly even when coexpressed with wild-type desmin. Using a proline screen, we showed that proline residues localized at nearly each of the positions in this stretch cause filament assembly defects. By modeling the tetrameric structure of desmin, we demonstrated that specific heptad positions, as well as positions of intramolecular and intermolecular ion bridge sites, are particularly susceptible to mutations that promote desmin aggregation. CONCLUSIONS: In summary, our study demonstrated that the highly conserved stretch at the C terminus of the coil-2 subdomain is a hotspot region, where several pathogenic DES mutations cause an aberrant desmin aggregation. Based on our molecular data, we suggest reclassifying the aggregate-forming variants as likely pathogenic mutations rather than variants of unknown significance. Our study may have relevance for the genetic counseling of cardiomyopathy patients with similar DES variants.

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, while 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 qPCR 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.