Cell-based Clostridal Neurotoxin Assays

Abstract

The present invention is directed to a method for identifying a gene that regulates clostridial neurotoxin activity, the method comprising: a. providing a sample of human neuronal cells expressing a polypeptide that comprises a C-terminal detectable label, wherein the polypeptide is cleavable by a clostridial neurotoxin; b. altering expression of a target gene of the cells; c. contacting the cells with the clostridial neurotoxin; d. measuring an amount of C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; and e. identifying the target gene as a regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is different to the quantified clostridial neurotoxin activity when expression of the target gene is unaltered; or f. identifying that the target gene is not a regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is equivalent to the quantified clostridial neurotoxin activity when expression of the target gene is unaltered. Also provided are related methods for identifying an agent that regulates clostridial neurotoxin activity, as well as human neuronal cells, nucleotides, vectors, polypeptides, kits, and compositions suitable for use in the methods of the invention.

Claims

1. A method for identifying a gene that regulates clostridial neurotoxin activity, the method comprising: a. providing a sample of human neuronal cells expressing a polypeptide that comprises a C-terminal detectable label, wherein the polypeptide is cleavable by a clostridial neurotoxin; b. altering expression of a target gene of the cells; c. contacting the cells with the clostridial neurotoxin; d. measuring an amount of C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; and e. identifying the target gene as a regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is different to the quantified clostridial neurotoxin activity when expression of the target gene is unaltered; or f. identifying that the target gene is not a regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is equivalent to the quantified clostridial neurotoxin activity when expression of the target gene is unaltered.

2. The method according to claim 1, wherein expression is altered by downregulating expression of the target gene.

3. The method according to claim 2, wherein the target gene is identified as a positive regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is less than the quantified clostridial neurotoxin activity when expression of the target gene is unaltered.

4. The method according to claim 2 or 3, wherein the target gene is identified as a negative regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is greater than the quantified clostridial neurotoxin activity when expression of the target gene is unaltered.

5. The method according to any one of the preceding claims, wherein a plurality of samples of human neuronal cells are provided, and wherein the expression of a different target gene is altered in each sample of human neuronal cells.

6. The method according to claim 5, wherein the expression of a different target gene is altered in each sample using a human RNAi library.

7. The method according to any one of the preceding claims, further comprising determining whether the target gene is a direct regulator of clostridial neurotoxin activity or an indirect regulator of clostridial neurotoxin activity.

8. The method according to claim 7, wherein the direct regulator regulates clostridial neurotoxin activity at the level of: i. binding of the clostridial neurotoxin to the cell; ii. internalisation of the clostridial neurotoxin; iii. translocation of the clostridial neurotoxin L-chain out of the endosome; iv. catalysis; and/or v. persistence of the L-chain activity within the cell cytoplasm.

9. The method according to claim 7 or 8, wherein the indirect regulator regulates cellular trafficking of a clostridial neurotoxin receptor.

10. The method according to any one of claims 7-9, wherein the determining comprises detecting the presence or absence of a clostridial neurotoxin receptor of the cell when expression of the target gene has been altered.

11. The method according to any one of claims 7-10, wherein: detecting a decreased amount of a clostridial neurotoxin receptor on the surface of the cell when expression of the target gene has been altered (preferably downregulated) when compared to an equivalent cell contacted with clostridial neurotoxin in which the expression of the target gene is unaltered indicates that the target gene indirectly regulates clostridial neurotoxin activity; or detecting an equivalent or greater (preferably greater) amount of a clostridial neurotoxin receptor on the surface of the cell when expression of the target gene has been altered (preferably downregulated) when compared to an equivalent cell contacted with clostridial neurotoxin in which the expression of the target gene is unaltered indicates that the target gene directly regulates clostridial neurotoxin activity.

12. The method according to any one of claims 9-11, wherein the clostridial neurotoxin receptor is Synaptic Vesicle Glycoprotein 2A (SV2).

13. A method for identifying an agent that regulates clostridial neurotoxin activity, the method comprising: a. providing a sample of human neuronal cells expressing a polypeptide that comprises a C-terminal detectable label, wherein the polypeptide is cleavable by a clostridial neurotoxin; b. contacting the cells with the clostridial neurotoxin and an agent, wherein the contacting is sequential or simultaneous; c. measuring an amount of C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; and d. identifying the agent as a regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is different to the quantified clostridial neurotoxin activity in the absence of the agent; or e. identifying that the agent is not a regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is equivalent to the quantified clostridial neurotoxin activity in the absence of the agent.

14. The method according to claim 13, wherein the agent is identified as a negative regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is less than the quantified clostridial neurotoxin activity in the absence of the agent.

15. The method according to claim 13 or 14, wherein the agent is identified as a positive regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is greater than the quantified clostridial neurotoxin activity in the absence of the agent.

