Biology subjects

Mietzsch, M.

Publications and source records attributed to Mietzsch, M..

8 recordsLinked to original sources

bioRxiv2026

AAV2 Crosslinks Actin Filaments: Implications for AAV Gene Therapy Vector Design

Adeno-associated virus (AAV) capsids are important gene therapy vectors, allowing for the one-time treatment of monogenetic disorders, with durable gene expression lasting for years. However, despite the clinical success of AAV usage, low transduction efficiencies require high dosage to achieve therapeutic efficacy, resulting in prohibitive costs and rare, but life-threatening immune responses. One key knowledge gap is how the capsids traffic to the nucleus following endocytosis. Here, we identify a direct interaction between AAV2 and actin filaments. Our results show that AAV2 bundles multiple actin filaments in a highly periodic hexagonal lattice. Therefore, we propose that AAV2 is the founding member of a new class of actin binding proteins, the actin bundling viruses (ABVs). Using cryogenic electron microscopy, we determined the structure of AAV2-actin filaments, identified the interaction interfaces, and engineered capsid variants that no longer bundle actin. Together, our results suggest that the AAV2-actin interaction may be responsible for trapping capsids and mediating peri-nuclear accumulation. Overall, this interaction expands the current understanding of AAV2 biology and offers new directions for capsid engineering.

biochemistry↗

bioRxiv2026

Identification of Human Transferrin Receptor as an Entry Co-receptor for Parvovirus B19 Infection of Human Erythroid Progenitor Cells

Parvovirus B19 (B19V), a member of the genus Erythroparvovirus within the Parvoviridae family, infects human erythroid progenitor cells (EPCs) of bone marrow and fetal liver, and causes various hematological disorders. The minor capsid protein VP1 of B19V contains a unique N-terminal region (VP1u) that facilitates virus binding and internalization into EPCs via its receptor-binding domain (RBD). We previously identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. In this study, we employed an ascorbate peroxidase 2 (APEX2)-based proximity labeling method to identify host proteins that are associated with B19V VP1u during entry. This analysis revealed human transferrin receptor 1 (hTfR) as a key host protein associated with VP1u. hTfR knockdown in UT7/Epo-S1 cells, a B19V-permissive human megakaryoblastoid leukemia cell line, showed significantly reduced B19V internalization and replication. Biolayer interferometry (BLI) assays confirmed a direct interaction between B19V VP1u and hTfR extracellular domain (ECD). Inhibition of VP1u interaction with hTfR ECD, either by a monoclonal antibody targeting the apical domain of the ECD or human ferritin, a natural ligand of hTfR that binds the apical domain, significantly reduced VP1u binding to hTfR, as well as B19V internalization and B19V replication in ex vivo-expanded EPCs. Furthermore, mutant RBD proteins that bear amino acid substitutions in the three helical domains nearly abolished RBD binding to hTfR and significantly reduced the ability to inhibit B19V infection of EPCs. Collectively, our findings establish hTfR as a B19V entry co-receptor that mediates B19V internalization into its natural host EPCs. SignificanceB19V causes severe hematological disorders, including transient aplastic crisis, chronic pure red cell aplasia, and hydrops fetalis, by selectively infecting erythroid progenitor cells (EPCs). Despite its clinical impact, no approved antivirals or vaccines exist, largely due to limited understanding of viral entry mechanisms. A unique feature of B19V is the externalization of the VP1 unique region (VP1u) from the viral capsid, which mediates receptor engagement. Our prior studies identified AXL as an attachment receptor for B19V. Here, we identify that human transferrin receptor 1 (hTfR) acts as a critical co-receptor that directly binds VP1u and promotes viral internalization. Inhibition of the VP1u-hTfR interaction by competitive binding of hTfR with either an anti-hTfR monoclonal antibody or human ferritin significantly reduces B19V internalization and replication in ex vivo-expanded EPCs, highlighting a link between VP1u binding to the apical domain of hTfR and viral internalization. RBD mutants that disrupt its interaction with hTfR barely inhibited B19V infection in EPCs. These findings support a receptor-switch model in which AXL mediates attachment and hTfR drives internalization. Defining these mechanisms provides a foundation for developing antiviral strategies targeting B19V entry into EPCs.

microbiology↗

bioRxiv2024

Structural characterization of antibody-responses from Zolgensma treatment provides the blueprint for the engineering of an AAV capsid suitable for redosing

