Volkmar Weissig
Midwestern University, USA
Title: DQAsome-based delivery of Phosphorothioate gapmer antisense oligonucleotides as therapy for Clostridium difficile
Biography
Biography: Volkmar Weissig
Abstract
Statement of the Problem: C. difficile infection (CDI) is a major healthcare burden due its prevalence, its communicable nature within healthcare settings, its frequent need for multiple rounds of conventional antibiotics and its predilection for severe forms of colitis.Unpredictable responses associated with conventional antibiotics have raised significant interest in designing alternative CDI therapies, among which “antisense antibiotics” (1) able to prevent the expression of bacterial genes through posttranscriptional mechanisms appeared of particular interest to us.
The purpose of this study was to test DQAsome (2-4) -like cationic nanovesicles (Figure 1, left) composed of bolaamphiphiles (Figure 1, right) as a delivery system for 2’-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO) in order to target the expression of essential genes of C. difficile. Methodology: The ASO were assessed for their ability to inhibit mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Bolasomes were prepared as described (2-4) and characterized by particle size distribution, zeta potential and oligonucleotide binding capacity. Anaerobic C. difficile log phase cultures were treated with serial doses of nanocomplexes obtained from incubating the cationic bolasomes with ASO’s. Results: Antisense gapmers for four chosen genetargets achieved nanomolar minimum inhibitory concentrations for C. difficile. No inhibition of bacterial growth was found in treatments at matched dosages of scrambled gapmers or plain oligonucleotide-free bolasomes compared to untreated cultures (5). Conclusion & Significance: Cationic bolasomes originally developed for the delivery of biologically active molecules to mammalian mitochondria (2-4) can successfully be used to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit growth of C. difficile. The efficient delivery of antisense molecules via DQAsome-like nanovesicles into bacteria will allow the advantageous targeting of virulence functions essential for infection, disease, and recurrence.