Biology subjects

Kunach, P.

Publications and source records attributed to Kunach, P..

6 recordsLinked to original sources

bioRxiv2026

A minimally modified AAV9 variant (AAV9fyhr) for enhanced CNS gene delivery by intra-CSF administration.

Adeno-associated virus (AAV) mediated gene therapies continue to show promise for the treatment of genetic diseases. However, delivery and safety still represent significant prohibitive barriers restricting translation of these delivery modalities to humans. We describe a novel AAV9 variant, AAV9fyhr or FYHR, identified from screening a controlled randomization point mutation AAV9 library in mice and nonhuman primates followed by directed evolution of in vivo CNS-derived libraries screened in human iPSC differentiated cortical neurons. Capsid variants of interest were identified by coupling long read sequencing and computational structure modeling. Structure-function analysis of our lead candidate reveal that two targeted amino acid changes are sufficient to result in beneficial pleiotropic effects across viral stability, cell surface attachment, and gene delivery. We developed a novel multiplexed barcoding reporter system, enabling a time and cost-effective option to gather spatially resolved single cell capsid validation data in mice and nonhuman primates. When administered intrathecally, AAV9fyhr exhibits 2-to-6-fold higher gene delivery across several tissues in the CNS while minimally altering the tropism to peripheral tissues in comparison to AAV9. These findings demonstrate the potential of whole capsid engineering for the rational development of translational next generation AAV capsids.

bioengineering↗

bioRxiv2026

A functional amyloid scaffold shapes insect egg coats

Functional amyloids serve as structural scaffolds across biology, yet the molecular architecture and assembly principles of many remain unresolved. The lepidopteran egg coat, or chorion, presents a striking example: hundreds of paralogous proteins sharing a conserved central domain form a mechanically resilient amyloid matrix essential for embryo protection. Here, combining evolutionary analysis of more than 500 sequences with cryo-electron microscopy and biophysical assays, we determine the atomic structure of chorion amyloid filaments and uncover the principles governing their assembly. Contrary to previous structural predictions, chorion filaments adopt a {beta}-serpentine fold stabilized by a short hexapeptide motif that forms homotypic steric-zipper interfaces, self-assembles autonomously, and seeds full-length filament growth. Amyloid formation proceeds through secondary nucleation, while thermodynamic and structural analyses support a hierarchical assembly mechanism in which motif-driven interactions nucleate filament formation prior to consolidation of the mature yet lalbile protofilament core. These findings establish the molecular basis of insect egg-coat assembly and demonstrate that mechanisms commonly associated with pathological aggregation can also operate within a biologically regulated functional amyloid framework.

biophysics↗

bioRxiv2024

Functional classification of tauopathy strains reveals the role of protofilament core residues.

Distinct tau amyloid assemblies underlie diverse tauopathies but defy rapid classification. Cell and animal experiments indicate tau functions as a prion, as different strains propagated in cells cause unique, transmissible neuropathology after inoculation. Strain amplification requires compatibility of the monomer and amyloid template. We used cryo-EM to study one cell-based YFP-tagged strain, resolving its amyloid nature. We then used sequential alanine (Ala) substitution (scan) within tau repeat domain (RD) to measure incorporation to pre-existing tau RD-YFP aggregates. This robustly discriminated strains, defining sequences critical for monomer incorporation. We then created 3R/4R or 4R WT RD (aa 246-408) biosensors. Ala scan of recombinant tau seeds with the Alzheimers Disease fold matched that of AD homogenate. We scanned 22 brain lysates comprising 4 tauopathies. This clustered cases by neuropathological syndrome, revealed the role of amino acids in protofilament folds, and allowed strain discrimination based on amino acid requirements for prion replication. TeaserDiscrimination of tau strains based on the relative contribution of each amino acid to templated propagation of the amyloid.

neuroscience↗

bioRxiv2024

ATTRv-V30M Type A amyloid fibrils from the heart and nerves exhibit structural homogeneity.

ATTR amyloidosis is a systemic disease characterized by the deposition of amyloid fibrils made of transthyretin, a protein integral to transporting retinol and thyroid hormones. Transthyretin is primarily produced by the liver and circulates in blood as a tetramer. The retinal epithelium also secretes transthyretin, which is secreted to the vitreous humor of the eye. Because of mutations or aging, transthyretin can dissociate into amyloidogenic monomers triggering amyloid fibril formation. The deposition of transthyretin amyloid fibrils in the myocardium and peripheral nerves causes cardiomyopathies and neuropathies, respectively. Using cryo-electron microscopy, here we determined the structures of amyloid fibrils extracted from cardiac and nerve tissues of an ATTRv-V30M patient. We found that fibrils from both tissues share a consistent structural conformation, similar to the previously described structure of cardiac fibrils from an individual with the same genotype, but different from the fibril structure obtained from the vitreous humor. Our study hints to a uniform fibrillar architecture across different tissues within the same individual, only when the source of transthyretin is the liver. Moreover, this study provides the first description of ATTR fibrils from the nerves of a patient and enhances our understanding of the role of deposition site and protein production site in shaping the fibril structure in ATTRv-V30M amyloidosis.

biophysics↗

bioRxiv2023

Docking for molecules that bind in a symmetric stack with SymDOCK

Discovering ligands for amyloid fibrils, such as those formed by the tau protein, is an area of much current interest. In recent structures, ligands bind in stacks in the tau fibrils to reflect the rotational and translational symmetry of the fibril itself; in these structures the ligands make few interactions with the protein but interact extensively with each other. To exploit this symmetry and stacking, we developed SymDOCK, a method to dock molecules that follow the proteins symmetry. For each prospective ligand pose, we apply the symmetry operation of the fibril to generate a self-interacting and fibril-interacting stack, checking that doing so will not cause a clash between the original molecule and its image. Absent a clash, we retain that pose and add the ligand-ligand van der Waals energy to the ligands docking score (here using DOCK3.8). We can check these geometries and energies using an implementation of ANI, a neural network-based quantum-mechanical evaluation of the ligand stacking energies. In retrospective calculations, symmetry docking can reproduce the poses of three tau PET tracers whose structures have been determined. More convincingly, in a prospective study SymDOCK predicted the structure of the PET tracer MK-6240 bound in a symmetrical stack to AD PHF tau before that structure was determined; the docked pose was used to determine how MK-6240 fit the cryo-EM density. In proof-of-concept studies, SymDOCK enriched known ligands over property-matched decoys in retrospective screens without sacrificing docking speed, and can address large library screens that seek new symmetrical stackers. Future applications of this approach will be considered.

biophysics↗

bioRxiv2023

Cryo-EM structure of Alzheimer disease tau filaments with PET ligand MK-6240

Positron Emission Tomography (PET) ligands have advanced Alzheimers disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain tissue with elevated tau burden, purified filaments, and determined the structure of second-generation high avidity PET ligand MK-6240 at 2.31 [A] resolution, which bound at a 1:1 ratio within the cleft of tau paired-helical filament (PHF), engaging with glutamine 351, lysine K353, and isoleucine 360. This information elucidates the basis of MK-6240 PET in quantifying PHF deposits in AD and may facilitate the structure-based design of superior ligands against tau amyloids.

neuroscience↗