Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients.

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or "empty" capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.

Oral Triphenylmethane Food Dye Analog, Brilliant Blue G, Prevents Neuronal Loss in APPSwDI/NOS2-/- Mouse Model.

Reducing amyloid-ß (Aß) accumulation is a promising strategy for developing Alzheimer's Disease (AD) therapeutics. We recently reported that a triphenylmethane food dye analog, Brilliant Blue G (BBG), is a dose-dependent modulator of in vitro amyloid-ß aggregation and cytotoxicity in cell-based assays. Following up on this recent work, we sought to further evaluate this novel modulator in a therapeutically-relevant AD transgenic mouse model. BBG was orally administered to APPSwDI/NOS2-/- mice for three months in order to assess its biocompatibility, its permeability across the blood-brain barrier, and its efficacy at rescuing AD pathology. The results showed that BBG was well-tolerated, caused no significant weight change/unusual behavior, and was able to significantly cross the AD blood-brain barrier in APPSwDI/NOS2-/- mice. Immunohistochemical and electron microscopic analysis of the brain sections revealed that BBG was able to significantly prevent neuronal loss and reduce intracellular APP/Aß in hippocampal neurons. This is the first report of 1) the effect of Brilliant Blue G on neuronal loss in a transgenic animal model of AD, 2) oral administration of BBG to affect a protein conformation/aggregation disease, and 3) electron microscopic ultrastructural analysis of AD pathology in APPSwDI/NOS2-/- mice.

Determining binding sites of polycyclic aromatic small molecule-based amyloid-beta peptide aggregation modulators using sequence-specific antibodies.

Numerous aromatic small molecule modulators of amyloid-beta peptide (Aß) monomer aggregation and neurotoxicity have been identified with the ultimate goal of Alzheimer's disease (AD) treatment. Determining binding sites of these modulators on Aß monomer is an important topic in the mechanistic understanding of AD pathology and drug development. However, Aß monomer binding sites have been reported for only a very limited number of Aß modulators. In this article, we present a convenient method for determining aggregation-modulating polycyclic aromatic small molecule ligand binding sites on Aß monomer using immunostaining with a panel of Aß sequence-specific antibodies. To validate our technique, we first examined one modulating aromatic ligand, Congo Red, with known binding sites, which yielded consistent results with previous findings. Then, using the same technique, binding sites on Aß of four known Aß monomer aggregation modulators, Erythrosin B, Eosin Y, Phloxine B, and Rose Bengal, were determined. The identified ligand binding sites were also confirmed by a separate fluorescence quenching-based assay using a panel of overlapping Aß sub-fragments. The technique described here greatly increases researchers' ability to determine the Aß monomer binding site(s) of aggregation-modulating aromatic small molecule ligands and to screen for new ligands that bind specific regions on Aß.

Halogenation generates effective modulators of amyloid-Beta aggregation and neurotoxicity.

Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates. Moreover, halogenation of aromatic molecules greatly affects aromatic interaction-mediated self-assembly processes, including amyloid fibril formation. Perturbation of the aromatic interaction caused by halogenation of peptide building blocks is known to affect the morphology and other physical properties of the fibrillar structure. Consequently, in this article, we investigated the ability of halogenated ligands to modulate the self-assembly of amyloidogenic peptide/protein. As a model system, we chose amyloid-beta peptide (Aß), which is implicated in Alzheimer's disease, and a novel modulator of Aß aggregation, erythrosine B (ERB). Considering that four halogen atoms are attached to the xanthene benzoate group in ERB, we hypothesized that halogenation of the xanthene benzoate plays a critical role in modulating Aß aggregation and cytotoxicity. Therefore, we evaluated the modulating capacities of four ERB analogs containing different types and numbers of halogen atoms as well as fluorescein as a negative control. We found that fluorescein is not an effective modulator of Aß aggregation and cytotoxicity. However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on Aß aggregation. Such Aß aggregation inhibition by ERB analogs except rose bengal correlated well to the inhibition of Aß cytotoxicity. To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on Aß-associated neurotoxicity via Aß aggregation modulation.

Targeting of heparanase-modified syndecan-1 by prosecretory mitogen lacritin requires conserved core GAGAL plus heparan and chondroitin sulfate as a novel hybrid binding site that enhances selectivity.

Cell surface heparan sulfate (HS) proteoglycans shape organogenesis and homeostasis by capture and release of morphogens through mechanisms largely thought to exclude the core protein domain. Nevertheless, heparanase deglycanation of the N-terminal HS-rich domain of syndecan-1 (SDC1), but not SDC2 or -4, is a prerequisite for binding of the prosecretory mitogen lacritin (Ma, P., Beck, S. L., Raab, R. W., McKown, R. L., Coffman, G. L., Utani, A., Chirico, W. J., Rapraeger, A. C., and Laurie, G. W. (2006) Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin. J. Cell Biol. 174, 1097-1106). We now report that the conserved and hydrophobic GAGAL domain in SDC1, adjacent to predicted HS substitution sites, is necessary to ligate and substantially enhance the α-helicity of the amphipathic C terminus of lacritin. Swapping out GAGAL for GADED in SDC2 or for GDLDD in SDC4 (both less hydrophobic) abrogated binding. HS and chondroitin sulfate are also essential. Both are detected in the N terminus, and when incubated with antibodies HS4C3 (anti-HS) or IO3H10 (anti-chondroitin sulfate), binding was absent, as occurred when all three N-terminal glycosaminoglycan substitution sites were mutated to alanine or when cells were treated with 4-methylumbelliferyl-ß-d-xylopyranoside or chlorate to suppress glycosaminoglycan substitution or sulfation, respectively. SDC1 interacts with the hydrophobic face of lacritin via Leu-108/Leu-109/Phe-112 as well as with Glu-103/Lys-107 and Lys-111 of the largely cationic face. Carving a hybrid hydrophobic/electrostatic docking site out of SDC1 in a manner dependent on endogenous heparanase is a dynamic process appropriate for subtle or broad epithelial regulation in morphogenesis, health, and disease.

Different fates of Alzheimer's disease amyloid-ß fibrils remodeled by biocompatible small molecules.

Amyloid fibrils implicated in numerous human diseases are thermodynamically very stable. Stringent conditions that would not be possible in a physiological environment are often required to disrupt the stable fibrils. Recently, there is increasing evidence that small molecules can remodel amyloid fibrils in a physiologically relevant manner. In order to investigate possible fibril remodeling mechanisms using this approach, we performed comparative studies on the structural features of the different amyloid-ß (Aß) aggregates remodeled from Aß fibrils by three biocompatible small molecules: methylene blue; brilliant blue G; and erythrosine B. Combined with circular dichroism (CD), immuno-blotting, transmission electron microscopy (TEM), and atomic force microscopy (AFM) results, it was found that brilliant blue G- and erythrosine B-treatment generate fragmented Aß fibrils and protofibrils, respectively. In contrast, incubation of the Aß fibrils with methylene blue perturbs fibrillar structure, leading to amorphous Aß aggregates. Our findings provide insights on the molecular mechanism of amyloid fibril formation and remodeling and also illustrate the possibility of controlled changes in biomolecule nanostructures.