Dyer, Paul D.R., Shepherd, Thomas R., Gollings, Alexander S., Shorter, Susan A., Gorringe-Pattrick, Monique A. M., Tang, Chun-Kit, Cattoz, Beatrice N., Baillie, Les, Griffiths, Peter C. ORCID: https://orcid.org/0000-0002-6686-1271 and Richardson, Simon C.W. ORCID: https://orcid.org/0000-0002-7927-0649 (2015) Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity. Journal of Controlled Release, 220 (A). pp. 316-328. ISSN 0168-3659 (doi:10.1016/j.jconrel.2015.10.054)

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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 ~45.9KDa ~37KDa ~83KDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0±1.0nm 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 30pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes were low (IC50 >100μg/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 72h time course. In HeLa cells, at 200pmol ASO (with PA:LFN-GAL4), 5.4±2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200pmol 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) Synth expression after treatment with 200pmol 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% Synth 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.

Item Type:	Article
Additional Information:	© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Gold Open Access funded by Cardiff University.
Uncontrolled Keywords:	Antisense; Anthrax toxin; siRNA; Endocytosis; Cell biology; RNAi; PEG-dilemma; Non-viral
Subjects:	Q Science > QR Microbiology > QR180 Immunology Q Science > QR Microbiology > QR355 Virology
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Education, Health & Human Sciences > School of Human Sciences (HUM)
Related URLs:	Publisher
Last Modified:	09 Oct 2021 04:45
URI:	http://gala.gre.ac.uk/id/eprint/14012

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