Leucine-based motif and clostridial neurotoxins

Abstract

Modified neurotoxin comprising neurotoxin including structural modification, wherein the structural modification alters the biological persistence, such as the biological half-life and/or a biological activity of the modified neurotoxin relative to an identical neurotoxin without the structural modification. In one embodiment, methods of making the modified neurotoxin include using recombinant techniques. In another embodiment, methods of using the modified neurotoxin to treat conditions include treating various disorders, neuromuscular aliments and pain.

What is claimed is:  
     
         1 . A modified neurotoxin comprising: 
 a neurotoxin including a structural modification, wherein said structural modification is effective to alter a biological persistence of said modified neurotoxin relative to an identical neurotoxin without said structural modification, and wherein said modified neurotoxin is structurally different from a naturally existing neurotoxin.    
     
     
         2 . The modified neurotoxin of  claim 1 , wherein said structural modification is effective to enhance a biological persistence of said modified neurotoxin.  
     
     
         3 . The modified neurotoxin of  claim 1  wherein said biological persistence of said modified neurotoxin is reduced relative to an identical neurotoxin without said structural modification.  
     
     
         4 . The modified neurotoxin of  claim 1  wherein said structural modification comprises 1 to about 22 amino acids.  
     
     
         5 . The modified neurotoxin of  claim 1  wherein said structural modification comprises an amino acid, said amino acid comprising an R group of 1 to about 12 carbon atoms.  
     
     
         6 . The modified neurotoxin of  claim 1  wherein said structural modification comprises a leucine-based motif (SEQ ID NO: 1).  
     
     
         7 . The modified neurotoxin of  claim 1  wherein said structural modification comprises a tyrosine-based motif.  
     
     
         8 . The modified neurotoxin of  claim 1  wherein said structural modification comprises an amino acid sequence of a botulinum type A light chain and of an amino acid sequence of a type B light chain.  
     
     
         9 . The modified neurotoxin of  claim 8  wherein said structural modification comprises the amino acid sequence KAFK.  
     
     
         10 . The modified neurotoxin of  claim 8  wherein said structural modification comprises the amino acid sequence YYD in combination with the amino acid sequence YYL in combination with the amino acid sequence T.  
     
     
         11 . The modified neurotoxin of  claim 1  wherein said structural modification comprises an amino acid derivative.  
     
     
         12 . The modified neurotoxin of  claim 1  wherein said neurotoxin is selected from the group consisting of botulinum toxin type A, B, C 1 , C 2 , D, E, F and G.  
     
     
         13 . The modified neurotoxin of  claim 1  wherein said neurotoxin is botulinum toxin type A.  
     
     
         14 . A modified neurotoxin comprising a neurotoxin including a structural modification, wherein said neurotoxin comprises three amino acid sequence regions:  
       
         
           
                 
                 
               
                     
                 
                     
                 
                   a) 
                   a first region effective as a cellular 
                 
                     
                   binding moiety; 
                 
                   b) 
                   a second region effective to translocate 
                 
                     
                   a modified neurotoxin or a part thereof 
                 
                     
                   across an endosome membrane; and 
                 
                   c) 
                   a third region effective to inhibit 
                 
                     
                   exocytosis when released into a cytoplasm 
                 
                     
                   of a target cell, 
                 
                     
                 
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       wherein at least one of said first, said second and said third regions is substantially derived from a Clostridial neurotoxin, said third region includes said structural modification, a modified neurotoxin is structurally different from a naturally existing neurotoxin, and said structural modification is effective to alter a biological persistence of said modified neurotoxin relative to an identical neurotoxin without said structural modification.  
     
     
         15 . The modified neurotoxin of  claim 14 , wherein said neurotoxin is a member selected from a group consisting of botulinum toxin serotypes A, B, C 1 , C 2 , D, E, F, G, tetanus toxin and mixtures thereof.  
     
     
         16 . The modified neurotoxin of  claim 14 , wherein said neurotoxin is a member selected from a group consisting of botulinum toxin serotypes A, B, C 1 , C 2 , D, E, F and G.  
     
     
         17 . The modified neurotoxin of  claim 14 , wherein said neurotoxin comprises botulinum toxin serotypes A.  
     
     
         18 . The modified neurotoxin of  claim 14 , wherein said third region is derived from botulinum toxin serotype A.  
     
     
         19 . The modified neurotoxin of  claim 14 , wherein said third region is not derived from botulinum toxin serotype A.  
     
