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Intraluminal Vesicles as Transfection Intermediaries.

Nourhan A M Mahmoud1,2, Hadeer K S Abdelrahman1,2, Benedita K L Feron1,3

  • 1The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK.

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Summary
This summary is machine-generated.

Attenuated anthrax toxin (aATx) facilitates enhanced cytosolic delivery of small interfering RNA (siRNA) via intraluminal vesicles (ILVs), improving gene silencing. Inhibition of ILVs significantly reduces aATx-mediated siRNA translocation and gene knockdown.

Keywords:
anthrax toxindrug deliveryendocytosisintraluminal vesiclesiRNA

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Protein architecture-based transfection systems are explored to manage hepatotropic distribution and toxicity.
  • Attenuated anthrax toxin (aATx) serves as a scaffold for a novel transfection system utilizing intraluminal vesicles (ILVs) for cytosolic delivery.
  • Small interfering RNA (siRNA) attached to LFn-PKR is not a predicted substrate for the aATx translocase (protective antigen (PA)).

Purpose of the Study:

  • To quantify the intracellular localization of siRNA delivered by aATx and correlate it with gene silencing activity.
  • To investigate the role of ILVs in aATx-mediated cytosolic translocation of siRNA.
  • To assess the efficacy of aATx-based siRNA delivery compared to traditional transfection methods.

Main Methods:

  • Radioactive (32P-labeled) siRNA was used to track intracellular distribution after transfection with aATx in MCF-7 cells.
  • Gene silencing efficacy was measured by targeting STAT3 gene expression via Western analysis.
  • Inhibition of ILV formation using hypertonic sucrose or wheat germ agglutinin (WGA) was employed to study its effect on siRNA translocation.

Main Results:

  • aATx-mediated transfection resulted in 77% ± 2.5% of cell-associated siRNA in the cytosol, compared to 45% ± 3.2% with Lipofectamine.
  • aATx delivery led to a significant reduction in STAT3 expression (64.04% ± 14.17%).
  • Inhibition of ILV formation by WGA (75.23% ± 0.06%) or sucrose (74.58% ± 7.76%) markedly reduced STAT3 gene knockdown.

Conclusions:

  • The protective antigen (PA) pore insertion into endosomal membranes does not compromise membrane integrity, preventing vesicular bursting.
  • Intraluminal vesicles (ILVs) play a critical role in the translocase activity of the aATx system.
  • The aATx system demonstrates efficient cytosolic delivery of siRNA, leading to effective gene silencing, with ILVs being essential for this process.