Cell-based clostridial neurotoxin assay

Abstract

Cell-based clostridial neurotoxin assay The present invention relates to a method for identifying a gene that regulates clostridial neurotoxin activity, the method comprising the steps of: (a) providing a sample of human neuronal cells expressing a polypeptide comprising a C-terminal detectable label, where the polypeptide is cleavable by a clostridial neurotoxin; (b) altering expression of a target gene in the cells; (c) contacting the cells with a clostridial neurotoxin; (d) measuring the amount of the C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; (e) identifying the target gene as a regulator of clostridial neurotoxin activity if the quantified clostridial neurotoxin activity is different from the clostridial neurotoxin activity quantified when expression of the target gene is unaltered; or (f) identifying the target gene as not a regulator of clostridial neurotoxin activity if the quantified clostridial neurotoxin activity is equivalent to the clostridial neurotoxin activity quantified when expression of the target gene is unaltered. Related methods for identifying agents that regulate clostridial neurotoxin activity are also provided, as well as human neuronal cells, nucleotides, vectors, polypeptides, kits, and compositions suitable for use in the methods of the invention.

Description

[Technical field]

[0001] The present invention relates to a cell-based Clostridial neurotoxin assay. [Background technology]

[0002] Bacteria of the genus Clostridium produce highly potent and specific protein toxins that can poison neurons and other cells to which they are delivered. Examples of such Clostridial neurotoxins include those produced by Clostridium tetani (TeNT) and by Clostridium botulinum (BoNT) serotypes A-G and X (see WO 2018 / 009903 A2), as well as those produced by Clostridium baratii and Clostridium butyricum.

[0003] Clostridial neurotoxins are among the most potent toxins known. For example, botulinum neurotoxins have median lethal doses (LD ) in mice ranging from 0.5 to 5 ng / kg depending on the serotype. 50 ) values ​​are shown. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, whereas tetanus toxin acts in the central nervous system.

[0004] Clostridial neurotoxins are naturally synthesized as single-chain polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked by disulfide bonds. Cleavage occurs at a specific cleavage site, often called the activation site, located between multiple cysteine ​​residues that provide interchain disulfide bonds. This two-chain form is the active form of the toxin. The two chains are called the heavy chain (H chain) (having a molecular mass of approximately 100 kDa) and the light chain (L chain) (having a molecular mass of approximately 50 kDa). The H chain is bound to an N-terminal cartwheel component (H N domain) and C-terminal targeting component (H CThe cleavage site is located between the L chain and the translocation domain component. C Following binding of the domain to its target neuron and internalization of the bound toxin into the cell via endosomes, H N The domain translocates the L chain across the membrane of the endosome into the cytosol, and the L chain provides the protease function (also known as a non-cytotoxic protease).

[0005] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin) (Gerald K (2002) "Cells and Molecular Biology" (4th edition) John Wiley & Sons, Inc.). The acronym SNARE stands for Soluble NSF Attachment Receptor, and NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and thus secretion of molecules from cells via vesicle trafficking. This protease function is a zinc-dependent endopeptidase activity and shows high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cell, non-cytotoxic proteases can inhibit cellular secretion from that target cell. The light chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave the SNARE proteins.

[0006] Mouse LD 50 The assay is currently the only FDA-approved assay for the release of botulinum toxin. The assay simultaneously tests the activity of all three domains of the botulinum neurotoxin (i.e., binding, translocation, and protease). More specifically, the assay defines the intraperitoneal median lethal dose of the toxin (activity is measured at the mouse LD ) at defined time points, usually 2-4 days after administration. 50Unfortunately, the LD 50 The assay uses a large number of animals. 50 The units are not biological constants and therefore are highly dependent on the assay conditions and therefore cannot be considered absolute measurements, particularly as the interlaboratory variation associated with this assay can be as high as 60% (Sesardic et al. 2003; Biologicals 31(4):265-276).

[0007] The mouse flaccid paralysis assay, also known as the "mouse visceroptosis assay," relates the activity of botulinum toxin to the degree of abdominal distention observed after subcutaneous injection of the toxin into the inguinal thigh of mice, and the magnitude of paralysis is dose-dependent. This method relies on a humane endpoint and is therefore not suitable for use in mice with LD. 50 This assay has been proposed as an improved method for testing LD 50 It is approximately 10 times more sensitive than the LD assay, uses sublethal doses of toxin, and has a typical LD 50 Results are obtained in 24-48 hours compared to 72-96 hours for LD assays 50 The results from this assay are more rapid than the LD 50 The animals used in this assay were LD 50 Although it is 20% of the animals used in the assay, it still requires the use of animals.