16. The method according to any one of claims 13-15, further comprising determining whether the agent is a direct regulator of clostridial neurotoxin activity or an indirect regulator of clostridial neurotoxin activity.

17. The method according to claim 16, wherein the direct regulator regulates clostridial neurotoxin activity at the level of: i. binding of the clostridial neurotoxin to the cell; ii. internalisation of the clostridial neurotoxin; iii. translocation of the clostridial neurotoxin L-chain out of the endosome; iv. catalysis; and/or v. persistence of the L-chain activity within the cell cytoplasm.

18. The method according to claim 16 or 17 wherein the indirect regulator regulates cellular trafficking of a clostridial neurotoxin receptor.

19. The method according to any one of claims 16-18, wherein the determining comprises detecting the presence or absence of a clostridial neurotoxin receptor of the cell when the cell has been targeted with the agent.

20. The method according to any one of claims 16-19, wherein: detecting a decreased amount of a clostridial neurotoxin receptor on the surface of the cell when the cell has been contacted with the agent when compared to an equivalent cell contacted with clostridial neurotoxin that has not been contacted with the agent indicates that the agent indirectly regulates clostridial neurotoxin activity; or detecting an equivalent or greater (preferably greater) amount of a clostridial neurotoxin receptor on the surface of the cell when the cell has been contacted with the agent when compared to an equivalent cell contacted with clostridial neurotoxin that has not been contacted with the agent indicates that the agent directly regulates clostridial neurotoxin activity.

21. The method according to any one of claims 18-20, wherein the clostridial neurotoxin receptor is Synaptic Vesicle Glycoprotein 2A (SV2).

22. A human neuronal cell expressing a polypeptide, wherein the polypeptide is cleavable by a clostridial neurotoxin and comprises a C-terminal detectable label.

23. The method according to any one of claims 1-21 or the cell according to claim 22, wherein the human neuronal cell is a non-cancer cell.

24. The method or cell according to any one of the preceding claims, wherein the human neuronal cell is an immortalized human neural progenitor cell, or preferably wherein the human neuronal cell has been derived (e.g. differentiated) from an immortalized human neural progenitor cell.

25. The method or cell according to any one of the preceding claims, wherein the polypeptide further comprises an N-terminal detectable label, and wherein the N-terminal detectable label is different to the C-terminal detectable label.

26. The method or cell according to claim 25, wherein one of the detectable labels is red fluorescent protein (RFP) and one of the detectable labels is selected from green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP).

27. The method or cell according to any one of the preceding claims, wherein the polypeptide comprises an N-terminal RFP and a C-terminal GFP.

28. The method or cell according to any one of the preceding claims, wherein the polypeptide: a. is encoded by a nucleotide sequence comprising: i. a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 3; ii. a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 9; and/or iii. a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 11; or b. is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1; or c. comprises a polypeptide sequence comprising: i. a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4; ii. a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 10; and/or iii. a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 12; or d. comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2.

29. The method or cell according to any one of the preceding claims, wherein the polypeptide: a. is encoded by a nucleotide sequence comprising: i. a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3; ii. a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9; and/or iii. a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 11; or b. is encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1; or c. comprises a polypeptide sequence comprising: i. a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 4; ii. a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 10; and/or iii. a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 12; or d. comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2.

30. The method or cell according to any one of the preceding claims, wherein the polypeptide: a. is encoded by a nucleotide sequence comprising: i. a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3; ii. a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9; and/or iii. a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11; or b. is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1; or c. comprises a polypeptide sequence comprising: i. a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 4; ii. a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 10; and/or iii. a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 12; or d. comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 2.

31. The method or cell according to any one of the preceding claims, wherein the polypeptide: a. is encoded by a nucleotide sequence comprising: i. SEQ ID NO: 3; ii. SEQ ID NO: 9; and iii. SEQ ID NO: 11; or b. is encoded by a nucleotide sequence comprising SEQ ID NO: 1; or c. comprises: i. SEQ ID NO: 4; ii. SEQ ID NO: 10; and iii. SEQ ID NO: 12; or d. comprises SEQ ID NO: 2.

32. A nucleotide sequence encoding a polypeptide, wherein the polypeptide is cleavable by a clostridial neurotoxin and comprises an N-terminal RFP and a C-terminal GFP, wherein the nucleotide sequence comprises: a. a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 3; b. a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 9; and c. a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 11.

33. The nucleotide sequence according to claim 32, wherein the nucleotide sequence has at least 70% sequence identity to SEQ ID NO: 1.

34. The nucleotide sequence according to claim 32 or 33, wherein the nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 1.

35. The nucleotide sequence according to any one of claims 32-34, wherein the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 1.

36. The nucleotide sequence according to any one of claims 32-35, wherein the nucleotide sequence comprises SEQ ID NO: 1.

37. The method or cell according to any one of claims 1-31 or the nucleotide sequence according to any one of claims 32-36, wherein the nucleotide sequence consists of SEQ ID NO: 1.

