Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity

Abstract

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50>100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted.

The Ins and Outs of Anthrax Toxin

“…The method seems to be efficient and of low toxicity. Coupling this delivery system with the targeting properties of engineered PA could be of great use in in vivo downregulation of target genes [ 104 ]. Another study showed that PA was also capable of translocating non-canonical polypeptides, nicely illustrating the broad translocation ability of the PA pore [ 105 ].…”

Green fluorescent protein (GFP): is seeing believing and is that enough?

“…Vero cells (ATCC number CCL-81), were cultured as previously described (Dyer et al, 2015). Purified GST-GFP in PBS was filter sterilised using a 0.2 micron filter (Milipore Ltd, Hertfordshire, UK) and added to cultured cells at a concentration of 1 mg/mL in the presence of 200 µM Leupeptin (Sigma-Aldrich Company Ltd., Dorset, UK).…”

“…This "pulse" of GST-GFP was added to complete cell culture media and incubated for 4 hours at 37 o C. The cells were then washed three times with fresh sterile PBS and returned to incubate in complete media for an additional 20h at 37 o C in 5% (v/v) CO2. The cells were then washed 3 times in PBS and fixed with either cold methanol or formaldehyde (as noted) and as previously described (Dyer et al, 2013;Dyer et al, 2015). Immunostaining was performed as previously described (Richardson et al, 2004;Richardson et al, 2008;Dyer et al, 2013;Dyer et al, 2015) using EEA1 (BD Bioscience,) LAMP1 or LAMP2 specific monoclonal antibodies (DHSB hybridoma bank, University of Iowa, USA) as previously described (Richardson et al, 2004;Richardson et al, 2008;Dyer et al, 2015).…”

The Delivery of Personalised, Precision MedicinesviaSynthetic Proteins

“…The activated PA can oligomerise into heptamers, or the less abundant octamers either in solution or at the cell surface after receptor binding [52]. The exact and specific order of events is controversial as one report details a lack of furinlike proteases at the cell surface and another shows that PA63 can oligomerise into annular structures in 50% serum in the absence of receptors [53]. Further, oligomerisation has been documented in serum after the administration of PA83 in vivo [54].…”

“…It is of note that after translocation into the ILV lumen, the LF and EF are effectively protected from lysosomal hydrolases. Given that the multimeric PA pore is described as a cation selective channel [58] that requires cargo to unfold in order to undergo translocation [62], the observation that supramolecular assemblies containing large anionic components can traverse the PA pore to access the cytosol is of direct interest here [53]. Recently published data describe the cytosolic translocation of both siRNA, antisense oligonucleotides [53], plasmid DNA [62] and plasmid DNA polylysine complexes [63], though it should be noted that plasmid polylysine complexes have been known to rupture endosomal membranes without anthrax toxin components [64].…”