Laboratory evolution of botulinum neurotoxins for therapeutic applications

Abstract

The goal of this thesis is to expand the therapeutic potential of botulinum neurotoxins (BoNTs) by using directed evolution to reprogram both major functional domains. Specifically, we aimed to engineer the protease domain to cleave novel, disease-relevant substrates and to reprogram the receptor-binding domain towards targeting of non-neuronal cell types, such as cancer cells. Through this work, BoNTs are transformed from neurotoxins into programmable delivery systems for targeted therapies, with applications ranging from cancer treatment to broader modulation of cell fate. Cancers evolve numerous mechanisms to evade both cell death and immune surveillance. Pyroptosis, an inflammatory form of programmed cell death, offers a promising therapeutic strategy by eliminating cancer cells while simultaneously activating immune responses. In this work, phage-assisted evolution was used to reprogram BoNT proteases to cleave two key activators of pyroptosis: procaspase-1 and gasdermin D. A substrate profiling platform was developed to assess the specificity of both wild-type and engineered proteases. While the wild-type protease failed to induce cell death, evolved variants triggered robust cytotoxicity in multiple cancer cell lines. The gasdermin D-cleaving variant induced strictly pyroptotic death, while the procaspase-1-cleaving variant triggered both pyroptosis and apoptosis, suggesting broader caspase-like activity. To enable targeted delivery, evolved proteases were reconstituted into BoNT toxins containing the native translocation domain, resulting in selective death of cancer cells but not non-cancerous cells. These findings demonstrate that BoNT proteases can be reprogrammed to modulate inflammatory cell death and serve as programmable tools for targeted cancer therapy. In addition to protease engineering, we reprogrammed the BoNT receptor-binding domain as a potential strategy for cell-specific targeting. Using two target nomination strategies—manual selection and a weighted alignment tool—we successfully evolved binders against all nominated targets. A bacterial two-hybrid circuit paired with phage-assisted evolution supported the evolution of binders to CD44v6, CD30, ACHA3, CD1b, RXFP1, and TSN8. Notably, evolved variants targeting ACHA3 and RXFP1 bound to full-length ectodomains with single-digit nanomolar affinity, exceeding the affinity of the wild-type BoNT HC for its native receptor. While in-cell and in vivo validations are ongoing, this work lays the foundation for BoNT HC evolution, analogous to antibody engineering strategies. Collectively, these studies demonstrate that BoNTs are highly reprogrammable and can be evolved for diverse therapeutic applications. By combining protease and receptor-binding domain engineering, this platform offers a blueprint for developing programmable protein-based therapeutics that modulate cell fate with precision.