38. A vector comprising the nucleotide sequence according to any one of claims 32-37.

39. A polypeptide that is cleavable by a clostridial neurotoxin and comprises an N-terminal RFP and a C-terminal GFP, wherein the polypeptide comprises: a. a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4; b. a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 10; and c. a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 12.

40. The polypeptide according to claim 39, wherein the polypeptide sequence has at least 70% sequence identity to SEQ ID NO: 2.

41. The polypeptide according to claim 39 or 40, wherein the polypeptide sequence has at least 80% sequence identity to SEQ ID NO: 2.

42. The polypeptide according to any one of claims 39-41, wherein the polypeptide sequence has at least 90% sequence identity to SEQ ID NO: 2.

43. The polypeptide according to any one of claims 39-42, wherein the polypeptide sequence comprises SEQ ID NO: 2.

44. The method or cell according to any one of claim 1-31 or 37 or the polypeptide according to any one of claims 39-43, wherein the polypeptide sequence consists of SEQ ID NO: 2.

45. The method according to any one of claim 1-31, 37 or 44, wherein the method comprises contacting the cells with a buffer comprising glial cell-derived neurotrophic factor (GDNF), cell-permeable cyclic adenosine monophosphate (cAMP), CaCl.sub.2 and KCl.

46. The method according to claim 45, wherein the GDNF is present in the buffer at a concentration of 1-100 ng/ml, cAMP is present in the buffer at a concentration of 0.1-5 mM, CaCl.sub.2 is present in the buffer at a concentration of 0.1-7 mM and/or KCl is present in the buffer at a concentration of 1-100 mM.

47. A kit comprising: a. the cell according to any one of claim 22-31, 37 or 44; or b. the nucleotide sequence according to any one of claims 32-37; or c. the vector according to claim 38; and d. optionally instructions for use of the same.

48. The kit according to claim 47, further comprising a buffer, said buffer comprising GDNF, cell-permeable cyclic adenosine monophosphate (cAMP), CaCl.sub.2 and KCl.

49. A composition comprising: a. a clostridial neurotoxin; and b. a buffer, the buffer comprising GDNF, cell-permeable cyclic adenosine monophosphate (cAMP), CaCl.sub.2 and KCl.

50. The kit according to claim 48 or the composition according to claim 49, wherein the GDNF is present in the buffer at a concentration of 1-100 ng/ml, cAMP is present in the buffer at a concentration of 0.1-5 mM, CaCl.sub.2 is present in the buffer at a concentration of 0.1-7 mM and KCl is present in the buffer at a concentration of 1-100 mM.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0193] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0194] FIG. 1 shows a method for generating ReNcell VM stably expressing TagRFPT-SNAP25-TagGFP. The images (lower) show red fluorescence (right) and green fluorescence (right).

[0195] FIG. 2 shows BoNT/A-induced degradation of the C-terminal fragment of the construct. ReD SNAPR cells were differentiated for 14 days without growth factors and 100 nM of BoNT/A was then added to the cells and intoxicated for 48 hours. (A) Cells were imaged using fluorescence microscopy and the corresponding GFP, RFP and GFP/RFP merged channels are as shown. (B) Lysates were subjected to SDS-PAGE for western blot analysis. Rabbit antibodies against tagRFPT and tagGFP were used for the western blot.

[0196] FIG. 3 shows a diagram summarising degradation of the construct by BoNT/A.

[0197] FIG. 4 shows that the enhanced differentiation and stimulation buffer sensitised ReD SNAPR cells to BoNT/A. (A) ReD SNAPR cells were subjected to ReNcell differentiation media supplemented with and without GDNF and d-cAMP during differentiation and high potassium buffer during intoxication. The resulting modified media is known as ReDS (ReNcell enhanced differentiation and stimulation) media. The loss of fluorescence of tagGFP upon cleavage of the dual-tagged SNAP25 construct was observed in a dose-dependent manner. ReD SNAPR cells in ReDS media exhibited enhanced sensitivity towards BoNT/A as compared to normal ReNcell media as detected using confocal microscopy. (B) Quantification of BoNT/A-mediated cleavage in both conditions showed the EC.sub.50 was improved from 43 nM to 6 nM in ReD SNAPR exposed to ReDS media. (C) Western blot detection of BoNT/A-mediated SNAP25 construct cleavage in ReD SNAPR cells in normal and ReDS media. The upper blot shows cell lysates probed with tRFP antibody and the lower blot probed with tGFP antibody.

[0198] FIG. 5 shows siRNA against TrxR rescues BoNT/A-mediated construct cleavage. Graphs of TrxR1 knockdown levels and construct cleavage are shown. The upper panel (Red SNAPR) shows green and red fluorescence (siNT3, −BoNT/A), red fluorescence only (siNR3, +BoNT/A), green and red fluorescence (siTrxR, −BoNT/A), and green and red fluorescence (siTrxR, −BoNT/A). The lower panel shows staining for TrxR1 in the presence of siNT3 (for both −BoNT/A and +BoNT/A conditions) and the absence of staining for TrxR1 in the presence of siTrxR (for both −BoNT/A and +BoNT/A conditions).

