Treatment methods using atoxic neurotoxin derivatives

1. A treatment method comprising:

selecting a subject in need of therapeutic treatment involving induction of muscle paralysis and

contacting the subject with an isolated, physiologically active derivative of a wild type Clostridium botulinum neurotoxin, wherein the derivative of a Clostridium botulinum neurotoxin comprises one or more amino acid substitutions relative to the wild type Clostridium botulinum neurotoxin that reduces the metalloprotease activity responsible for the toxicity of wild type Clostridium botulinum neurotoxin and wherein the neurotoxin derivative comprises:

a light chain region and

a heavy chain region, wherein the light and heavy chain regions are linked by a disulfide bond, and wherein the light and heavy chain regions are not truncated,

said contacting being carried out to induce muscle paralysis in the subject to treat the subject, with the proviso that the neurotoxin derivative does not possess a cargo attachment peptide sequence at its N-terminus.

2. The method according to claim 1 , wherein the derivative of a Clostridium botulinum neurotoxin is a derivative of Clostridium botulinum serotype A, Clostridium botulinum serotype B, Clostridium botulinum serotype C, Clostridium botulinum serotype D, Clostridium botulinum serotype E, Clostridium botulinum serotype F, or Clostridium botulinum serotype G.

3. The method according to claim 1 , wherein the derivative of a Clostridium botulinum neurotoxin is a recombinant protein.

4. The method according to claim 1 , wherein the treatment is for a dermatologic or aesthetic condition selected from the group consisting of Rhytides, hypertrophic masseter muscles, and focal hyperhydrosis.

5. The method according to claim 1 , wherein the treatment is for a gastroenterological condition selected from the group consisting of esophageal motility disorders, pharyngeal-esophageal spasm, and anal fissure.

6. The method according to claim 1 , wherein the treatment is for a genitourinaric condition selected from the group consisting of neurogenic dysfunction of the urinary tract, overactive bladder, and neuromodulation of urinary urge incontinence.

7. The method according to claim 1 , wherein the treatment is for a neurologic condition selected from the group consisting of tourettes syndrome, focal muscle spasticity or dystonias, cervical dystonia, primary blepharospasm, hemifacial spasm, spasmodic dysphonia, facial nerve disorders, Rasmussen syndrome, amputation pain, voice tremor, crocodile tear syndrome, marginal mandibular nerve paralysis, pain, chest pain of esophageal origin, headache, cerebral palsy, hip adductor muscle dysfunction in multiple sclerosis, neurogenic pain and inflammation, arthritis, iatrogenic parotid sialocele, and chronic TMJ pain and displacement.

8. The method according to claim 1 , wherein the derivative of a Clostridium botulinum neurotoxin has an LD 50 that is at least 1,000-fold higher than the LD 50 of the corresponding wild-type Clostridium botulinum neurotoxin.

9. The method according to claim 1 , wherein the derivative of a Clostridium botulinum neurotoxin accumulates within neuronal cytosol in higher amounts than the corresponding wild-type Clostridium botulinum neurotoxin.

10. The method according to claim 1 , wherein the derivative of a wild type Clostridium botulinum neurotoxin is produced by cleaving a propeptide, wherein the propeptide comprises:

a light chain region;

a heavy chain region; and

an intermediate region connecting the light and heavy chain regions and comprising a highly specific protease cleavage site, wherein said highly specific protease cleavage site has three or more specific adjacent amino acid residues that are recognized by the highly specific protease in order to enable cleavage.

11. The method according to claim 10 , wherein the highly specific protease cleavage site is selected from an enterokinase cleavage site and a tobacco etch virus protease recognition (TEV) sequence.

12. The method according to claim 10 , wherein the propeptide has no low-specificity protease cleavage sites in the intermediate region, said low-specificity protease cleavage sites having two or less adjacent amino acid residues that are recognized by a protease in order to permit cleavage.

13. The method according to claim 10 , wherein the propeptide further comprises a signal peptide coupled to the light chain region, wherein the signal peptide is suitable to permit secretion of the neurotoxin propeptide from a eukaryotic cell to a medium.

14. The method according to claim 13 , wherein the signal peptide is a gp64 signal peptide.

15. The method according to claim 13 , wherein the propeptide further comprises an affinity tag located between the signal peptide and the light chain region.

16. The method according to claim 15 , wherein the affinity tag has a sequence of SEQ ID NO:10.

17. The method according to claim 1 , wherein the heavy chain has no trypsin-susceptible recognition sequences.

18. The method according to claim 1 , wherein the wild type Clostridium botulinum neurotoxin is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO: 7.

19. The method according to claim 1 , wherein the derivative of a Clostridium botulinum neurotoxin is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprising an amino acid substitution in the light chain region.

20. The method according to claim 19 , wherein the amino acid substitution is in a metalloprotease site.

21. The method according to claim 1 , wherein the derivative of a Clostridium botulinum neurotoxin is selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprising a non-native motif in the light chain region.