Journal Article

Intraluminal Vesicles as Transfection Intermediaries

Tripathi, Vineeta, Gomez, Timothy, Mahmoud, Nourhan A M, Feron, Benedita K L, Miest-Bray, Joanna J, Youens, Natalie, Richardson, Simon C W, Pintilie, Andra, Abdelrahman, Hadeer K S, Fivaz, Marc

2025

Overview

To address hepatotropic body distribution and toxicity, transfection systems based on protein architecture have been proposed. Attenuated anthrax toxin (aATx) has provided the backbone for a first in class transfection system that, in the wild, uses intraluminal vesicles (ILVs) as an intermediary compartment during the translocation of large molecules into the cytosol. Small interfering (si)RNA molecules non-covalently attached to a carrier (LFn-PKR) would not be predicted to be an aATx translocase (protective antigen (PA)) substrate. Previously, siRNA has been shown to be delivered to the cytosol using this system. Here, the localisation of P-labelled siRNA delivered using aATx was quantified directly and related to siRNA activity. In addition, inhibition of ILV formation by hypertonic sucrose or wheatgerm agglutinin (WGA) was shown to inhibit the aATx-mediated cytosolic translocation of siRNA. MCF-7 cells were used to establish siRNA intracellular distribution in relation to pharmacological activity by targeting STAT3 gene expression. After Lipofectamine-mediated transfection using 100 nM P-labelled siRNA, 45 ± 3.2% (±SD; = 3) of the cell associated siRNA was found in the cytosol. After the transfection of 100 nM P-labelled siRNA using aATx, 77 ± 2.5% (±SD; = 3) of the cell associated siRNA was found in the cytosol and resulted in a reduction in STAT3 expression of 64.04 ± 14.17% (±SD; = 3) relative to an untreated control by Western analysis. Further, 25 μg/mL of WGA inhibited 75.23 ± 0.06% (±SD; = 3) of the knockdown attributed to a non-WGA-treated control. Relative to the control, treatment with 200 mM sucrose resulted in a reduction of 74.58 ± 7.76% (±SD; = 3) of target gene knockdown. These data indicated that the insertion of the PA pore into endosomal membrane did not weaken the endosomal limiting membrane, leading to vesicular bursting during transfection and ILVs played critical role in translocase activity.

• Publisher: MDPI AG,Multidisciplinary Digital Publishing Institute (MDPI)
• Subject: anthrax toxin / Cell culture / drug delivery / endocytosis / Ethylenediaminetetraacetic acid / Gene expression / Infection / intraluminal vesicle / Kinases / Proteins / Regulatory approval / RNA / siRNA / Sucrose / Toxicity / Toxins