Cationic Amphiphilic Bolaamphiphile-based Delivery of Antisense Oligonucleotides Provides a Potentially Microbiome Sparing Treatment for C. difficile

Conventional antibiotics for C. difficile infection (CDI) have mechanisms of action without organismal specificity, potentially perpetuating the dysbiosis contributing to CDI, making antisense approaches an attractive alternative. Here, three (APDE-8, CODE-9, CYDE-21) novel cationic amphiphilic bolaamphiphiles (CABs) were synthesized and tested for their ability to form nano-sized vesicles or vesicle-like aggregates (CABVs) which were characterized based on their physiochemical properties, their antibacterial activities, and their toxicity toward colonocyte (Caco-2) cell cultures. The antibacterial activity of empty CABVs were tested against cultures of E. coli, B. fragilis and E. faecalis, and against C. difficile by “loading” CABVs with 25-mer antisense oligonucleotides (ASO) targeting dnaE. Our results demonstrate that empty CABVs have minimal colonocyte toxicity until concentrations of 71 μM, with CODE-9 demonstrating the least toxicity. Empty CABVs had little effect on C. difficile growth in culture (MIC90 ≥160 μM). While APDE-8 and CODE-9 nanocomplexes demonstrated high MIC(90) against C. difficile cultures (>300 μM), CYDE-21 nanocomplexes demonstrated MIC(90) at CABV concentrations of 19 μM. Empty CABVs formed from APDE-8 and CODE-9 had virtually no effect on E. coli, B. fragilis and E. faecalis across all tested concentrations, while empty CYDE-21 demonstrated MIC(90) of >160 μM against E. coli and >40μM against B. fragilis and E. faecalis. Empty CABVs have limited antibacterial activity and they can deliver an amount of ASO effective against C. difficile at CABV concentrations associated with limited colonocyte toxicity, while sparing other bacteria. With further refinement, antisense therapies for CDI may become a viable alternative to conventional antibiotic treatment.