     
         20 . The modified neurotoxin of  claim 14 , wherein said structural modification includes a biological persistence enhancing component effective to enhance said biological persistence of said modified neurotoxin.  
     
     
         21 . The modified neurotoxin of  claim 20 , wherein said biological persistence enhancing component comprises a leucine-based motif of SEQ ID NO: 1.  
     
     
         22 . The modified neurotoxin of  claim 20 , wherein said leucine-based motif comprises a run of seven amino acids, wherein said run comprises a quintet of amino acids and a duplet of amino acids, wherein said quintet of amino acids defines the amino terminal end of said leucine-based motif and said duplet of amino acids defines the carboxyl end of said leucine-based motif.  
     
     
         23 . The modified neurotoxin of  claim 22 , wherein said quintet of amino acids comprises an acidic amino acid, wherein said acidic amino acid is selected from a group consisting of a glutamate and an aspartate.  
     
     
         24 . The modified neurotoxin of  claim 22 , wherein said quintet of amino acids comprises a hydroxyl containing amino acid, wherein said hydroxyl containing amino acid is selected from the group consisting of a serine, a threonine and a tyrosine.  
     
     
         25 . The modified neurotoxin of  claim 24 , wherein said hydroxyl containing amino acid can be phophorylated.  
     
     
         26 . The modified neurotoxin of  claim 22 , wherein said duplet of amino acids comprises at least one amino acid, wherein said amino acid is selected from the group consisting of leucine, isoleucine, methionine, alanine, phenylalanine, tryptophan, valine and tyrosine.  
     
     
         27 . The modified neurotoxin of  claim 22 , wherein said duplet of amino acids is selected from a group consisting of leucine-leucine, leucine-isoleucine, isoleucine-leucine, isoleucine-isoleucine and leucine-methionine.  
     
     
         28 . The modified neurotoxin of  claim 21 , wherein said leucine-based motif comprises an amino acid sequence phenylalanine-glutamate-phenylalanine-tyrosine-lysine-leucine-leucine of SEQ ID NO: 1.  
     
     
         29 . A modified neurotoxin wherein said modification comprises a tyrosine-based motif.  
     
     
         30 . The modified neurotoxin of  claim 29  wherein said tyrosine-based motif comprises a run of four amino acids, wherein an amino acid comprising an N-terminal end of said run comprises a tyrosine residue and an amino acid comprising a C-terminal end of said run comprises a hydrophobic amino acid.  
     
     
         31 . The modified neurotoxin of  claim 14 , wherein said biological persistence of said modified neurotoxin is reduced relative to an identical neurotoxin without said structural modification.  
     
     
         32 . The modified neurotoxin of  claim 31 , wherein said structural modification includes a leucine-based motif with mutation to one or more amino acids comprising said leucine-based motif of SEQ ID NO: 1.  
     
     
         33 . The modified neurotoxin of  claim 31 , wherein said structural modification includes a tyrosine-based motif with a mutation to one or more amino acids comprising said tyrosine-based motif.  
     
     
         34 . The modified neurotoxin of  claim 31 , wherein said structural modification comprises an amino acid derivative with a mutation to one or more amino acids comprising said amino acid derivative.  
     
     
         35 . A method for enhancing the biological persistence of a neurotoxin of  claim 14 , wherein a structural modification is fused or added to said neurotoxin.  
     
     
         36 . The modified neurotoxin of  claim 35  wherein said structural modification comprises a leucine-based motif.  
     
     
         37 . The modified neurotoxin of  claim 35  wherein said structural modification comprises a tyrosine-based motif.  
     
     
         38 . The modified neurotoxin of  claim 35  wherein said structural modification comprises an amino acid derivative.  
     
     
         39 . A modified neurotoxin comprising: a botulinum type A neurotoxin including a structural modification, wherein said structural modification is effective to alter a biological persistence of said modified neurotoxin relative to an identical neurotoxin without said structural modification, wherein said structural modification comprises a deletion of amino acids 1 to 8 and 416 to 437 from a light chain of said neurotoxin.  
     
     
         40 . A modified neurotoxin comprising: 
 a botulinum type A neurotoxin including a structural modification, wherein said structural modification is effective to alter a biological persistence of said modified neurotoxin relative to an identical neurotoxin without said structural modification, wherein said structural modification comprises substitution of leucine at position 427 for an alanine and leucine at position 428 for an alanine in a light chain of said neurotoxin.    
     
     
         41 . A method for reducing the biological persistence of a neurotoxin comprising the step of mutating an amino acid of the neurotoxin.  
     
