Transport protein which is used to introduce chemical compounds into nerve cells
The invention relates to a transport protein which can be obtained by modifying the heavy chain of the neurotoxin formed by Clostridium botulinum . The protein binds specifically to nerve cells with a higher affinity as the native neurotoxin. The invention also relates to a method for the production of transport protein, the nucleic acids coding for the transport protein, the transport protein containing pharmaceutical and cosmetic compositions and use thereof.
1. An isolated, non-naturally occurring botulinum neurotoxin type A (BoNT/A) transport protein which binds to the nerve cells with a higher affinity than the native neurotoxin, wherein at least one amino acid of the native neurotoxin corresponding to positions 1117, 1252 and 1253 of the native botulinum neurotoxin type A protein sequence has been substituted by a naturally occurring amino acid, wherein the native botulinum neurotoxin type A protein sequence has protein database Accession Number AAA23262 (SEQ ID No. 1).
2. The transport protein according to claim 1 , wherein the amino acid corresponding to position 1117 of the native botulinum neurotoxin type A protein sequence has been removed substituted by a naturally occurring amino acid.
3. The transport protein according to claim 2 , wherein the amino acid corresponding to position 1117 of the native botulinum neurotoxin type A protein sequence has been substituted with an amino acid selected from the group consisting of alanine, cysteine, serine, threonine and valine.
4. The transport protein according to claim 2 , wherein the amino acid corresponding to position 1117 of the native botulinum neurotoxin type A protein sequence has been substituted with an amino acid selected from the group consisting of alanine, cysteine and valine.
5. The transport protein according to claim 1 , wherein the amino acid corresponding to position 1252 of the botulinum neurotoxin type A protein sequence is substituted by tyrosine or wherein the amino acid corresponding to position 1253 of the botulinum neurotoxin type A protein sequence is substituted by lysine.
6. The transport protein according to claim 1 , wherein the protein binds specifically to nerve cells and enters the cells by endocytosis.
7. The transport protein according to claim 1 , wherein the protein binds specifically to complex gangliosides of cholinergic motor neurons, localised in the plasma membrane, preferably GT1b.
8. A composition comprising a transport protein according to claim 1 and a protease, wherein the protease includes a light chain of a neurotoxin protein of Clostridium botulinum type A, B, C1, D, E, F or G, and wherein the protease and the transport protein are covalently bonded by an amino acid sequence, which is specifically recognized and split by an endopeptidase.
9. The composition according to claim 8 , wherein after splitting by the endopeptidase a disulphide-bridge interlinks the protease and the transport protein, which, in turn, results in the formation of an active holotoxin.
10. The composition according to claim 8 , wherein the protease has the sequence His-Glu-Leu-Xaa-His-(Xaa) 33-35 -Glu-(Xaa) 84-90 -Glu-(Xaa) 11 -Arg-Xaa-Xaa-Tyr, wherein Xaa may be any amino acid (SEQ ID NO:11).
11. The composition according to claim 8 , wherein the protease splits specific substrates within cholinergic motor neurons.
12. The composition according to claim 11 , wherein the substrates are selected from proteins involved in the release of neurotransmitters in peripheral nerves, or from proteins capable of catalytic reactions within the nerve cell.
13. A pharmaceutical composition, comprising the composition according to claim 8 , as well as optionally a pharmaceutically acceptable excipient, diluent and/or additive.
14. A cosmetic composition comprising the composition according to claim 8 , as well as optionally a pharmaceutically acceptable excipient, diluent and/or additive.
15. A method for treating a disorder or disease for which a therapy with botulinum neurotoxin is indicated, said method comprising administration of an effective amount of the pharmaceutical composition according to claim 13 .
16. A method of claim 15 , wherein the disorder or disease is selected from the group consisting of hemi-facial spasm, spasmodic torticollis, spasticities, dystonias, migraine, pain, disorders of the neck-and lumber vertebral column, strabism, and hypersalivation.
17. A method for treating a cosmetic indication selected from hyperhydrosis and pronounced facial wrinkles, said method comprising administration of an effective amount of the cosmetic composition according to claim 14 .