Degradable clostridial toxins

Abstract

The specification discloses modified Clostridial toxins comprising a PAR ligand domain, a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and a Clostridial toxin binding domain; polynucleotide molecules encoding modified Clostridial toxins comprising a PAR ligand domain, a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and a Clostridial toxin binding domain; and method of producing modified Clostridial toxins comprising a PAR ligand domain, a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and a Clostridial toxin binding domain.

1 . A method of treating a human in need thereof comprising administration of a therapeutically effective amount of modified Clostridial toxin, said modified Clostridial toxin comprising:
 a) a protease-activated G protein-coupled receptor (PAR) ligand domain, wherein the PAR ligand domain comprises a masked or unmasked PAR ligand domain, and wherein the unmasked PAR ligand domain binds to a PAR ligand binding domain of a target cell under physiological conditions;   b) a Clostridial toxin enzymatic domain that proteolytically cleaves a substrate of a Clostridial toxin;   c) a Clostridial toxin translocation domain that executes a translocation step of a Clostridial toxin intoxication process; and   d) a Clostridial toxin binding domain that that executes a cell binding step of a Clostridial toxin intoxication process.   
     
     
         2 . The method according to  claim 1 , wherein the PAR ligand domain is operationally-linked to the amino terminus of the Clostridial toxin enzymatic domain. 
     
     
         3 . The method according to  claim 2 , wherein the modified Clostridial toxin comprises an amino to carboxyl single polypeptide linear order comprising the PAR ligand domain, the Clostridial toxin enzymatic domain, the Clostridial toxin translocation domain and the Clostridial toxin binding domain. 
     
     
         4 . The method according to  claim 2 , wherein the modified Clostridial toxin comprises an amino to carboxyl single polypeptide linear order comprising the PAR ligand domain, the Clostridial toxin enzymatic domain, the Clostridial toxin binding domain and the Clostridial toxin translocation domain. 
     
     
         5 . The method according to  claim 1 , wherein the PAR ligand domain is operationally-linked to the amino terminus of the Clostridial toxin translocation domain. 
     
     
         6 . The method according to  claim 5 , wherein the modified Clostridial toxin comprises an amino to carboxyl single polypeptide linear order comprising the Clostridial toxin binding domain, the Clostridial toxin enzymatic domain, the PAR ligand domain and the Clostridial toxin translocation domain. 
     
     
         7 . The method according to  claim 5 , wherein the modified Clostridial toxin comprises an amino to carboxyl single polypeptide linear order comprising the Clostridial toxin enzymatic domain, the PAR ligand domain, the Clostridial toxin translocation domain and the Clostridial toxin binding domain. 
     
     
         8 . The method according to  claim 1 , wherein the PAR ligand domain is operationally-linked to the amino terminus of the Clostridial toxin binding domain. 
     
     
         9 . The method according to  claim 8 , wherein the modified Clostridial toxin comprises an amino to carboxyl single polypeptide linear order comprising the Clostridial toxin enzymatic domain, the PAR ligand domain, the Clostridial toxin binding domain and the Clostridial toxin translocation domain. 
     
     
         10 . The method according to  claim 1 , wherein the modified Clostridial toxin further comprises a protease cleavage site in the masked PAR ligand domain; and wherein cleavage of the protease cleavage site unmasks the PAR ligand domain. 
     
     
         11 . The method according to  claim 1 , wherein the PAR ligand domain comprises a PAR1 ligand domain. 
     
     
         12 . The method according to  claim 11 , wherein the PAR1 ligand Domain comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 133. 
     
     
         13 . The method according to  claim 1 , wherein the modified Clostridial toxin is a modified  Botulinum  toxin comprising a PAR ligand domain, a  Botulinum  toxin enzymatic domain, a  Botulinum  toxin translocation domain and a  Botulinum  toxin binding domain. 
     
     
         14 . The method according to  claim 13 , wherein the modified  Botulinum  toxin is a modified BoNT/A comprising a PAR ligand domain, a BoNT/A enzymatic domain, a BoNT/A translocation domain and a BoNT/A binding domain. 
     
     
         15 . The method of  claim 1 , wherein the human is treated for achalasia, adult spasticity, anal fissure, back pain, blepharospasm, bruxism, cervical dystonia, essential tremor, glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismus and/or VII nerve disorder.