Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

Abstract

Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe the novel application of cationic bolasomes to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit the growth of C. difficile.

Keywords: Clostridium difficile; antisense; bacteria; cationic bolaamphiphiles; dequalinium derivatives; gapmers; nanocomplex.

Figure 1

Cationic bolaamphiphile chemical structures. Notes: (A) Dequalinium: 1,1′-decane-1,10-diylbis(4-amino-2-methylquinolinium) decyl-2-methyl-4-quinolin-1-iumamine dichloride. C₃₀H₄₀Cl₂N₄ MW =527.58. (B) 10-cyclohexyl-DQA: 10-10′-(decane-1,10-diyl)bis(9-amino-1,2,3,4-tetrahydroacridinium) dichloride. C₃₆H₄₈N₄CL₂ MW =607.71. (C) 12-cyclohexyl-DQA: 10-10′-(dodecane-1,12-diyl) bis(9-amino-1,2,3,4-tetrahydroacridinium) dichloride. C₃₈H₅₂N₄CL₂ MW =635.76.

Figure 2

Hypothetical cationic bolaamphiphile nanovesicle structure.

Figure 3

Representative antisense sequence targeting open loop local secondary structures within the 5′UTR of an mRNA transcript (eg, C. difficile rpoB). Note: Antisense gapmer is depicted in red. Abbreviation: 5′-UTR, 5-untranslated region.

Figure 4

Luciferase reporter and protein expression plasmids. Notes: (A) Representative 5′UTR-modified luciferase reporter plasmid (eg, dnaE); (B) Full-length dnaE expression plasmid. Abbreviation: 5′-UTR, 5-untranslated region.

Figure 5

Antisense gapmer luciferase reporter assays. Notes: Inhibition of luciferase translation expressed as relative luciferase units in cell-free reactions programmed with matched 5′UTR-modified luciferase reporter plasmid by antisense gapmers (0–5 µM; shown as log₁₀ nM) targeting C. difficile 5′UTR sequences and a scrambled gapmer control. Standard error of the mean for each set of samples is indicated. Abbreviation: 5′-UTR, 5-untranslated region.

Figure 6

Immunoblot densitometry of triplicate antisense and scrambled gapmer (0–1 µM) on cell-free translation of C. difficile dnaE protein. Notes: Protein (dnaE) levels are expressed as percent of untreated controls. Standard error of the mean for each set of samples is indicated.

Figure 7

Image of compound synthesis. Note: Tricyclic aromatic amine 1 was heated together with alkyl dichloride 2, linking the two acridine moieties with an alkyl chain.

Figure 8

(A) Oligreen dye-exclusion assays to determine bolasome binding capacity using 2 µg gapmer ASO. (B) Bolasome complexation and retention of phosphorothioate antisense gapmers following metaphor electrophoresis and SYBR Green II staining. Lanes 1–4: Amine-to-phosphate (N/P) ratio =0, 0.5, 2.5, 5.0. Abbreviation: ASO, antisense oligonucleotides.

Figure 9

Dose effects of 12-cyclohexyl-DQA gapmer nanocomplexes (0–0.8 µM gapmer) and matching levels of gapmer-free 12-cyclohexyl-DQA bolasomes (1.2–9.6 µM) on the growth C. difficile ribotype 027 in 24-hour endpoint BHIS broth cultures (n=3). Notes: The data are expressed as average CFU/mL values normalized to the initial inoculum CFU. Error bars represent standard deviation observed for each group of experiments. Abbreviation: CFU, colony-forming units.