Kristian Mark Müller

Research, production and application of the recombinant adeno-associated virus (rAAV) require versatile tools for rapid and cost-effective advances. We developed fusion constructs with the pan-serotype, AAV-binding VHH-antibody-fragment named nanobody AAVX and devised procedures for AAV purification, quantification, and detection. Starting from an amino acid sequence, we cytosolically expressed in E. coli nanobody AAVX carrying various tags and fusion partners. Very efficient soluble production followed by a one-step immobilized metal-ion affinity chromatography (IMAC) yielded mostly >90% pure protein. Crosslinking AAVX with an agarose matrix enabled efficient purification of intact AAV2 and AAV5 capsids from cell lysates, as assessed by qPCR and ELISA. AAVX also supported effective AAV quantification by ELISA, as capture reagent and, when fused to luciferase, as detection reagent. Using tagged AAVX and streptavidin biosensors, we developed a rapid and label-free quantification assay of AAV capsids by biolayer interferometry (BLI). Finally, immunostaining with an AAVX-eGFP fusion visualized AAV capsids in fixed HEK-293 cells by fluorescence microscopy. Collectively, these results establish nanobody AAVX fusion proteins as versatile and readily customizable tools for AAV research and bioprocessing.

Tumor-specific targeting remains a major obstacle for the development of precision cancer therapies. For several tumors and specifically glioblastoma (GBM), an aggressive brain tumor with poor prognosis, more effective treatments are urgently needed. Recombinant adeno-associated viruses (rAAVs), with their established clinical utility and mutational capsid plasticity, offer a promising platform for targeted delivery. We engineered and evaluated modified AAV capsids derived from the established AAV2 with HSPG-tropism knockdown and two less-characterized, tropism-reduced AAV9 variants. The epidermal growth factor receptor (EGFR), a tumor marker often overexpressed in GBM, was targeted by inserting an affibody (Z _EGFR:1907 ). Also chlorotoxin (CLTX) was inserted, a peptide from scorpion venom reported to bind GBM. Transduction efficiencies were initially assessed with established cell lines (A431, HeLa, U251MG, MCF7). Affibody-displaying capsids exhibited EGFR-dependent transduction, with AAV9-affibody variants surpassing AAV9 wild-type. Several affibody-displaying capsids also transduced patient-derived GBM explants, as confirmed by fluorescence microscopy. These findings highlight the potential of retargeting AAV9 variants and the use of human surgical tissue samples for the initial evaluation of newly designed AAV capsids.

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.

Recombinant adeno‐associated virus (rAAV) production lags demand with respect to quality and quantity. We report insights from producer plasmid engineering aimed at increasing yield and homogeneity of rAAV vectors obtained by HEK‐293 triple transfection. Miniaturized production and same‐day quantification streamlined the investigation. We demonstrate that modifications of the AAV2 Rep gene cluster reduces titers of currently circulating packaging plasmids. Revertants to wild type yielded 116‐fold higher titers of about 10 ⁶ particles per cell and reduced mispackaging. Modifications of predicted Rep post‐translational modification sites decreased the empty capsid titer burden. A 7 kbp minimal helper plasmid lacking L4 22k maintained production capability upon optimized Rep expression for AAV2 but not for AAV6 and AAV9. Knockout of the egress protein MAAP increased rAAV yield from the cell pellet for convenient lysate processing. Together, these findings highlight the importance and potential tradeoffs of designing producer plasmids to obtain high titer systems.

Cystic fibrosis (CF) is the most prevalent inherited disease. Inactivating mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene lead to the accumulation of viscous mucus and subsequent respiratory complications. This study optimized a prime editing (PE) approach to correct CFTR mutations focusing on the F508del mutation. Prime editing allowed to introduce missing bases without double-strand breaks using a Cas9-nickase fused with a reverse transcriptase in combination with a prime editing guide RNA (pegRNA). Various self-designed pegRNAs were compared. For delivery, various lipid nanoparticles (LNP) were tested, which were optimized for stability and lung cells targeting based on lipid selection or chitosan complexion. A fluorescence reporter system, pPEAR_CFTR, was developed mimicking F508del for validation. The five pegRNAs yielding the highest efficiency were used for genomic CFTR correction in a CF bronchial cell line. Nanopore sequencing of genomic DNA revealed approximate 5% edited reads. These results highlight the promise of prime editing-LNP systems for precise and lung-specific gene correction, paving the way for novel therapies in cystic fibrosis and other pulmonary genetic disorders.

Recombinant adeno-associated virus (rAAV) is the leading vector for gene replacement therapy; however, the roles and regulation of host proteins in rAAV production remain incompletely understood. In this comparative proteomic analysis, we focused on proteins in the nucleus, the epicenter of DNA uptake, transcription, capsid assembly, and packaging. HEK-293 cells were analyzed under the following three conditions: (i) untransfected, (ii) mock-transfected with the ITR and an unrelated plasmid, and (iii) triple-transfected with rAAV2 production plasmids. Cells were harvested at 24 and 72 h post-transfection, and nuclear fractions were processed using filter-aided sample preparation (FASP) followed by nano-scale liquid chromatography–tandem mass spectrometry (nLC-Orbitrap MS/MS). Across all samples, we identified 3384 proteins, revealing significant regulatory changes associated with transfection and rAAV production. Transfection alone accounted for some of the most substantial proteomic shifts, while rAAV production induced diverse regulatory changes linked to cell cycle control, structure, and metabolism. STRING analysis of significantly regulated proteins also identified an enrichment of those associated with the Gene Ontology (GO) term ‘response to virus’. Additionally, we examined proteins with reported relation to adenoviral components. Our findings help to unravel the complexity of rAAV production, identify interesting targets for further investigation, and may contribute to improving rAAV yield.

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.

Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.

PH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK′ = 6.6) and EGF-CoGFP-mCRISPRed (pK′ = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein–drug conjugates in cancer therapy.