Cell-specific targeting of extracellular vesicles though engineering the glycocalyx

Extracellular vesicles (EVs) are promising carriers for the delivery of a variety of chemical and biological drugs. However, their efficacy is limited by the lack of cellular specificity. Available methods to improve the tissue specificity of EVs predominantly rely on surface display of proteins and peptides, largely overlooking the dense glycocalyx that constitutes the outermost layer of EVs. I

Researchers from the Karolinska Institutet have developed a reconfigurable glycoengineering strategy that can endogenously display glycans of interest on EV surface. Briefly, EV producer cells are genetically engineered to co-express a glycosylation domain (GD) inserted into the large extracellular loop of CD63 (a well-studied EV scaffold protein) and fucosyltransferase VII (FUT7) or IX (FUT9), so that the engineered EVs display the glycan of interest. Through this strategy, the researchers showcase surface display of two types of glycan ligands, sialyl Lewis X (sLeX) and Lewis X, on EVs and achieve high specificity towards activated endothelial cells and dendritic cells, respectively. Moreover, the endothelial cell-targeting properties of sLeX-EVs were combined with the intrinsic therapeutic effects of mesenchymal stem cells (MSCs), leading to enhanced attenuation of endothelial damage. In summary, this study presents a reconfigurable glycoengineering strategy to produce EVs with strong cellular specificity and highlights the glycocalyx as an exploitable trait for engineering EVs.

Production and cellular uptake of LeX-EVs

(a) Scheme of surface display of LeX on CD63. A glycosylation domain (GD) was inserted to the large extracellular loop of CD63 to form a novel sLeX protein carrier. Fucosyltransferase IX (FUT9) subsequently catalyzed the synthesis of LeX glycan. The luciferase Nluc was fused to the C-terminal of CD63 for labelling engineered EVs. (b) Detection of LeX on EVs captured by CD63-coated beads. (c) Size distribution profile of EVs. (d) Elution profile of Nluc-labelled EVs in size exclusion chromatography. Nluc in each fraction was normalized to total input. (e) Binding of Nluc-labelled EVs to recombinant human DC-SIGN. Microplates were coated with human DC-SIGN and incubated with EVs for 2 h. The amount of bound EVs were presented as fold-change over vehicle-coated plates. N = 3. (f) Expression of DC-SIGN on mouse bone marrow-derived dendritic cells (mBMDCs). (g) Relative quantification of cellular Nluc in mBMDCs after treatment with 2e (Kwon et al., 2021)/ml EVs for 6 h. N = 4. (h) Histogram of mBMDCs after treatment with the same amount of HiBiT-mNG-labeled EVs for 6 h. (i) Relative quantification of cellular MFI after treatment with the same amount of HiBiT-mNG-labeled EVs for 6 h or 16 h. N = 5. The amount of cellular EVs in (g) and (i) were presented as fold-change over non-targeting EVs. Data are shown as mean ± standard deviation. CN: CD63-Nluc; F9CPN: FUT9 + CD63-P19-Nluc; F9CCN: FUT9+ CD63-CTP-Nluc. CH: CD63-mNG-HiBiT; F9CPH: FUT9 + CD63-P19-mNG-HiBiT; F9CCH: FUT9+ CD63-CTP-mNG-HiBiT.Two-tailed unpaired t-test. n.s.: non-significant; **p < 0.01; ***p < 0.001

Zheng W, He R, Liang X, Roudi S, Bost J, Coly PM, Niel GV, Andaloussi SEL. (2022) Cell-specific targeting of extracellular vesicles though engineering the glycocalyx. J Extracell Vesicles 11(12):e12290. [article]

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