Person: György, Bence
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György
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Bence
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György, Bence
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Publication Mutant torsinA in the heterozygous DYT1 state compromises HSV propagation in infected neurons and fibroblasts(Nature Publishing Group UK, 2018) György, Bence; Cruz, Lilian; Yellen, David; Aufiero, Massimo; Alland, Isabel; Zhang, Xuan; Ericsson, Maria; Fraefel, Cornel; Li, Yu-Ching; Takeda, Shuko; Hyman, Bradley; Breakefield, XandraMost cases of early onset torsion dystonia (DYT1) are caused by a 3-base pair deletion in one allele of the TOR1A gene causing loss of a glutamate in torsinA, a luminal protein in the nuclear envelope. This dominantly inherited neurologic disease has reduced penetrance and no other medical manifestations. It has been challenging to understand the neuronal abnormalities as cells and mouse models which are heterozygous (Het) for the mutant allele are quite similar to wild-type (WT) controls. Here we found that patient fibroblasts and mouse neurons Het for this mutation showed significant differences from WT cells in several parameters revealed by infection with herpes simplex virus type 1 (HSV) which replicates in the nucleus and egresses out through the nuclear envelope. Using a red fluorescent protein capsid to monitor HSV infection, patient fibroblasts showed decreased viral plaque formation as compared to controls. Mouse Het neurons had a decrease in cytoplasmic, but not nuclear HSV fluorescence, and reduced numbers of capsids entering axons as compared to infected WT neurons. These findings point to altered dynamics of the nuclear envelope in cells with the patient genotype, which can provide assays to screen for therapeutic agents that can normalize these cells.Publication Exosome-associated AAV vector as a robust and convenient neuroscience tool(2016) Hudry, Eloise; Martin, Courtney; Gandhi, Sheetal; György, Bence; Scheffer, Deborah I.; Mu, Dakai; Merkel, Steven F.; Mingozzi, Federico; Fitzpatrick, Zachary; Dimant, Hemi; Masek, Marissa; Ragan, Tim; Tan, Sisareuth; Brisson, Alain R.; Ramirez, Servio H.; Hyman, Bradley; Maguire, CaseyAdeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill, and time intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared to standard AAV. Here we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation, results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing 3-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex-vivo serial 2-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.Publication Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties(Public Library of Science, 2015) Osteikoetxea, Xabier; Balogh, Andrea; Szabó-Taylor, Katalin; Németh, Andrea; Szabó, Tamás Géza; Pálóczi, Krisztina; Sódar, Barbara; Kittel, Ágnes; György, Bence; Pállinger, Éva; Matkó, János; Buzás, Edit IrénIn recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.Publication Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection(Nature Publishing Group, 2017) Wassmer, Sarah J.; Carvalho, Livia S.; György, Bence; Vandenberghe, Luk; Maguire, CaseyWidespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye.