Treatment of STEC infection via CRISPR-Cas targeted cleavage of the Shiga toxin gene in animal models

Abstract Escherichia coli is a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extra-intestinal infections. Shiga toxin-producing E. coli (STEC) pose a significant public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome (HUS), a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections due to their potential to induce prophages carrying Shiga toxin (stx) genes, triggering toxin production. Here, we present a CRISPR-based antimicrobial strategy that selectively targets and eliminates O157 STEC clinical isolates while preventing toxin release. We designed a Cas12 nuclease to cleave >99% of all stx variants found in O157 strains, leading to bacterial killing and suppression of toxin production. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a non-replicative DNA payload to E. coli O157, preventing its dissemination. In a mouse STEC colonization model, our therapeutic candidate, EB003, reduced bacterial burden by a factor of 3×103. In an infant rabbit disease model, EB003 mitigated clinical symptoms, abrogated stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens. Competing Interest Statement All authors are current or former employees, or paid advisors, of Eligo Bioscience. Eligo Bioscience owns US patent nos. US11,905,516, US10,808,254, US11,078,490, US11,946,056, US11,661,443, US11,236,133, US11,512,116, US11584918, US11970716, US11746352, US12,098,372, and US11584781; Japanese patent No. JP7250702; Japanese patent No. JP7627223; Korean patent No. KR10-2563835; Israel patent No. 267932; and international patent application Nos. WO2018/141907, WO2020/109339, WO2020/187836, WO2022/144381 and WO2022/144382 relating to certain research described in this article.