Non-neuronal snare-cleaving botulinum neurotoxins

Abstract

The present invention provides a modified botulinum neurotoxin A (BoNT/A) L-chain protease that demonstrates enhanced cleaveage of human SNAP-23 (hSNAP-23) relative to unmodified (wild-type) BoNT/A L-chain protease, together with the use thereof for cleaving hSNAP-23.

1 . A modified botulinum neurotoxin A (BoNT/A) L-chain protease that demonstrates increased human SNAP-23 (hSNAP-23) cleavage relative to wild-type BoNT/A L-chain (SEQ ID NO: 1) and having a modified amino acid sequence relative to the wild-type BoNT/A L-chain (SEQ ID NO: 1), that comprises:
 a) at least one amino acid residue change located within a BoNT/A L-chain protease binding pocket for binding to the I198 binding site of hSNAP-23;   b) wherein said BoNT/A L-chain protease binding pocket for binding to the I198 binding site of hSNAP-23 is defined by amino acid residue K166 of wild-type BoNT/A L-chain (SEQ ID NO: 1);   c) and wherein said at least one amino acid residue change comprises an amino acid residue selected from the group consisting of valine, phenylalanine, leucine, and isoleucine at the position on the modified L-chain protease amino acid sequence that corresponds to amino acid residue K166 of wild-type BoNT/A L-chain (SEQ ID NO: 1).   
     
     
         2 . The modified BoNT/A L-chain protease according to  claim 1 , further comprising:
 a) an amino acid residue change located within a BoNT/A L-chain protease binding pocket for binding to the D189/D192 binding site of hSNAP-23;   b) wherein said BoNT/A L-chain protease binding pocket for binding to the D189/D192 binding site of hSNAP-23 is defined by amino acid residue Q29 of wild-type BoNT/A L-chain (SEQ ID NO: 1);   c) and wherein said amino acid residue change comprises an amino acid residue selected from the group consisting of alanine at the position on the modified L-chain protease amino acid sequence that corresponds to amino acid residue Q29 of wild-type BoNT/A L-chain (SEQ ID NO: 1).   
     
     
         3 . (canceled) 
     
     
         4 . The modified BoNT/A L-chain protease according to  claim 1 , further comprising:
 a) at least one amino acid residue change located within a BoNT/A L-chain protease binding pocket for binding to the K185 binding site of hSNAP-23;   b) wherein said BoNT/A L-chain protease binding pocket for binding to the K185 binding site of hSNAP-23 is defined by amino acid residue G305 of wild-type BoNT/A L-chain (SEQ ID NO: 1);   c) and wherein said at least one amino acid residue change comprises an amino acid residue selected from the group consisting of glutamate and aspartate at the position on the modified L-chain protease amino acid sequence that corresponds to amino acid residue G305 of wild-type BoNT/A L-chain (SEQ ID NO: 1).   
     
     
         5 . The modified BoNT/A L-chain protease according to  claim 1 , further comprising:
 a) an amino acid residue change located within a BoNT/A L-chain protease binding pocket for binding to the R186 binding site of hSNAP-23;   b) wherein said BoNT/A L-chain protease binding pocket for binding to the R186 binding site of hSNAP-23 is defined by amino acid residue S143 of wild-type BoNT/A L-chain (SEQ ID NO: 1);   c) and wherein said amino acid residue change comprises an amino acid residue selected from the group consisting of glutamine, glutamate and aspartate at the position on the modified L-chain protease amino acid sequence that corresponds to amino acid residue S143 of wild-type BoNT/A L-chain (SEQ ID NO: 1).   
     
     
         6 . The modified BoNT/A L-chain protease for  claim 1 , further comprising:
 a) at least one amino acid residue change located within a BoNT/A L-chain protease binding pocket for binding to the K206 binding site of hSNAP-23;   b) wherein said BoNT/A L-chain protease binding pocket for binding to the K206 binding site of hSNAP-23 is defined by amino acid residue Y251 of wild-type BoNT/A L-chain (SEQ ID NO: 1);   c) and wherein said at least one amino acid residue change comprises an amino acid residue selected from the group consisting of glutamate and aspartate at the position on the modified L-chain protease amino acid sequence that corresponds to amino acid residue Y251 of wild-type BoNT/A L-chain (SEQ ID NO: 1).   
     
     
         7 . A nucleic acid construct comprising or consisting of a nucleic acid sequence encoding the modified BoNT/A L-chain protease as defined in  claim 1 . 
     
     
         8 . A delivery vehicle, comprising:
 a) the modified BoNT/A L chain protease as defined in  claim 1 ; and   b) a means for delivering said modified BoNT/A L-chain protease, or said nucleic acid construct, into a target cell, preferably into a non-neuronal target cell.   
     
     
         9 . The delivery vehicle according to  claim 8 , wherein the means b) for delivering said modified BoNT/A L chain protease to a target cell comprises:
 i) a targeting moiety that binds the delivery vehicle to the target cell; and   ii) a translocation peptide that translocates the modified BoNT/A L-chain protease or the nucleic acid construct into the target cell, preferably into a non-neuronal target cell.   
     
     
         10 . A method of cleaving hSNAP-23, comprising contacting hSNAP-23 with the (BoNT/A) L-chain protease according to  claim 1 . 
     
     
         11 - 12 . (canceled) 
     
     
         13 . The modified BoNT/A L-chain protease according to  claim 1 , wherein said at least one amino acid residue change comprises the amino acid residue phenylalanine at the position on the modified L-chain protease amino acid sequence that corresponds to amino acid residue K166 of wild-type BoNT/A L-chain (SEQ ID NO: 1). 
     
     
         14 . A method of cleaving hSNAP-23, comprising contacting hSNAP-23 with the delivery vehicle of  claim 8 . 
     
     
         15 . The modified BoNT/A L-chain protease according to  claim 1 , further comprising a S254A substitution relative to the wild-type BoNT/A L-chain (SEQ ID NO: 1).