[0199] FIG. 6 shows that BoNT/A intoxication prevents trafficking of SV2 to the cell surface A) ReNcell VM cells were treated with siNT3, siVAMP2 and siTrxR1 for 72 hours before addition of BoNT/A. Cells were fixed and stained with a primary antibody against SV2A (without permeabilisation). An Alexa-488 secondary antibody was used against the primary antibody. (B) An illustration of BoNT/A-mediated decreased trafficking of SV2 to the cell surface.

[0200] FIG. 7 shows a schematic for performing a genome-wide siRNA screen using the cell line of the invention.

TABLE-US-00002 SEQUENCE LISTING Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon may be optional. (Nucleotide Sequence of Construct TaqRFPT-SNAP25-TaqGFP) SEQ ID NO: 1 ATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAAC CACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTC GAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAGAACCTTCATCAAC CACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACCACATAC GAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATC AGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCAACACCGAGATG CTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAACCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATC TGCAACTTCAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGAC CACAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATAC TGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATGGCATGGACGAGCTGTACAAGGGCTCGGGCTCGGGCTCG GGCGTGGCCGAAGACGCAGACATGCGCAATGAGCTGGAGGAGATGCAGCGAAGGGCTGACCAGTTGGCTGATGAG TCGCTGGAAAGCACCCGTCGTATGCTGCAACTGGTTGAAGAGAGTAAAGATGCTGGTATCAGGACTTTGGTTATG TTGGATGAACAAGGAGAACAACTCGATCGTGTCGAAGAAGGCATGAACCATATCAACCAAGACATGAAGGAGGCT GAGAAAAATTTAAAAGATTTAGGGAAATGCTGTGGCCTTTTCATATGTCCTTGTAACAAGCTTAAATCAAGTGAT GCTTACAAAAAAGCCTGGGGCAATAATCAGGACGGAGTGGTGGCCAGCCAGCCTGCTCGTGTAGTGGACGAACGG GAGCAGATGGCCATCAGTGGCGGCTTCATCCGCAGGGTAACAAATGATGCCCGAGAAAATGAAATGGATGAAAAC CTAGAGCAGGTGAGCGGCATCATCGGGAACCTCCGTCACATGGCCCTGGATATGGGCAATGAGATCGATACACAG AATCGCCAGATCGACAGGATCATGGAGAAGGCTGATTCCAACAAAACCAGAATTGATGAGGCCAACCAACGTGCA ACAAAGATGCTGGGAAGTGGTTACGGCGGCTCGGGCTCGGGCGTGAGCGGGGGCGAGGAGCTGTTCGCCGGCATC GTGCCCGTGCTGATCGAGCTGGACGGCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGAC GCCGACTACGGCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTG ACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCACATGAAGATGAACGACTTCTTCAAGAGC GCCATGCCCGAGGGCTACATCCAGGAGCGCACCATCCAGTTCCAGGACGACGGCAAGTACAAGACCCGCGGCGAG GTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATC CTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCCAACAACGGCCTG GAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCGACCACTACCAGACCAACGTG CCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGACCAAGATCAGCAAGGACCGC AACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTG TACAGGTAA (Polypeptide Sequence of Construct TaqRFPT-SNAP25-TaqGFP) SEQ ID NO: 2 MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSRTFIN HTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEM LYPADGGLEGRTDMALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARY CDLPSKLGHKLNGMDELYKGSGSGSGVAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVM LDEQGEQLDRVEEGMNHINQDMKEAEKNLKDLGKCCGLFICPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDER EQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRA TKMLGSGYGGSGSGVSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICTTGKLPVPWPTLV TTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVKFEGDTLVNRIELKGKDFKEDGNI LGHKLEYSFNSHNVYIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDR NEARDHMVLLESFSACCHTHGMDELYR* (Nucleotide Sequence of TagRFPT) SEQ ID NO: 3 ATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAAC CACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTC GAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAGAACCTTCATCAAC CACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACCACATAC GAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATC AGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCAACACCGAGATG CTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAACCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATC TGCAACTTCAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGAC CACAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATAC TGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATGGCATGGACGAGCTGTACAAG (Polypeptide Sequence of TagRFPT) SEQ ID NO: 4 MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSRTFIN HTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEM LYPADGGLEGRTDMALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARY CDLPSKLGHKLNGMDELYK (Nucleotide Sequence of Glycine-Serine rich linker 1) SEQ ID NO: 5 GGCTCGGGCTCGGGCTCGGGC (Polypeptide Sequence of Glycine-Serine rich linker 1) SEQ ID NO: 6 GSGSGSG (Nucleotide Sequence of Glycine-Serine rich linker 2) SEQ ID NO: 7 GGCGGCTCGGGCTCGGGC (Polypeptide Sequence of Glycine-Serine rich linker 2) SEQ ID NO: 8 GGSGSG (Nucleotide Sequence of SNAP25) SEQ ID NO: 9 GTGGCCGAAGACGCAGACATGCGCAATGAGCTGGAGGAGATGCAGCGAAGGGCTGACCAGTTGGCTGATGAGTCG CTGGAAAGCACCCGTCGTATGCTGCAACTGGTTGAAGAGAGTAAAGATGCTGGTATCAGGACTTTGGTTATGTTG GATGAACAAGGAGAACAACTCGATCGTGTCGAAGAAGGCATGAACCATATCAACCAAGACATGAAGGAGGCTGAG AAAAATTTAAAAGATTTAGGGAAATGCTGTGGCCTTTTCATATGTCCTTGTAACAAGCTTAAATCAAGTGATGCT TACAAAAAAGCCTGGGGCAATAATCAGGACGGAGTGGTGGCCAGCCAGCCTGCTCGTGTAGTGGACGAACGGGAG CAGATGGCCATCAGTGGCGGCTTCATCCGCAGGGTAACAAATGATGCCCGAGAAAATGAAATGGATGAAAACCTA GAGCAGGTGAGCGGCATCATCGGGAACCTCCGTCACATGGCCCTGGATATGGGCAATGAGATCGATACACAGAAT CGCCAGATCGACAGGATCATGGAGAAGGCTGATTCCAACAAAACCAGAATTGATGAGGCCAACCAACGTGCAACA AAGATGCTGGGAAGTGGTTAC (Polypeptide Sequence of SNAP25) SEQ ID NO: 10 VAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLDRVEEGMNHINQDMKEAE KNLKDLGKCCGLFICPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENL EQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSGY (Nucleotide Sequence of TaqGFP) SEQ ID NO: 11 GTGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACGGCGACGTGCACGGCCAC AAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACGGCAAGCTGGAGATCAAGTTCATCTGCACCACC GGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCC GAGCACATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCGCACCATCCAGTTC CAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCACAACGTG TACATCCGCCCCGACAAGGCCAACAACGGCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGC GTGCAGCTGGCCGACCACTACCAGACCAACGTGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACTAC CTGAGCACTCAGACCAAGATCAGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTTCAGC GCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAA (Polypeptide Sequence of TagGFP) SEQ ID NO: 12 VSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICTTGKLPVPWPTLVTTLCYGIQCFARYP EHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVKFEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNV YIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLESFS ACCHTHGMDELYR* (BoNT/A - UniProt P10845) SEQ ID NO: 13 MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELN EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAK LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL (BoNT/B - UniProt P10844) SEQ ID NO: 14 MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSY SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE (BoNT/C - UniProt P18640) SEQ ID NO: 15 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRK DINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQN VDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLM WANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILL EAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQF NNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNIN KFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSF QNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQ LNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIID SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNM KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKEL DGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNN DFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYA IGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYR HNYLVPTVKQGNYASLLESTSTHWGFVPVSE (BoNT/D - UniProt P19321) SEQ ID NO: 16 MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDS STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSE VVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA LQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYL NSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANE VVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFP EFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHIN YQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIR ECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTM PFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTI YTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS GWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYIN GELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQI LRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSV SDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNY GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLS TSSFWKFISRDPGWVE (BoNT/E - UniProt Q00496) SEQ ID NO: 17 MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGS IAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKL SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE SKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKIN KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYF NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNF KNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK (BoNT/F - UniProt A7GBG3) SEQ ID NO: 18 MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFD PPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGN EHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVY DPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGAR GVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATR LSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKF KVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKG LVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLN NNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFF YLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIR DFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELL IPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERF ITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNN SIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIY STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDC IRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGN SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETL YSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSN TRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKL IRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTS SNGCFWSFISKEHGWQEN (BoNT/G - UniProt Q60393) SEQ ID NO: 19 MPVNIKXFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFN ASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLG NASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGH SPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLY GIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIA NRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNL AGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAY EEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYN TQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGD SLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVK GVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFI PELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTI KERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFI NQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDS IPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFND IGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTII SCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLG NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVS SLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMONIYIKYFSKASMGETAPRTNFNNAAI NYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQ LFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYD NYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE (Polypeptide Sequence of BoNT/X) SEQ ID NO: 20 MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIP LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEG TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRD QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAK IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNL GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWS SFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMG ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED (TeNT - UniProt P04958) SEQ ID NO: 21 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFN PPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGN SYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDN KNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLH GLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYK AIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTE IELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNT NAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAE KNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAP EYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVI SKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGN FIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYK LVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKN KLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIG ITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDIS GFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPK VSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLP DKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNN NQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDV QLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYV SYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDD KNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