     
         42 . The method of  claim 41  which comprises the step of deleting or substituting said amino acid of a leucine-based motif within the neurotoxin.  
     
     
         43 . The method of  claim 41  which comprises the step of deleting or substituting said amino acid from a tyrosine-based motif within the neurotoxin.  
     
     
         44 . The method of  claim 41  which comprises the step of deleting or substituting an amino acid derivative within the neurotoxin.  
     
     
         45 . A method of treating a condition, comprising a step of administering an effective dose of a modified neurotoxin to a mammal to treat a condition, wherein said modified neurotoxin comprises a neurotoxin including a structural modification, and wherein said structural modification is effective to alter a biological persistence of said neurotoxin.  
     
     
         46 . The method of treating a condition of  claim 45 , wherein said neurotoxin does not comprise a leucine-based motif.  
     
     
         47 . The method of treating said condition of  claim 46 , wherein said structural modification includes a biological persistence enhancing component.  
     
     
         48 . The method of  claim 47  wherein said biological persistence enhancing component comprises a leucine-based motif.  
     
     
         49 . The method of  claim 47  wherein said biological persistence enhancing component comprises a tyrosine-based motif.  
     
     
         50 . The method of  claim 47  wherein said biological persistence enhancing component comprises an amino acid derivative.  
     
     
         51 . The method of treating said condition of  claim 45 , wherein said condition comprises a condition selected from the group consisting of a neuromuscular disorder, an autonomic disorder and pain.  
     
     
         52 . The method of treating said condition of  claim 51 , wherein treatment of said neuromuscular disorder comprises a step of locally administering an effective amount of said modified neurotoxin to a muscle or group of muscles.  
     
     
         53 . The method of treating said condition of  claim 51 , wherein treatment of said autonomic disorder comprises a step of locally administering an effective amount of said modified neurotoxin to a gland.  
     
     
         54 . The method of treating said condition  claim 51 , wherein treatment of pain comprises a step of administering an effective amount of said modified neurotoxin to a site of pain.  
     
     
         55 . The method of treating said condition of  claim 51 , wherein treatment of pain comprises a step of administering an effective amount of said modified neurotoxin to a spinal cord.  
     
     
         56 . The method of treating said condition of  claim 45 , wherein said condition is selected from the group consisting of spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, pain from muscle spasms, headache pain, brow furrows and skin wrinkles.  
     
     
         57 . A modified neurotoxin comprising: 
 a neurotoxin including a structural modification, wherein said structural modification is effective to alter a biological activity of said modified neurotoxin relative to an identical neurotoxin without said structural modification, and wherein said modified neurotoxin is structurally different from a naturally existing neurotoxin.    
     
     
         58 . The modified neurotoxin of  claim 57 , wherein said structural modification is effective to reduce an exocytosis from a target cell by more than the amount of the exocytosis reduced from the target cell by an identical neurotoxin without said structural modification.  
     
     
         59 . The modified neurotoxin of  claim 57 , wherein said structural modification is effective to reduce an exocytosis from a target cell by less than the amount of the exocytosis reduced from the cell by an identical neurotoxin without said structural modification.  
     
     
         60 . The modified neurotoxin of  claim 58  or  claim 59  wherein the exocytosis is exocytosis of a neurotransmitter.  
     
     
         61 . The modified neurotoxin of  claim 57 , where the modified neurotoxin exhibits an altered biological activity without exhibiting an altered biological persistence.  
     
     
         62 . The modified neurotoxin of  claim 57 , where the modified neurotoxin exhibits an altered biological activity and an altered biological persistence.  
     
     
         63 . The modified neurotoxin of  claim 57 , where the modified neurotoxin exhibits an increased biological activity and an increased biological persistence.  
     
     
         64 . The modified neurotoxin of  claim 57 , where the modified neurotoxin exhibits an increased biological activity and a reduced biological persistence.  
     
     
         65 . The modified neurotoxin of  claim 57 , where the modified neurotoxin exhibits a decreased biological activity and a decreased biological persistence.  
     
     
         66 . The modified neurotoxin of  claim 57 , where the modified neurotoxin exhibits an decreased biological activity and an increased biological persistence.  
     
     
         67 . The modified neurotoxin of  claim 57  wherein said structural modification comprises a leucine-based motif.  
     
     
         68 . The modified neurotoxin of  claim 57 , wherein a unit amount of the modified neurotoxin is more efficient to reduce an exocytosis from a cell than is a unit amount of the naturally existing neurotoxin.