Research ArticleAPPLIED SCIENCES AND ENGINEERING

Attenuated diphtheria toxin mediates siRNA delivery

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Science Advances  01 May 2020:
Vol. 6, no. 18, eaaz4848
DOI: 10.1126/sciadv.aaz4848
  • Fig. 1 Using aDT for siRNA delivery.

    (A) Attenuated AB toxins, such as attenuated DT (aDT), consist of three main components: a receptor binding domain (R) that binds to a receptor on the cell surface, a translocation domain (T) that allows for endosomal escape, and a mutated active domain (a) for the protein to retain its trafficking functions but be no longer toxic to cells. Cargo, such as small interfering RNA (siRNA), can be attached to this a domain. (B) siRNA delivered using aDT occurs in five main steps: (1) binding to the heparin-binding epidermal growth factor (HBEGF) precursor cell surface receptor; (2) endocytosis of the aDT-siRNA cargo; (3) translocation through the endosomal membrane, inserting the a domain and cargo into the cytoplasm; (4) cleavage of the a domain from the rest of the protein; and (5) release of the siRNA into the cytoplasm where it down-regulates the relevant gene.

  • Fig. 2 siRNAs can be conjugated to aDT.

    (A) aDT was engineered to contain a free cysteine as a functional handle (aDT-SH), protected by a SUMO tag and purified using a histidine (His) tag. (B) aDT was reacted with a PEG cross-linker containing both maleimide and DBCO functional groups to obtain DBCO-modified aDT (aDT-DBCO). (C) The presence of the DBCO modification on aDT was confirmed by measuring the absorbance at 280 nm (aDT) and 309 nm (DBCO). Curves shown are aDT before modification (blue) and after DBCO modification (red). A280, absorbance at 280 nm. (D) Azide-modified siRNA was reacted with the DBCO-functionalized aDT to obtain the aDT-siRNA conjugate. RT, room temperature. (E) Modification of the aDT with the siRNA was confirmed via polyacrylamide gel electrophoresis (PAGE) stained with Coomassie blue to localize the DT protein. Lane 1 shows the aDT-DBCO starting material (Mw, ~72,000), and lane 2 shows the aDT-siRNA conjugate (Mw, ~90,000) alone with some unreacted starting material. (F) Purification of the excess siRNA was confirmed via PAGE stained with GelRed to localize the siRNA. Lane 3 shows the aDT-siRNA conjugate along with unreacted siRNA; lane 4 shows the aDT-siRNA conjugate after nickel column purification, with only a small amount of excess siRNA left over.

  • Fig. 3 GSCs express HBEGF, the native receptor for DT.

    Representative confocal images are shown for HBEGF (anti-HBEGF antibody; green) and nucleus (Hoechst; blue). The secondary antibody–only control confirms lack of nonspecific binding. Scale bars, 50 μm.

  • Fig. 4 aDT-siRNA down-regulates ITGB1 expression in GSCs and reduces cellular invasion.

    (A) aDT-ITGB1 (light red bars) down-regulates ITGB1 mRNA expression compared to negative controls (CTL): aDT-NT (black bars) and ITGB1 siRNA only without Lipofectamine (blue bar) at 24 hours after treatment. Positive control is transfected ITGB1 siRNA with Lipofectamine (dark red bar). Data are shown as n = 3, are means ± SD, and are normalized to an untreated control. Data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s correction on the logarithmic data (*P < 0.05 and **P < 0.01). (B) Cells were plated in a 3D hydrogel assay on the surface of preformed hydrogels and treated with aDT-ITGB1 conjugates at the beginning of the experiment. Invasion depth was measured after 5 days. (C) aDT-ITGB1 reduces invasion compared to controls (no treatment and aDT-NT) in a 3D hydrogel model. Representative images are shown. Fifteen-micrometer red beads label the top of the hydrogel; blue cell nuclei are labeled using Hoechst. Scale bar, 50 μm. (D) Invasion depth was quantified as a percentage of the untreated control. Data were analyzed using one-way ANOVA, followed by Tukey’s correction (*P < 0.05 and **P < 0.01). ns, not significant. (E) aDT-ITGB1 did not reduce the number of adhered cells in a 3D hydrogel model. Representative images are shown. Scale bar, 150 μm. (F) Number of adherent cells was quantified by counting the number of cell nuclei; no significant difference was observed, demonstrating that differences in invasion were due to ITGB1 down-regulation. Data were analyzed using one-way ANOVA, followed by Tukey’s correction.

  • Fig. 5 aDT-siRNA down-regulates eIF-3b expression in GSCs and reduces cell viability.

    (A) aDT–eIF-3b (light red bar) down-regulates eIF-3b mRNA expression compared to negative controls: aDT-NT (black bar) and eIF-3b siRNA only without Lipofectamine (blue bar) at 24 hours after treatment. Positive control is transfected siRNA with Lipofectamine (dark red bar). Data are shown as n = 3, are means ± SD, and are normalized to an untreated control. Data were analyzed using one-way ANOVA, followed by Tukey’s correction on the logarithmic data (*P < 0.05 and **P < 0.01). (B) aDT–eIF-3b (red bars) reduces cell viability of GSCs at 48 hours after treatment compared to aDT-NT (black bars) at 100 nM. Data are shown as n = 3, are means + SD, and are normalized to an untreated control. Data were analyzed using one-way ANOVA, followed by Sidak’s correction (*P < 0.05).

Supplementary Materials

  • Supplementary Materials

    Attenuated diphtheria toxin mediates siRNA delivery

    Amy E. Arnold, Laura J. Smith, Greg Beilhartz, Laura C. Bahlmann, Emma Jameson, Roman Melnyk, Molly S. Shoichet

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