EXAMPLES

Example 1—Generation of a Stable Cell Line

[0201] Gene Synthesis and Subcloning

[0202] The nucleotide sequences of tagRFPT and tagGFP were derived from Evrogen and synthesized by GeneArt (Thermo Fisher Scientific). The gene product, tRFPT-SNAP25-tGFP, was flanked with attB sequences for Gateway® cloning. The synthesized gene product was then subcloned into a lentivirus vector, pLenti6.3/V5-dest using the BP clonase enzyme kit (Thermo Fisher) according to manufacturer's protocol. The resulting vector, pLenti6.3-tRFPT-SNAP25-tGFP, was transformed in E. coli BL21 cells and selected using Ampicillin antibiotic. Positive bacteria clones were maxi-prepped using Machery-Nagel Endotoxin-free Maxiprep kit according to manufacturer's protocol.

[0203] Generation of Lentivirus from HEK293FT Cells

[0204] To prepare for generation of lentivirus, HEK293FT cells were cultured in high glucose Dulbecco's modified Eagle's media with 4500 mg/L glucose, supplemented with 10% Fetal Bovine Serum (FBS) (Gibco) then seeded into T75 cm.sup.2 flask at 80% confluence and incubated overnight at 37° C. with 5% CO2. The cells were then co-transfected with plenti6.3-tRFPT-SNAP25-tGFP plasmid and ViraPower Lentiviral Packaging Mix (Invitrogen Cat No.K497000) using Lipofactamine 3000 reagent (Invitrogen) according to the manual provided by supplier and incubated flask for 6 hours at 37° C. with 5% CO.sub.2. After 6 hours post-transfection, medium that contains lipid-DNA complexes were carefully removed and discarded from the flask, and replaced with 10 ml of pre-warmed medium. The cells were incubated overnight at 37° C. with 5% CO.sub.2. 10 ml of cell supernatant (first batch of virus) was collected after 24 hours post-transfection, and stored in 15 ml conical tubes at 4° C. The collected medium was replaced with 10 ml of pre-warmed medium and the flask was incubated overnight at 37° C. with 5% CO.sub.2. A second batch of virus was collected 48 hours post-transfection. Both batches of supernatant were centrifuged at 2000 rpm for 10 minutes at room temperature to remove cellular debris. The clarified lentiviral supernatant was collected after centrifugation and filtered using a 0.45 μm pore filter to remove any remaining cellular debris. Virus was aliquoted into 1 ml and stored at −80° C.

[0205] Measurement of Lentivirus Titre by GFP Selection

[0206] HEK293FT cells were seeded in a 96 wells plate (Nunc) at a density of 10000 cells/well in 100 μl of culture medium. Serial dilutions from 10.sup.−1 to 10.sup.−4 of virus were made using fresh culture medium with 8 mg/ml (final concentration) Polybrene reagent (Sigma cat no. H9268). Cells were transduced by removing the existing medium and replaced with 100 μl of the prepared dilutions to corresponding well. The plates were incubated overnight at 37° C. with 5% CO2. Culture medium was changed to fresh medium without polybrene the next day. Cells were incubated for additional 3 days before the titer of virus was calculated. The appropriate dilution factor used to calculate the titer in transducing units (TU) per ml based on the percentage of GFP positive cells. The desired transduction range was 1-20%. Hence, titer of virus was determined with the following formula: Titer=(F×C/V)×D, where F=frequency of GFP-positive cells (percent GFP-positive cells/100), C=cell number per well at the time of transduction, V=volume of inoculum in ml (0.1 ml) and D=lentivirus dilution factor.

[0207] Generation of ReNcell VM Stable Cell Line from Lentivirus

[0208] ReNcell VM (Millipore) cells were seeded in 24 wells coated with laminin (final concentration 20 μg/ml) at 80% confluence and incubated overnight at 37° C. with 5% CO.sub.2. Medium was removed and 500 μl of lentivirus added per well with Polybrene reagent at a final concentration of 8 mg/ml. Cells were incubated overnight at 37° C. with 5% CO.sub.2. Medium was replaced with fresh medium without polybrene the next day. Transduced cells were expanded and FAC-sorted using the GFP wavelength.

[0209] Results

[0210] The assay construct consisting of full-length SNAP25 flanked by tagRFPT and tagGFP was cloned into a lentivirus vector backbone. Generation of stable cell line was achieved using a modified lentivirus generation protocol consisting of lipofection of construct with lentiviral packaging plasmids into the HEK293T cell line. The resulting lentivirus was purified and added onto ReNcell VM cells, which were eventually sorted using FACS (see FIG. 1), and the generation of the stable cell line confirmed. This v-myc immortalised cell line is derived from human neural progenitor cells (NPCs), which is genetically closer to native human neurons as compared to cancer cell lines, and is therefore a better neuronal cell model for use in the assays described herein.