[0008] Assays such as the mouse / rat phrenic nerve hemidiaphragm assay (based on the use of an ex vivo muscle preparation) relate the activity of botulinum neurotoxins to the reduction in the amplitude of the twitch response of the preparation after application to a maintenance medium. The usual endpoint of this assay is the time required to observe a 50% reduction in amplitude. Unfortunately, the hemidiaphragm assay (LD 50This assay leads to the use of large numbers of animals (as well as the conventional ELISA assay). Also, this assay requires highly skilled personnel trained in the use of sophisticated and expensive equipment. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Sesardic et al. 2003; Biologicals 31(4):265-276 [Non-Patent Document 2] Sesardic et al.,1996; Pharmacol Toxicol:78(5): 283-8 Summary of the Invention [Problem to be solved by the invention]

[0010] All of the above assays have certain drawbacks, especially animal welfare issues. Moreover, none of the above assays are suitable for high-throughput testing, for example to detect genetic or chemical regulators of Clostridial neurotoxins. Thus, there is a need in the art for alternative and / or improved Clostridial neurotoxin assays. [Means for solving the problem]

[0011] The present invention overcomes one or more of the problems set forth above.

[0012] In one aspect, the present invention provides a method for identifying a gene that controls Clostridial neurotoxin activity, comprising the steps of: a. providing a sample of human neuronal cells expressing a polypeptide comprising a C-terminal detectable label, said polypeptide being cleavable by a Clostridial neurotoxin; b. altering expression of a target gene in said cell; c. contacting the cell with the Clostridial neurotoxin; d. measuring the amount of C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; e. identifying the target gene as a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity differs from the Clostridial neurotoxin activity quantified when expression of the target gene is unaltered; The present invention provides a method comprising:

[0013] Alternatively, the method may include identifying the target gene as not being a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is equivalent to the Clostridial neurotoxin activity quantified when expression of the target gene is unaltered.

[0014] In one embodiment, the cell may be contacted with a Clostridial neurotoxin prior to altering expression of the target gene.

[0015] In a related aspect, the invention provides a method for identifying an agent that regulates Clostridial neurotoxin activity, comprising the steps of: a. providing a sample of human neuronal cells expressing a polypeptide comprising a C-terminal detectable label, said polypeptide being cleavable by a Clostridial neurotoxin; b. contacting said cell with said Clostridial neurotoxin and agent, said contacting being sequential or simultaneous; c. measuring the amount of C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; d. identifying the agent as a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity differs from the Clostridial neurotoxin activity quantified in the absence of the agent; The present invention provides a method comprising:

[0016] Alternatively, the method may include identifying the agent as not being a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is equivalent to the Clostridial neurotoxin activity quantified in the absence of the agent.

[0017] An agent may be identified as a negative regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is less than the Clostridial neurotoxin activity quantified in the absence of the agent, or an agent may be identified as a positive regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is greater than the Clostridial neurotoxin activity quantified in the absence of the agent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention employs the use of human neuronal cells, which entails a number of advantages not present in conventional cell-based assays, such as those employing the use of mouse cells. Human neuronal cells allow the use of human gene silencing libraries (e.g., human siRNA libraries) or agent libraries (e.g., human drug compound libraries). Thus, the present invention has higher predictive power than conventional cell-based assays, and improved human therapeutic relevance.

[0019] Thus, in one aspect, the invention provides a human neuronal cell expressing a polypeptide that is cleavable by a Clostridial neurotoxin and comprises a C-terminal detectable label, said cell preferably being a stable cell line comprising a nucleotide sequence or vector of the invention.

[0020] The human neural cell of the present invention is preferably a non-cancer cell. Preferably, the human neural cell of the present invention is an immortalized human neural progenitor cell or an equivalent (e.g., functionally equivalent) cell. For example, the cell may be ReNcell® Human Neural Progenitor (commercially available from Sigma-Aldrich) expressing the polypeptide of the present invention. Advantageously, non-cancer cells are genetically closer to native human neurons than cancer cell lines (e.g., neuroblastoma cells), and thus provide improved neural cell models for use in the assays described herein.

[0021] The human neural progenitor cells are preferably differentiated prior to use in the methods of the invention. Thus, in one embodiment, the human neural cells of the invention or for use in the methods of the invention (e.g., human neurons or precursors thereof) are derived from human neural progenitor cells. More preferably, the human neural cells of the invention or for use in the methods of the invention are human neurons or equivalent (e.g., functionally equivalent) cells thereof.

[0022] The polypeptides expressed by the human neural cells described herein can include both an N-terminal detectable label and a C-terminal detectable label. In one embodiment, the N-terminal detectable label and the C-terminal detectable label are different.

