Dr.

Burger-Kentischer, Anke

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  • Publication
    Drug repurposing for the treatment of COVID-19: Targeting nafamostat to the lungs by a liposomal delivery system
    ( 2023)
    Reus, Philipp
    ;
    Guthmann, Hadar
    ;
    Uhlig, Nadja
    ;
    Agbaria, Majd
    ;
    Issmail, Leila
    ;
    Eberlein, Valentina
    ;
    Nordling-David, Mirjam M.
    ;
    Jbara-Agbaria, Doaa
    ;
    Ciesek, Sandra
    ;
    Bojkova, Denisa
    ;
    Cinatl, Jindrich
    ;
    Burger-Kentischer, Anke
    ;
    Rupp, Steffen
    ;
    Zaliani, Andrea
    ;
    Grunwald, Thomas
    ;
    Gribbon, Philip
    ;
    Kannt, Aimo
    ;
    Golomb, Gershon
    Despite tremendous global efforts since the beginning of the COVID-19 pandemic, still only a limited number of prophylactic and therapeutic options are available. Although vaccination is the most effective measure in preventing morbidity and mortality, there is a need for safe and effective post-infection treatment medication. In this study, we explored a pipeline of 21 potential candidates, examined in the Calu-3 cell line for their antiviral efficacy, for drug repurposing. Ralimetinib and nafamostat, clinically used drugs, have emerged as attractive candidates. Due to the inherent limitations of the selected drugs, we formulated targeted liposomes suitable for both systemic and intranasal administration. Non-targeted and targeted nafamostat liposomes (LipNaf) decorated with an Apolipoprotein B peptide (ApoB-P) as a specific lung-targeting ligand were successfully developed. The developed liposomal formulations of nafamostat were found to possess favorable physicochemical properties including nano size (119-147 nm), long-term stability of the normally rapidly degrading compound in aqueous solution, negligible leakage from the liposomes upon storage, and a neutral surface charge with low polydispersity index (PDI). Both nafamostat and ralimetinib liposomes showed good cellular uptake and lack of cytotoxicity, and non-targeted LipNaf demonstrated enhanced accumulation in the lungs following intranasal (IN) administration in non-infected mice. LipNaf retained its anti-SARS-CoV 2 activity in Calu 3 cells with only a modest decrease, exhibiting complete inhibition at concentrations >100 nM. IN, but not intraperitoneal (IP) treatment with targeted LipNaf resulted in a trend to reduced viral load in the lungs of K18-hACE2 mice compared to targeted empty Lip. Nevertheless, upon removal of outlier data, a statistically significant 1.9-fold reduction in viral load was achieved. This observation further highlights the importance of a targeted delivery into the respiratory tract. In summary, we were able to demonstrate a proof-of-concept of drug repurposing by liposomal formulations with anti-SARS-CoV-2 activity. The biodistribution and bioactivity studies with LipNaf suggest an IN or inhalation route of administration for optimal therapeutic efficacy.
  • Publication
    Liposomal siRNA Formulations for the Treatment of Herpes Simplex Virus-1: In Vitro Characterization of Physicochemical Properties and Activity, and In Vivo Biodistribution and Toxicity Studies
    ( 2022)
    Jbara-Agbaria, D.
    ;
    Blondzik, Saskia
    ;
    Burger-Kentischer, Anke
    ;
    Agbaria, M.
    ;
    Nordling-David, M.M.
    ;
    Giterman, A.
    ;
    Aizik, G.
    ;
    Rupp, Steffen
    ;
    Golomb, G.
    Herpes simplex virus-1 (HSV-1) is highly contagious, and there is a need for a therapeutic means to eradicate it. We have identified an siRNA (siHSV) that knocks down gene expression of the infected cell protein 0 (ICP0), which is important in the regulation of HSV infection. The selected siHSV was encapsulated in liposomes to overcome its poor stability, increase cell permeability, and prolonging siRNA circulation time. Several siRNAs against ICP0 have been designed and identified. We examined the role of various parameters, including formulation technique, lipids composition, and ratio. An optimal liposomal siHSV formulation (LipDOPE-siHSV) was characterized with desirable physiochemical properties, in terms of nano-size, low polydispersity index (PDI), neutral surface charge, high siHSV loading, spherical shape, high stability in physiologic conditions in vitro, and long-term shelf-life stability (>1 year, 4â—¦C). The liposomes exhibited profound internalization by human keratinocytes, no cytotoxicity in cell cultures, no detrimental effect on mice liver enzymes, and a gradual endo-lysosomal escape. Mice biodistribution studies in intact mice revealed accumulation, mainly in visceral organs but also in the trigeminal ganglion. The therapeutic potential of siHSV liposomes was demonstrated by significant antiviral activity both in the plaque reduction assay and in the 3D epidermis model, and the mechanism of action was validated by the reduction of ICP0 expression levels.
  • Publication
    The E. coli S30 lysate proteome: A prototype for cell-free protein production
    ( 2018)
    Foshag, D.
    ;
    Henrich, E.
    ;
    Hiller, Ekkehard
    ;
    Schäfer, Miriam
    ;
    Kerger, Christian
    ;
    Burger-Kentischer, Anke
    ;
    Diaz-Moreno, I.
    ;
    García-Mauriño, S.M.
    ;
    Dötsch, V.
    ;
    Rupp, Steffen
    ;
    Bernhard, F.
    Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.