Example 2—the Construct is Sensitive to BoNT/A Cleavage Materials and Methods

[0211] Perkin Elmer CellCarrier 384 Ultra™ imaging plates and Nunc 24-well tissue culture dishes were incubated with 20 μg/mL laminin (Invitrogen) overnight at 4° C.

[0212] Imaging

[0213] The stable cell line of the invention (referred to as the ReD SNAPR cell line) was differentiated according to the ReNcell VM cell manufacturer's protocol. In brief, cells were seeded on pre-coated Perkin Elmer CellCarrier 384 Ultra™ imaging plates at 3000 cells per well. Cells were maintained in ReNcell NSC Maintenance Media without growth factors (EGF & FGF2) (differentiation media) for 14 days, with media change every 3 days. Cells were incubated with 100 nM BoNT/A in differentiation media for 48 hours. Cells were fixed with fixative (4% paraformaldehyde and 2% sucrose). Fixed cells were imaged using Opera™ Phenix.

[0214] Western Blot

[0215] ReD SNAPR cells were differentiated according to the ReNcell VM cell manufacturer's protocol. Briefly, cells were seeded on Nunc 24-well tissue culture dish at 30,000 cells per well. Cells were maintained in ReNcell NSC Maintenance Media without growth factors (EGF & FGF2) (differentiation media) for 14 days, with media change every 3 days. Cells were incubated with 100 nM BoNT/A in differentiation media for 48 hours. The medium was aspirated and cells were lysed with NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 nM Tris-Cl, pH 8.0). To prepare samples for loading into SDS-PAGE gel, 10% DTT and 6X loading buffer (BioRad) was added to the samples and boiled for 5 mins. 20 μL of samples were added into each lane of a NuPAGE Bis-tris 4-12% gel (Thermo Fisher) and run at 120V until the dye front ran out. The gel was transferred onto a nitrocellulose membrane and probed with anti-tRFP and anti-tag (CGY)FP (Evrogen) overnight.

[0216] Results

[0217] FIG. 2 demonstrates that the construct of the assay is sensitive to degradation by BoNT/A. Conventional cell-based assays focus on FRET interactions or direct western blot detection of cleaved SNAP25 in the cell lysate, which are not suitable for high-throughput screening (HTS) applications. In normal circumstances, the BoNT/A-cleaved C-terminal fragment of full-length SNAP25 is hardly detectable due to the small molecular weight, hence its fate is usually unknown. This previously-unrecognized observation shows that the C-terminal fragment is degraded along with the fluorophore that is attached with it. The relative ease of this methodology is highly suitable for a range low to high-throughput applications.

[0218] FIG. 3 presents a schematic showing BoNT/A-mediated degradation of the C-terminus of the SNAP25 construct. After internalisation of BoNT/A in ReD SNAPR cells, the light chain of BoNT/A enters the cytoplasm and cleaves tagRFPT-SNAP25-tagGFP (stably expressed in ReD SNAPR cells). This results in the degradation of the C-terminal fragment, while retaining the N-terminal construct. The degradation can be detected using fluorescence microscopy and Western blot.

Example 3—Improving Sensitivity of the ReD SNAPR Cells to BoNT/A

[0219] ReD SNAPR cells were seeded onto 384 well plates as described above. For enhanced differentiation, ReD SNAPR cells were cultured in normal ReNcell media with 10 ng/mL GDNF and 1 mM d-cAMP (cell permeable cAMP). Various concentrations (0-1 μM) of BoNT/A was added to normal and ReDS media where ReDS media contained 10 ng/mL GDNF, 1 mM d-cAMP, 2 mM CaCl.sub.2 and 56 mM KCl. Differentiated ReD SNAPR cells were intoxicated with BoNT/A-containing medium, fixed and imaged as described above.

[0220] Results

[0221] FIG. 4 shows that addition of GDNF and d-cAMP during differentiation, and high potassium conditions during intoxication improved sensitivity of the cell line to BoNT/A. The dose response curves and EC.sub.50 values of BoNT/A in the assay were determined (see FIG. 4B) with low nM values when the cells were exposed to ReDS medium.

Example 4—Thioredoxin Reductase (TrxR1) as an Assay Control

[0222] ReD SNAPR cells were seeded onto 384 well plates and differentiated as described above. Differentiated cells were treated with 25 nmol of either a siRNA non-targeting control, NT3 or siRNA against TrxR1 using Lipofectamine RNAimax according to the manufacturer's protocol and left on cells for 72 hours. ReDS medium containing 10 nM BoNT/A was added to cells for 48 hours and then fixed and imaged as described above. Briefly, cells were fixed and an antibody against TrxR1 was used to detect TrxR1 and fluorescence imaged using Opera Phenix. Mean fluorescence intensity levels of GFP, RFP and Far-red channels were captured and measured.

