Intraluminal Vesicles as Transfection Intermediaries

Background. 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 substrate. Previously siRNA has been shown to be delivered to the cytosol using this system. Methods. Here, the localisation of 32P-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. Results. To this end 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 100nM 32P-labelled siRNA, 45±3.2% (±SD; n=3) of the cell associated siRNA was found in the cytosol. After the transfection of 100nM 32P-labelled siRNA using aATx, 77±2.5% (±SD; n=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; n=3) relative to an untreated control by Western analysis. Further, 25μg/mL of WGA inhibited 75.23±0.06% (±SD; n=3) of the knockdown attributed to a non-WGA treated control. Relative to the control, treatment with 200mM sucrose resulted in a reduction of 74.58±7.76% (±SD; n=3) of target gene knockdown. Conclusions. These data indicated that the insertion of the PA pore into endosomal membrane was not weakening the endosomal limiting membrane leading to vesicular bursting during transfection.