[0023] The detectable label is preferably a fluorescent label. In some embodiments, the polypeptides of the invention do not include an additional non-fluorescent label.

[0024] In one embodiment, either the N-terminal detectable label or the C-terminal detectable label is red fluorescent protein (RFP). More preferably, one end of the protein has an RFP detectable label, and the other end of the protein has a detectable label selected from green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP). Advantageously, RFP is antigenically distinguishable from GFP, CFP, and YFP, so the method of the invention allows the use of (secondary / confirmatory) immunogenic detection techniques such as Western blotting. Polypeptides in which both labels are selected from GFP, CFP, and YFP are typically not suitable for use with such immunogenic detection techniques in terms of antibody cross-reactivity. Preferably, the polypeptides described herein comprise an N-terminal RFP detectable label and a C-terminal GFP detectable label.

[0025] The polypeptides of the invention preferably do not contain tags for immobilization and / or purification. Since the assay is cell-based, such tags are not necessary. In one embodiment, the polypeptides of the invention may not contain a His tag (e.g., a polyhistidine tag such as a 6-His tag), a FLAG tag, a Protein A tag, a maltose binding protein tag, and / or a Myc tag.

[0026] Thus, in one aspect, the invention provides a polypeptide that is cleavable by a Clostridial neurotoxin and comprises an N-terminal RFP detectable label and a C-terminal GFP detectable label. In a related aspect, the invention provides a nucleotide sequence encoding the polypeptide, as well as a vector (e.g., a plasmid) comprising the nucleotide sequence of the invention operably linked to a promoter. Any promoter suitable for expression in human neuronal cells may be used, such as the CMV promoter.

[0027] The polypeptide comprises a substrate or a portion thereof for a Clostridial neurotoxin. The substrate (or portion thereof) is suitably selected based on the Clostridial neurotoxin to be assayed.

[0028] In one embodiment, the polypeptide comprises a substrate (or a portion thereof) of a botulinum neurotoxin, such as a SNARE protein. Thus, the polypeptide may comprise a substrate (or a portion thereof) of BoNT / A, BoNT / B, BoNT / C1, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X, etc. Preferably, the polypeptide comprises a substrate (or a portion thereof) of BoNT / A.

[0029] Polypeptides of the present invention may include synaptosomal associated protein of 25 kDa (SNAP-25), synaptoplevin / vesicle-associated membrane protein (VAMP, e.g., VAMP1, VAMP2, VAMP3, VAMP4, or VAMP5), syntaxin (e.g., syntaxin 1, syntaxin 2, or syntaxin 3), Ykt6, or portions thereof.

[0030] Preferably, the polypeptide of the present invention comprises SNAP-25 or a portion thereof, more preferably full-length SNAP-25.

[0031] BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptoplegin / vesicle-associated membrane protein (VAMP), BoNT / C1, BoNT / A, and BoNT / E cleave synaptosomal associated protein of 25 kDa (SNAP-25), and BoNT / C1 cleaves syntaxin 1, syntaxin 2, and syntaxin 3. BoNT / X has been shown to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

[0032] The term "portion thereof" in reference to a substrate of a clostridial neurotoxin includes the site at which the clostridial neurotoxin cleaves and may further include, for example, amino acid residues surrounding the site, if those amino acid residues are necessary for cleavage by the clostridial neurotoxin cleavage of the substrate. For example, a fragment may be ≦50, ≦25, or ≦15 amino acids of the clostridial neurotoxin substrate.

[0033] Polypeptides of the invention may comprise one or more polypeptides having at least 70% sequence identity to SEQ ID NOs: 4, 6, 8, 10, and / or 12. In one embodiment, polypeptides of the invention comprise one or more polypeptides having at least 80% or 90% sequence identity to SEQ ID NOs: 4, 6, 8, 10, and / or 12. Preferably, polypeptides of the invention comprise one or more polypeptides set forth as SEQ ID NOs: 4, 6, 8, 10, and / or 12.

[0034] Polypeptides of the invention may include polypeptides having at least 70% sequence identity to SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:12. In one embodiment, polypeptides of the invention include polypeptides having at least 80% or 90% sequence identity to SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:12. Preferably, polypeptides of the invention include the polypeptides set forth as SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:12.

[0035] A polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2. In one embodiment, a polypeptide of the invention comprises a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 2. Preferably, a polypeptide of the invention comprises (more preferably consists of) the polypeptide sequence shown as SEQ ID NO:2.

