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  1. Article ; Online: AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.

    Theuerkauf, Samuel A / Herrera-Carrillo, Elena / John, Fabian / Zinser, Luca J / Molina, Mariano A / Riechert, Vanessa / Thalheimer, Frederic B / Börner, Kathleen / Grimm, Dirk / Chlanda, Petr / Berkhout, Ben / Buchholz, Christian J

    Biomaterials

    2023  Volume 303, Page(s) 122399

    Abstract: Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by ... ...

    Abstract Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.
    MeSH term(s) Mice ; Humans ; Animals ; Designed Ankyrin Repeat Proteins ; Transduction, Genetic ; Dependovirus/genetics ; Genetic Vectors/genetics ; Genetic Therapy ; HIV Infections
    Chemical Substances Designed Ankyrin Repeat Proteins
    Language English
    Publishing date 2023-11-16
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ZDB-ID 603079-8
    ISSN 1878-5905 ; 0142-9612
    ISSN (online) 1878-5905
    ISSN 0142-9612
    DOI 10.1016/j.biomaterials.2023.122399
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.B 2371: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)

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  2. Article ; Online: Quantitative assays reveal cell fusion at minimal levels of SARS-CoV-2 spike protein and fusion from without.

    Theuerkauf, Samuel A / Michels, Alexander / Riechert, Vanessa / Maier, Thorsten J / Flory, Egbert / Cichutek, Klaus / Buchholz, Christian J

    iScience

    2021  Volume 24, Issue 3, Page(s) 102170

    Abstract: Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates ...

    Abstract Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here, we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion but also fusion from without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of
    Language English
    Publishing date 2021-02-09
    Publishing country United States
    Document type Journal Article
    ISSN 2589-0042
    ISSN (online) 2589-0042
    DOI 10.1016/j.isci.2021.102170
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

    Riechert, Vanessa / Hein, Sascha / Visser, Mayken / Zimmermann, Mathias / Wesche, Jan / Adams, Philipp A / Theuerkauf, Samuel A / Jamali, Arezoo / Wangorsch, Andrea / Reuter, Andreas / Pasternak, Alexander O / Hartmann, Jessica / Greinacher, Andreas / Herrera-Carrillo, Elena / Berkhout, Ben / Cichutek, Klaus / Buchholz, Christian J

    The Journal of biological chemistry

    2023  Volume 299, Issue 6, Page(s) 104743

    Abstract: Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently ... ...

    Abstract Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA
    MeSH term(s) Humans ; Antibodies/metabolism ; Blood Platelets/metabolism ; Designed Ankyrin Repeat Proteins/metabolism ; HIV-1 ; Platelet Aggregation ; Protein Isoforms/metabolism ; Receptors, IgG/metabolism ; Virus Latency ; T-Lymphocytes/virology
    Chemical Substances Antibodies ; Designed Ankyrin Repeat Proteins ; Fc gamma receptor IIA ; Protein Isoforms ; Receptors, IgG
    Language English
    Publishing date 2023-04-24
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1016/j.jbc.2023.104743
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Uc I Zs.108: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)

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  4. Article ; Online: Quantitative assays reveal cell fusion at minimal levels of SARS-CoV-2 spike protein and fusion from without

    Samuel A. Theuerkauf / Alexander Michels / Vanessa Riechert / Thorsten J. Maier / Egbert Flory / Klaus Cichutek / Christian J. Buchholz

    iScience, Vol 24, Iss 3, Pp 102170- (2021)

    2021  

    Abstract: Summary: Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S ...

    Abstract Summary: Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here, we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion but also fusion from without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies, as well as sera from convalescent patients, inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during coronavirus disease 2019 (COVID-19) can proceed at low levels of S protein and may not be effectively prevented by antibodies.
    Keywords Membrane Architecture ; Cell Biology ; Biochemical Assay ; Science ; Q
    Language English
    Publishing date 2021-03-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

    Theuerkauf, Samuel A. / Michels, Alexander / Riechert, Vanessa / Maier, Thorsten J. / Flory, Egbert / Cichutek, Klaus / Buchholz, Christian J.

    bioRxiv

    Abstract: Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between ... ...

    Abstract Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split β-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.
    Keywords covid19
    Language English
    Publishing date 2020-10-15
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2020.10.15.340604
    Database COVID19

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  6. Article ; Online: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

    Theuerkauf, Samuel A. / Michels, Alexander / Riechert, Vanessa / Maier, Thorsten J. / Flory, Egbert / Cichutek, Klaus / Buchholz, Christian J.

    bioRxiv

    Abstract: Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between ... ...

    Abstract Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split β-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.
    Keywords covid19
    Publisher BioRxiv; WHO
    Document type Article ; Online
    DOI 10.1101/2020.10.15.340604
    Database COVID19

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    Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

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