Non-cytotoxic protein conjugates
The present invention is directed to non-cytotoxic protein conjugates for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell. The protein conjugates comprise: (i) a dynorphin Targeting Moiety (TM), wherein the TM is an agonist of a receptor present on a nociceptive sensory afferent cell, and wherein the receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell; (ii) a non-cytotoxic protease or a fragment thereof, wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of the nociceptive sensory afferent cell; and (iii) a Translocation Domain, wherein the Translocation Domain translocates the protease or protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell. Nucleic acid sequences encoding the protein conjugates, methods of preparing same and uses thereof are also described.
1. An isolated polynucleotide molecule comprising a polynucleotide encoding a non-cytotoxic protein conjugate for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell, comprising:
(i) a dynorphin Targeting Moiety (TM), wherein said TM is an agonist of a receptor present on a nociceptive sensory afferent cell, and wherein said receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell;
(ii) a non-cytotoxic protease or a fragment thereof, wherein the protease or protease fragment cleaves a protein of the exocytic fusion apparatus of said nociceptive sensory afferent cell; and
(iii) a Translocation Domain, wherein the Translocation Domain translocates the protease or protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell.
2. The isolated polynucleotide molecule of claim 1 , wherein the receptor is an opioid receptor-like 1 (ORL1) receptor.
3. The isolated polynucleotide molecule of claim 1 , wherein the dynorphin TM has at least 70% or at least 80% sequence identity to SEQ ID NO:101 or a fragment thereof.
4. The isolated polynucleotide molecule of claim 1 , wherein the dynorphin TM has at least 90% sequence identity to SEQ ID NO:101 or a fragment thereof.
5. The isolated polynucleotide molecule of claim 1 , wherein the dynorphin TM has at least 95% sequence identity to SEQ ID NO:101 or a fragment thereof.
6. The isolated polynucleotide molecule of claim 1 , wherein the dynorphin TM is SEQ ID NO:101 or a fragment thereof.
7. The isolated polynucleotide molecule of claim 1 , wherein the non-cytotoxic protease is selected from a clostridial neurotoxin, or an IgA protease.
8. The isolated polynucleotide molecule of claim 1 , wherein the Translocation Domain is a botulinum H N domain.
9. The isolated polynucleotide molecule of claim 1 , wherein the nociceptive sensory afferent cell is a primary nociceptive sensory afferent cell.
10. The isolated polynucleotide molecule of claim 1 , wherein the conjugate has the structure, arranged amino terminal to carboxy terminal, or vice versa: protease or fragment thereof—dynorphin TM—Translocation Domain.
11. An expression vector comprising a polynucleotide molecule of claim 1 .
12. A host cell comprising an expression vector of claim 11 .
13. A method of producing a polypeptide comprising (a) culturing a host cell of claim 12 under conditions promoting expression of a polypeptide encoded by the polynucleotide molecule, and (b) recovering said polypeptide from the cell culture.
14. An isolated polynucleotide molecule comprising a polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs:103, 104, 105, 106, 107, 108, and 109.
15. An expression vector comprising a polynucleotide molecule of claim 14 .
16. A host cell comprising an expression vector of claim 15 .
17. A method of producing a polypeptide comprising (a) culturing a host cell of claim 16 under conditions promoting expression of a polypeptide encoded by the polynucleotide molecule, and (b) recovering said polypeptide from the cell culture.
18. An isolated polynucleotide molecule comprising the nucleic acid sequence set forth in SEQ ID NO:102.
19. An expression vector comprising a polynucleotide molecule of claim 18 .
20. A host cell comprising an expression vector of claim 19 .
21. A method of producing a polypeptide comprising (a) culturing a host cell of claim 20 under conditions promoting expression of a polypeptide encoded by the polynucleotide molecule, and (b) recovering said polypeptide from the cell culture.