Carrier for targeting nerve cells
The present invention relates to a transport protein which can be obtained by modifying the heavy chain of the neurotoxin formed by Clostridium botulinum wherein (i) the protein binds specifically to nerve cells with a higher or lower affinity as the native neurotoxin; (ii) the protein has an increased or reduced neurotoxicity compared to the native neurotoxin, the neurotoxicity being preferably determined in the hemidiaphragm assay; and/or (iii) the protein comprises a lower affinity against neutralizing antibodies compared to the native neurotoxin. The invention also relates to methods for producing the same and the use thereof in cosmetic and pharmaceutical compositions.
1. A transport protein comprising a modified Clostridium botulinum serotype A neurotoxin heavy chain, the heavy chain comprising:
(A) a H N -fragment of Clostridium botulinum serotype A of SEQ ID NOs.: 2, 4 or 5; and
(B) a H C -fragment of Clostridium botulinum serotype A of SEQ ID NOs.: 2, 4 or 5, modified by the substitution of an amino acid at positions 1195, 1196, 1199, 1204, 1205, 1206, 1207, 1213, 1217, 1258, and/or 1260 with either a naturally occurring or a non-naturally occurring amino acid,
wherein the H C -fragment comprises a H CC -fragment and a H CN -fragment, and wherein the H CC -fragment binds to a nerve cell with a higher or lower affinity than native Clostridium botulinum serotype A neurotoxin.
2. The protein of claim 1 , which binds to a nerve cell with an affinity that is at least 15% higher or lower than its binding affinity for native Clostridium botulinum serotype A neurotoxin.
3. The protein of claim 1 , which binds to synaptic vesicle glycoprotein 2 (SV2).
4. The protein of claim 3 , which binds to SV2A, SV2B or SV2C.
5. The protein of claim 1 , wherein the H C -fragment is modified by the substitution of an amino acid at positions 1196, 1199, 1207, 1258, and/or 1260 of SEQ ID NOs.: 2, 4 or 5; and wherein the substitution is with either a naturally occurring or a non-naturally occurring amino acid.
6. The protein of claim 5 , wherein the amino acid at position 1207 is substituted with arginine or tyrosine.
7. The protein of claim 5 , wherein the amino acid at position 1260 is substituted with glutamate.
8. The protein of claim 1 , which protein is covalently linked to a clostridial translocation domain.
9. The protein of claim 8 , which protein is covalently linked to a clostridial neurotoxin protease.
10. The protein of claim 9 , which protein includes an amino acid sequence that is recognized by, and may be cleaved by, a protease to provide a di-chain polypeptide, wherein the clostridial translocation domain and the clostridial neurotoxin protease are linked together by a disulfide-bridge.
11. The protein of claim 10 , wherein the amino acid sequence is a native clostridial amino acid sequence or a non-clostridial amino acid sequence.
12. A method for treating an individual suffering from a disease or disorder selected from hemi-facial spasm, spasmodic torticollis, blepharospasm, spasticities, dystonias, migraine, pain, disorders of the neck and lumber vertebral column, strabism, hypersalivation, snoring, wound healing, and depressive disorders, the method comprising administering a therapeutically effective amount of a protein according to claim 1 to the individual.
13. A method for treating an individual suffering from a cosmetic indication selected from hyperhidrosis and facial wrinkles, the method comprising administering a therapeutically effective amount of a transport protein according to claim 1 to the individual.
14. A process for producing the protein of claim 1 , which protein is produced by recombinant expression.