Botulinum Neurotoxin Serotype B Activatable Botulinum Neurotoxin Serotype Bs

Abstract

The specification discloses modified Clostridial toxins comprising a Clostridial toxin substrate cleavage site located within the di-chain loop region; polynucleotide molecules encoding such modified Clostridial toxins comprising a Clostridial toxin substrate cleavage site located in the di-chain loop region; and method of producing modified Clostridial toxins comprising Clostridial toxin substrate cleavage site located within the di-chain loop region.

1 . A recombinantly modified Botulinum neurotoxin toxin serotype C1 (BoNT/C1) comprising an enzymatic domain having a zinc-dependent endopeptidase activity which targets a targets a synaptosomal-associated protein of 25 kDa (SNAP-25) or a Syntaxin, a translocation domain, a binding domain and a modified di-chain loop region, the modified di-chain loop region intervening between the enzymatic domain and the translocation domain;
 wherein the di-chain loop region modification comprises a BoNT/C1 substrate cleavage site from a SNAP-25 or a Syntaxin of at least six amino acids in length located within the di-chain loop region that is proteolytically cleaved by the enzymatic domain of the recombinantly modified BoNT/C1; and   wherein expression of the recombinantly modified BoNT/C1 within a cell results in single chain to di-chain conversion of the recombinantly modified BoNT/C1 due to proteolytic cleavage of the BoNT/C1 substrate cleavage site.   
     
     
         2 . The modified BoNT/C1 according to 2, wherein the BoNT/C1 substrate cleavage site comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Arg-Ala. 
     
     
         3 . The modified BoNT/C1 according to 2, wherein the BoNT/C1 substrate cleavage site comprises at least six consecutive residues of a Syntaxin, said six consecutive residues comprising Lys-Ala. 
     
     
         4 . The modified BoNT/C1 according to 2, wherein the BoNT/C1 substrate cleavage site comprises at least six consecutive residues of a Syntaxin, said six consecutive residues comprising Arg-Ala. 
     
     
         5 . The modified BoNT/C1 according to 2, wherein the BoNT/C1 substrate cleavage site comprises SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, or SEQ ID NO: 121. 
     
     
         6 . The modified BoNT/C1 according to  claim 1 , wherein the modification further comprises a flexible region comprising one or more flexible spacers located within the di-chain loop region. 
     
     
         7 . The modified BoNT/C1 according to  claim 6 , wherein the one or more flexible spacer are G-spacer, A-spacers or any combination thereof. 
     
     
         8 . A polynucleotide molecule encoding a modified BoNT/C1 of  claim 1 . 
     
     
         9 . A method of producing a modified BoNT/C1 comprising the step of expressing in a cell a polynucleotide molecule of  claim 8 , wherein expression from the polynucleotide molecule produces a modified BoNT/C1. 
     
     
         10 . A method of producing a modified BoNT/C1 comprising the steps of:
 a. introducing into a cell a polynucleotide molecule of  claim 8 ; and   b. expressing the polynucleotide molecule, wherein expression from the polynucleotide molecule produces a modified BoNT/C1.