[0223] Results

[0224] FIG. 5 shows that siTrxR-treated cells are more resistant to BoNT/A-mediated cleavage as compared to control. Thus, TrxR1 can be used as a suitable positive control to identify genes involved in the intracellular trafficking of BoNT/A. In particular TrXR1 knock-down can be used to show that BoNT/A intoxication can be rescued, thus further validating genes identified in an assay.

Example 5—Use of BoNT Receptor SV2

[0225] ReNcell VM cells were seeded onto 384 well plates and differentiated as previously described. Differentiated cells were treated with 25 nmol of either siNT3, siVAMP2 or siTrxR using Lipofectamine RNAimax according to manufacturer's protocol and left on cells for 72 hours. ReDS medium containing 10 nM BoNT/A was added to cells for 48 hours and then fixed. For immunostaining, the cells were blocked with 0.5% BSA/PBS for 1 hour and an antibody against SV2A (Cell Signaling, #66724) was added to cells and incubated for at least 1 hour. An Alexa-488 conjugated secondary antibody was added into cells for 1 hour and cells were imaged using Opera Phenix. Cells were then imaged using Opera Phenix with GFP and DAPI channels shown.

[0226] Results

[0227] Although SV2 is the major receptor for BoNT/A, it has not previously been further studied for its post-intoxication itinerary in the cell. FIG. 6 shows that BoNT/A intoxication leads to decreased SV2 at the cell surface which could be the typical consequence of BoNT/A-mediated defective trafficking to the cell surface. In this case, the recycling of SV2 back to the cell surface is blocked. Hence SV2 can be used as a readout for BoNT/A intoxication.

[0228] Many genes can regulate BoNT/A activity in the cell. An example of indirect regulation would be at the level of BoNT receptor SV2 trafficking (instead of modulation of toxin activity itself). To sieve out candidates involved in SV2 trafficking, the surface SV2 staining may be an ideal selection criteria. An example shown here are cells depleted with VAMP2, which upon BoNT/A intoxication resulted in lower surface SV2 staining. This could be due to the synergistic action of blocking vesicle exocytosis at the cell surface via decreased VAMP2 and BoNT/A intoxication.

[0229] Surface SV2 can be rescued via depleting TrxR, which shows that TrxR itself does not affect exocytosis of SV2 at the cell surface but directly modulating BoNT/A activity via release of its light chain (LC). This inadvertently results in the restoration of surface SV2 due to decreased BoNT/A LC in the cytoplasm.

[0230] Thus, SV2 is useful in an assay of the invention as it can be used to sieve out gene candidates directly involved in BoNT/A trafficking from those that modulate the trafficking of the BoNT/A receptor SV2.

Example 6—Genome-Wide siRNA Screen

[0231] FIG. 7 provides a diagram showing a method for carrying out a genome-wide siRNA screen with a cell line of the invention. The ReD SNAPR cells are plated and differentiated as described above. An siRNA library is prepared and complexed with Lipofectamine™ RNAiMAX using standard protocols, and the ReD SNAPR cells are transfected with the siRNA. BoNT/A in stimulation buffer is added to the cells prior to fixing and imaging using Opera™ Phenix and quantification with Columbus™ software.

[0232] Positive hits may be subjected to further validation by assessing recovery in the presence of siRNA against TrxR1. Confirmation that the genes directly regulate BoNT activity are confirmed by way of SV2 cell surface staining as described above.

Example 7—Identifying Prophylactic Anti-Botulism Therapeutics

[0233] The ReD SNAPR cells are plated and differentiated as described above and exposed to an agent (e.g. a small-molecule drug). BoNT/A in stimulation buffer is added to the cells prior to fixing and imaging using Opera™ Phenix and quantification with Columbus™ software.

[0234] An agent is identified as a prophylactic anti-botulism therapeutic if cleavage of the construct is inhibited.

Example 8—Identifying Post-Intoxication Anti-Botulism Therapeutics

[0235] The ReD SNAPR cells are plated and differentiated as described above and BoNT/A in stimulation buffer is added to the cells and cleavage of the construct (loss of GFP) is observed. Next, the cells are exposed to an agent (e.g. a small-molecule drug). Finally, cells are fixed and imaged using Opera™ Phenix and quantification with Columbus™ software.

[0236] An agent is identified as a post-intoxication anti-botulism therapeutic if recovery of GFP is observed.

Example 9—Identifying BoNT Sensitising Agents

[0237] The ReD SNAPR cells are plated and differentiated as described above and exposed to an agent (e.g. a small-molecule drug). BoNT/A in stimulation buffer is added to the cells prior to fixing and imaging using Opera™ Phenix and quantification with Columbus™ software.

[0238] An agent is identified as a BoNT sensitising agent if cleavage of the construct is improved (e.g. occurs faster or more cleavage is evident). The sensitising agent is taken forward for further study for use as a companion product to modulate local activity of clostridial neurotoxins (e.g. to allow reduced dosage and minimise spread to other tissues).

[0239] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.