US Patent 12060553 Evolution of BoNT peptidases

The disclosure provides amino acid sequence variants of Botulinum neurotoxin (BoNT) proteases that cleave (VAMP1, VAMP2, VAMP7, VAMP8, SNAP25, SNAP23, PTEN, etc.) and methods of evolving the same. In some embodiments, proteases described by the disclosure are useful for cleaving proteins found in a cell, that is in an intracellular environment. In some embodiments, proteases described by the disclosure are useful for treating diseases associated with increased or aberrant VAMP7, VAMP8, SNAP23 or PTEN expression or activity, for example, cancer and neurological disorders. Some aspects of this disclosure provide methods for generating BoNT protease variants by continuous directed evolution.