Engineering an Effective Human SNAP-23 Cleaving Botulinum Neurotoxin A Variant

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Botulinum neurotoxin serotype A (BoNT/A) cleaves SNAP-25 to treat nerve hyperactivity.
• Non-neuronal cell oversecretion, involving SNAP-23, contributes to diseases like COPD and asthma.
• Current BoNT/A is ineffective against human SNAP-23, limiting its therapeutic scope.

Purpose of the Study:

• To engineer BoNT/A to effectively cleave the non-neuronal SNAP-25 isoform, SNAP-23.
• To overcome the resistance of human SNAP-23 to BoNT/A for expanded therapeutic applications.

Main Methods:

• Utilized structural data and mutagenesis of SNAP-23 to optimize BoNT/A's enzymatic domain.
• Performed systematic mutagenesis and rational design to identify key mutations (E148Y, K166F, S254A, G305D).
• Assessed in vitro catalytic activity and in vivo efficacy in primary neurons using engineered BoNT/A.

Main Results:

• Individual mutations increased BoNT/A activity against SNAP-23 by 3- to 23-fold.
• The quadruple mutant exhibited a ~2000-fold increase in catalytic activity against human SNAP-23 in vitro.
• The full-length BoNT/A quadruple mutant showed enhanced activity in primary neurons expressing SNAP-23.

Conclusions:

• The engineered quadruple mutant BoNT/A effectively cleaves human SNAP-23.
• This enhanced BoNT/A holds promise for treating diseases involving non-neuronal cell oversecretion.
• Further testing in immune cells is warranted for potential therapeutic development.