Advancing a Platform Technology for C. difficile Infection Treatment: the Promise of Antisense Oligonucleotides - ABSTRACT Clostridioides difficile infection (CDI) is a debilitating diarrheal disease that is precipitated upon antibiotic-induced gastrointestinal tract dysbiosis. Currently, there is no vaccine to prevent CDI, and the primary FDA-approved treatment relies on substantial antibiotic use. In the hospital as well as community, the elderly and immune- compromised are at most risk; therefore, CDI is a significant problem worldwide. The causative agent, C. difficile, is an anaerobic, spore-forming bacterium designated as an “Urgent Threat” to US healthcare. To date, there is a paucity of organism-specific (precision) antibacterials. Therefore, we developed a novel, water-soluble, cationic bolaamphiphile (CAB) platform nanocarrier, and complexed it with an antisense oligonucleotide (ASO) targeting the essential C. difficile dnaE gene. In preliminary studies, this dnaE*964 nanocomplex was bactericidal, with a C. difficile Minimum Inhibitory Concentration (MIC) comparable to a standard-of-care CDI antibiotic, vancomycin. Crucially, dnaE*964 was inactive against a panel of human intestinal commensal organisms. Pharmacokinetic (PK) assessments in mice and Golden Syrian hamsters revealed that the CAB nanocarrier was safely tolerated, and that it preferentially accumulated in the rodent GI tract; thus, a dosing regimen was defined. In pilot efficacy studies, dnaE*964 ablated CDI pathology, cleared C. difficile from the rodent cecum and stool, and maintained microbiota restitution. dnaE*964 clinical development is therefore in progress. To expand the utility of our CAB platform and also address the inevitable clinical concern of single-target resistance development, we will now evaluate 3 additional ASO leads targeting C. difficile genes encoding cytoplasmic as well as cell wall-associated proteins; all are essential. Preliminary studies reveal that phosphorothioated ASOs customized for each gene completely ablate expression and also inhibit C. difficile growth in a dose-dependent manner. We thus hypothesize that ASO*CAB nanoparticles targeting essential genes (1) will specifically kill C. difficile in vitro and in vivo, and (2) can functionally synergize with each other and with standard-of-care antibiotics without significantly engendering resistance emergence. In vitro (Aim 1; MIC, resistance) and in vivo (Aim 2; efficacy, microbiota sparing) studies will test these hypotheses. Taken together, we thus prioritize key pre-clinical assessments of CAB-based bacterial anti-infectives. Studies in both Aims will be underpinned by quantitative milestones that will inform the translational pathway via a clear framework for go/no-go decisions.
