FUSION PROTEINS AND METHODS FOR TREATING, PREVENTING OR AMELIORATING PAIN
A single chain polypeptide fusion protein, comprising: a non-cytotoxic protease capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a galanin targeting moiety; a protease cleavage site at which site the fusion protein is cleavable by a protease; a translocation domain capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; a first spacer located between the non-cytotoxic protease and the protease cleavage site; and a second spacer located between the galanin targeting moiety and the translocation domain.
1 . A single chain polypeptide fusion protein, comprising:
a non-cytotoxic protease capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent;
a galanin-targeting moiety that binds to a binding site on the nociceptive sensory afferent, the binding site capable of incorporating into an endosome within the nociceptive sensory afferent;
a protease cleavage site at which site the fusion protein is cleavable by a protease;
a translocation domain capable of translocating the protease from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory
a first spacer located between the non-cytotoxic protease and the protease cleavage site, the first spacer comprising an amino acid sequence of from 4 to 25 amino acid residues; and
a second spacer located between the galanin-targeting moiety and the translocation domain, the second spacer comprising an amino acid sequence of from 4 to 35 amino acid residues;
wherein:
the protease cleavage site is located between the non-cytotoxic protease and the galanin-targeting moiety, and
the galanin-targeting moiety is located between the protease cleavage site and the translocation domain.
2 . The fusion protein of claim 1 , wherein the first spacer comprises an amino acid sequence of from 6 to 16 amino acid residues.
3 . The fusion protein of claim 1 , wherein the first spacer comprises amino acid residues selected from the group consisting of: glycine, threonine, arginine, serine, alanine, asparagine, glutamine, aspartic acid, proline, glutamic acid, and lysine.
4 . The fusion protein of claim 1 , wherein the first spacer comprises amino acid residues selected from the group consisting of: glycine, serine, and alanine.
5 . The fusion protein of claim 1 , wherein the first spacer is a GS5, GS10, GS15, GS18 or GS20 spacer.
6 . The fusion protein of claim 1 , wherein the galanin-targeting moiety binds specifically to the GALR1, GALR2 and/or the GALR3 receptor.
7 . The fusion protein of claim 1 , wherein the galanin-targeting moiety comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8.
8 . The fusion protein of claim 1 , wherein the galanin-targeting moiety comprises: the amino acid sequence of SEQ ID NO. 7 a fragment of the amino acid sequence of SEQ ID NO: 7 comprising at least 14 contiguous amino acid residues thereof, a variant amino acid sequence of the sequence of SEQ ID NO: 7 having a maximum of 5 or 6 conservative amino acid substitutions as compared to the sequence of SEQ ID NO: 7, or a variant amino acid sequence of the fragment of SEQ ID NO: 7 having a maximum of 5 or 6 conservative amino acid substitutions as compared to the fragment.
9 . The fusion protein of claim 1 , wherein the non-cytotoxic protease is a clostridial neurotoxin L-chain or an IgA protease.
10 . The fusion protein of claim 1 , wherein the translocation domain is the H N domain of a clostridial neurotoxin.
11 . The fusion protein of claim 1 comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41,42, 43,44, 45, 46, 47, 48,49, 50, 53, 56 and 59.
12 . A polynucleotide encoding the fusion protein of claim 1 .
13 . An expression vector comprising , a promoter, the polynucleotide of claim 12 located downstream of the promoter, and a terminator located downstream of the polynucleotide.
14 . A method for preparing a single-chain polypeptide fusion protein, comprising:
transfecting a host cell with the expression vector of claim 13 , and
culturing the host cell under conditions that promote the expression of the polypeptide fusion protein by the expression vector.
15 . A method of preparing a non-cytotoxic agent, comprising:
contacting a single-chain polypeptide fusion protein of claim 1 with a protease capable of cleaving the protease cleavage site;
cleaving the protease cleavage site, thereby forming a di-chain fusion protein.
16 . A non-cytotoxic polypeptide, obtained by the method of claim 15 , wherein:
the polypeptide is a di-chain polypeptide comprising a first and second chain joined together by a disulphide bond;
the first chain comprises the non-cytotoxic protease; and
the second chain comprises the galanin-targeting moiety and the translocation domain.
17 . A method of treating, preventing or ameliorating pain in a subject, comprising administering to the subject a therapeutically effective amount of the fusion protein of claim 1 .
18 . The method of claim 17 , wherein the pain is chronic pain selected from: neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
19 . A method of treating, preventing or ameliorating pain in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide of claim 16 .
20 . The method of claim 19 , wherein the pain is chronic pain selected from: neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.