Monoclonal antibodies (mAbs) are useful tools to dissect the neutralizing antibody response against the adeno-associated virus (AAV) capsids used as gene therapy delivery vectors. This study structurally characterizes the interactions of 21 human-derived antibodies from patients treated with the AAV9 vector, Zolgensma(R), utilizing high-resolution cryo-electron microscopy. The majority of the bound antibodies do not conform to the icosahedral symmetry of the capsid, thus requiring localized reconstructions. These complex structures provide unprecedented details of the mAbs binding interfaces, with some antibodies inducing structural perturbations of the capsid upon binding. Key surface capsid amino acid residues were identified facilitating the design of capsid variants with an antibody escape phenotype, with the potential to expand the patient cohort treatable with AAV9 vectors to include those that were previously excluded due to their pre-existing neutralizing antibodies, and possibly also to those requiring redosing.

immunology↗

bioRxiv2024

Hyperacetylation mimetics within the tau filament core inhibits prion-like propagation of misfolded tau

The two most prominent post-translational modifications of pathologic tau are Ser/Thr/Tyr phosphorylation and Lys acetylation. Whether acetylation impacts the susceptibility of tau to templated seeding in diseases like Alzheimers disease (AD) and Progressive Supranuclear Palsy (PSP) is largely uncharacterized. Towards this, we examined how acetylation mimicking or nullifying mutations on five sites of tau (K311, K353, K369, K370, K375), located within the tau filament core, influenced the susceptibility of P301L (PL) tau to seeds from AD (AD-tau) or PSP (PSP-tau) in HEK293T cells. Acetyl-mimicking substitutions of individual Lys sites to Glutamine as well as mutation of all 5 sites together (PL+5K-Q and PL+5K-R) had inconsistent effects on tau seeding by AD-tau or PSP-tau seeds. Unexpectedly, mutating all 5 sites to Alanine (PL+5K-A) resulted in a tau variant that spontaneously aggregated. These aggregates were amorphous and yet able to propagate to naive cells expressing P301L tau but not wild-type tau. Previously, we reported that a phospho-mimetic S305E mutation in PL tau abrogated seeding by AD-tau but not PSP-tau seeds in the HEK293T cells. To assess how changes in acetylation and phosphorylation together could influence seeding, we combined the S305E and the 5K-Q mutations in P301L tau, creating a variant that retained specificity for PSP-tau seeds over AD-tau seeds. Our findings indicate that phosphorylation of tau at Ser305 is a strong determinant of disease-specific tau templating, even in the presence of hyperacetylation within the fibril core domain. Overall, our findings suggest that acetylation could be a secondary modifier of misfolded hyperphosphorylated tau seeds.

neuroscience↗

bioRxiv2023

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60{degrees} are virtually indistinguishable. These strategies allowed the reconstruction to 3 [A] resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

biophysics↗

bioRxiv2021

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. ImportanceA major byproduct of all Adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

molecular biology↗

bioRxiv2020

AAV Capsid Chimeras with Enhanced Infectivity reveal a core element in the AAV Genome critical for both Cell Transduction and Capsid Assembly

Adeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking a novel AAV capable of enhanced transduction of human T cells for applications in immunotherapy, we have developed a unique capsid variant termed AAV X-Vivo (AAV-XV) that is a chimera of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. This AAV chimera showed enhanced infection of human primary T cells and hematopoietic stem cells, and superiority over wildtype AAV6 for the genomic integration of DNA sequences either by AAV alone or in combination with CRISPR gene editing. AAV-XV demonstrated transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. Analyzing the protein coding sequence of AAV-XV revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations reverting the AAP sequence back to wildtype had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show that AAV-XV is highly efficient at T cell engineering at low AAV dose and demonstrates the importance of AAP coding region in both viral particle assembly and cell infection. IMPORTANCEA major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated a new AAV variant possessing a unique combination of capsid proteins for ex-vivo application termed AAV-XV, which delivers high levels of cell transduction and gene delivery at a lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid protein and AAP gene is critical for both virus yield and the enhancement of infection/transduction.

cell biology↗

bioRxiv2018

Sub-2 A Ewald Curvature Corrected Single-Particle Cryo-EM

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we have exploited several technical solutions to improve resolution, including sub-Angstrom pixelation, per-particle CTF refinement, and most notably a correction for Ewald sphere curvature. The application of these methods on micrographs recorded on a base model Titan Krios enabled structure determination at [~]1.86-[A] resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle.

biophysics↗