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

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 .

Cationic amphiphilic bolaamphiphile-based delivery of antisense oligonucleotides provides a potentially microbiome sparing treatment for C. difficile

“…CABVs were prepared as previously 9 described. Briefly, all compounds were dissolved in a round-bottom flask using between 2 and 10 mL of methanol followed by removal of the solvent using a rotary evaporator.…”

“…Our group published the first 9 in vitro data for antisense therapies against CDI by complexing cyclohexyl-dequalinium analogues to various ASO targeting essential C. difficile genes. However, since dequalinium has both antibacterial activity as well as toxicity at higher doses, a better delivery compound for ASO is required if antisense approaches to CDI are to be further developed.…”

Mitochondrial Drug Delivery for the Liver Diseases: Comprehensive Review

“…Emulsi ication-solvent evaporation or nanoprecipitation are the methods used for the preparation of polymer particles. By using click chemistry, TPP and PEG are prepared (Hegarty et al, 2016). Mitochondrial membrane by the proteins 7…”

A Review of Experimental and Off-Label Therapies for Clostridium difficile Infection

“…A recent study demonstrated the efficacy of this novel therapy against C. difficile in vitro [59]. The authors observed significant inhibition of bacterial growth with the addition of phosphorothioate gapmer antisense oligonucleotides complementary to C. difficile mRNAs coding essential bacterial enzymes [59].…”