Compositions and methods for treatment of pain
Embodied herein are engineered fusion proteins that bind and target nociceptor neurons, compositions comprising these engineered fusion proteins, and methods for treatment of pain using these engineered fusion proteins or compositions containing the engineered fusion proteins. The engineered fusion proteins contain domains derived from protein toxins such as the anthrax toxin, clostridial botulinum family of toxins, disulphide-containing toxins, and AB component type toxins.
1. A nucleic acid encoding a fusion protein comprising:
(a) a botulinum neurotoxin (BTx) moiety that comprises an enzymatic moiety and a translocation domain; and
(b) a targeting moiety comprising a C-terminal receptor-binding domain of anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain,
wherein part (a) and (b) are linked or fused together.
2. The nucleic acid of claim 1 , wherein the enzymatic moiety or the translocation domain is selected from BTx light chain and heavy chain domains of any one of BTx/A, BTx/B, BTx/C, BTx/D, BTx/E, BTx/F, and BTx/G.
3. The nucleic acid of claim 1 , wherein the enzymatic moiety and the translocation domain are linked by a linker peptide, optionally wherein the linker peptide:
(i) is 1-20 amino acids long;
(ii) is stable in human serum for at least 1 minute;
(iii) comprises at least one amino acid that is Gly or Ser; and/or
(iv) lacks a Lys, Arg, or both.
4. The nucleic acid of claim 1 , wherein the BTx moiety and the targeting moiety are linked by a linker peptide, optionally wherein the linker peptide:
(i) is 1-20 amino acids long;
(ii) is stable in human serum for at least 1 minute;
(iii) comprises at least one amino acid that is Gly or Ser; and/or
(iv) lacks a Lys, Arg, or both.
5. The nucleic acid of claim 1 , wherein:
(i) the C-terminal receptor-binding domain of PA is resistant to cleavage by a protease; or
(ii) the C-terminal receptor-binding domain of PA is resistant to furin cleavage or Lys C cleavage or both.
6. The nucleic acid of claim 1 , wherein the C-terminal receptor-binding domain of PA comprises a furin cleavage site comprising amino acid residues RKKR that has been replaced by a furin-resistant amino acid sequence, wherein RKKR are residues 164-167 of SEQ ID NO: 1 minus the 29 amino acid signal peptide in SEQ ID NO: 1.
7. The nucleic acid of claim 6 , wherein the furin-resistant amino acid sequence is SSSR (SEQ ID NO: 32).
8. The nucleic acid of claim 6 , wherein the furin-resistant amino acid sequence is SSSS (SEQ ID NO: 33).
9. The nucleic acid of claim 1 , wherein the C-terminal receptor-binding domain of PA further comprises a PAd2 or comprises PA63.
10. The nucleic acid of claim 1 , wherein:
(i) the fusion protein comprises 2-10 PAd4 domains in tandem; or
(ii) 1-60 consecutive amino acids from the N-terminal side adjacent to the native PAd4 domain are incorporated between the BTx moiety and the PAd4.
11. The nucleic acid of claim 1 , wherein:
(i) the fusion protein further comprises an epitope tag; or
(ii) the fusion protein further comprises an epitope tag selected from the group consisting of FLAG, influenza virus haemagglutinin (HA), c-myc, and His.
12. The nucleic acid of claim 1 , wherein:
(i) the fusion protein comprises an amino acid substitution at one or more cysteine residues; or
(ii) the fusion protein comprises an amino acid substitution at one or more cysteine residues, wherein the one or more cysteine residues are substituted with serine.
13. A vector comprising the nucleic acid of claim 1 .
14. The vector of claim 2 , wherein the vector is a plasmid, a bacteriophage, a phagmid, a cosmid, a viral particle, or viral vector.
15. A cell comprising the vector of claim 13 .
16. A method of producing a fusion protein comprising steps of:
(a) culturing a cell comprising a nucleic acid sequence encoding a fusion protein comprising:
(i) a botulinum neurotoxin (BTx) moiety that comprises an enzymatic moiety and a translocation domain; and
(ii) a targeting moiety comprising a C-terminal receptor-binding domain of anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain,
wherein part (i) and (ii) are linked or fused together; and
(b) recovering the fusion protein.
17. The method of claim 16 , wherein said cell is a bacterial cell.
18. The method of claim 17 , wherein said bacterial cell is an Escherichia coli.
19. The method of claim 16 , wherein said cell is a yeast cell.
20. The method of claim 19 , wherein said yeast is Saccharomyces cerevisiae.
21. The method of claim 19 , wherein said yeast is cell glycosylation deficient.
22. The method of claim 19 , wherein said yeast is glycosylation and protease deficient.
23. The method of claim 16 , wherein said cell is a mammalian cell.
24. The method of claim 23 , wherein said mammalian cell is a COS cell, a CHO cell, or a NSO cell.