Engineering botulinum neurotoxin to extend therapeutic intervention

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Clostridium botulinum neurotoxins (BoNTs) are valuable therapeutics for neurological disorders due to their neuronal targeting and cleavage of SNARE proteins.
• Current therapeutic applications of BoNTs are limited to neuronal targets.
• Extending BoNTs' therapeutic scope to nonneuronal cells requires retargeting their catalytic activity to nonneuronal SNARE isoforms.

Purpose of the Study:

• To engineer a BoNT derivative capable of cleaving nonneuronal SNARE proteins.
• To investigate the potential of targeting SNAP23 for therapeutic applications in nonneuronal cells.

Main Methods:

• Genetic engineering of BoNT/E light chain (LC/E) to create a derivative (LC/E(K(224)D)) with altered substrate specificity.
• Testing the enzymatic activity of LC/E(K(224)D) against various SNARE proteins, including SNAP23, SNAP25, SNAP29, and SNAP47.
• Delivering the engineered BoNT derivative into cultured human epithelial cells to assess its effect on endogenous SNAP23 and downstream secretion processes.

Main Results:

• The engineered LC/E(K(224)D) demonstrated extended substrate specificity, cleaving both nonneuronal SNAP23 and neuronal SNAP25, but not SNAP29 or SNAP47.
• Direct delivery of LC/E(K(224)D) into human epithelial cells resulted in the cleavage of endogenous SNAP23.
• Cleavage of SNAP23 by LC/E(K(224)D) effectively inhibited the secretion of mucin and IL-8 in cultured cells.

Conclusions:

• Genetically modifying BoNT light chains is feasible to retarget their catalytic activity towards nonneuronal SNARE proteins like SNAP23.
• The engineered BoNT derivative LC/E(K(224)D) shows promise for developing novel therapeutics.
• This approach extends the therapeutic potential of BoNTs for treating human hypersecretion diseases.