[0036] A nucleotide sequence of the invention (e.g. encoding a polypeptide sequence of the invention) may comprise one or more nucleotide sequences having at least 70% sequence identity to SEQ ID NO: 3, 5, 7, 9, and / or 11. In one embodiment, a nucleotide sequence of the invention comprises one or more nucleotide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 3, 5, 7, 9, and / or 11. Preferably, a nucleotide sequence of the invention comprises one or more nucleotide sequences set forth as SEQ ID NO: 3, 5, 7, 9, and / or 11.

[0037] The nucleotide sequence of the invention may comprise one or more nucleotide sequences having at least 70% sequence identity to SEQ ID NO: 3, 9, and / or 11. In one embodiment, the nucleotide sequence of the invention comprises one or more nucleotide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 3, 9, and / or 11. Preferably, the nucleotide sequence of the invention comprises one or more nucleotide sequences set forth as SEQ ID NO: 3, 9, and / or 11.

[0038] The nucleotide sequence of the invention may comprise a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1. In one embodiment, the nucleotide sequence of the invention comprises a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 1. Preferably, the nucleotide sequence of the invention comprises (more preferably consists of) the nucleotide sequence shown as SEQ ID NO: 1.

[0039] As described above, the present invention provides a method for identifying regulators (e.g., genes or agents) of Clostridial neurotoxin activity. Regulation of Clostridial neurotoxin activity can be upregulation / positive regulation (e.g., increasing Clostridial neurotoxin activity) or downregulation / negative regulation (e.g., decreasing Clostridial neurotoxin activity) of Clostridial neurotoxin activity. For example, regulation of Clostridial neurotoxin activity can be i. Binding of Clostridial neurotoxins to cells; ii. internalization of clostridial neurotoxins; iii. translocation of clostridial neurotoxin L chains out of endosomes (including reduction of disulfide bonds between L and H chains); iv. catalysis (e.g., inhibition or activation of SNARE cleavage); or v. duration of effect (e.g., duration of L chain activity in the cytoplasm of a cell) It may be a direct control of the level of

[0040] In other embodiments, the control of Clostridial neurotoxin activity may be indirect control. Indirect control may include control of a pathway required for Clostridial neurotoxin activity. An example of indirect control is the control of cellular trafficking of Clostridial neurotoxin receptors, such as synaptic vesicle glycoprotein 2A (SV2).

[0041] Preferably, the control of Clostridial neurotoxin activity is direct control.

[0042] Knowledge of indirect regulators can be used to further validate any gene or agent identified by the methods of the invention. In one embodiment, the Clostridial neurotoxin receptor (preferably SV2) can be used to determine whether a gene or agent indirectly regulates the transport of the Clostridial neurotoxin receptor, rather than directly regulating Clostridial neurotoxin activity (e.g., by transport of the Clostridial neurotoxin).

[0043] Thus, in one embodiment, the method of the invention comprises further validating the gene or agent identified in the method of the invention, which further comprises detecting the presence or absence of a Clostridial neurotoxin receptor in a cell when expression of the target gene is altered in the cell or when the cell is contacted with an agent. A suitable validation method is provided in Example 5 herein.

[0044] The amount of detected clostridial neurotoxin receptor may be compared with a negative control in which the expression of the target gene is not altered or the cells are not contacted with the agent. Alternatively or additionally, the negative control may include cells that are not contacted with clostridial neurotoxin. When the cells are not contacted with clostridial neurotoxin, the clostridial neurotoxin receptor is internalized and then recycled to the cell surface. In contrast, when the cells are contacted with clostridial neurotoxin, the SNARE protein is cleaved by the clostridial neurotoxin, preventing the effective recycling of the receptor to the cell surface.

[0045] Thus, in one embodiment, the amount of Clostridial neurotoxin receptor detected is less than the amount detected on the surface of cells that have not been contacted with a Clostridial neurotoxin.

[0046] In one embodiment, detecting a decreased amount of Clostridial neurotoxin receptor on the surface of a cell in which a) expression of a target gene has been altered and b) contacted with a Clostridial neurotoxin can indicate that the target gene indirectly controls Clostridial neurotoxin activity.

[0047] The above-mentioned reduction may be in comparison to comparable cells in which expression of the target gene is unaltered.

[0048] By further employing the use of a known direct inhibitor of Clostridial neurotoxin activity, it can be confirmed that the target gene indirectly controls Clostridial neurotoxin activity at the level of Clostridial neurotoxin receptor trafficking. For example, indirect control can be confirmed when the amount of cell surface receptor detected in the presence of an inhibitor is less than the amount of receptor detected in a comparable cell (where expression of the target gene is unaltered) that has also been contacted with Clostridial neurotoxin and the inhibitor (and vice versa).

[0049] In one embodiment, a) altered expression of a target gene and b) detecting equal or greater amounts of Clostridial neurotoxin receptors on the surface of cells contacted with a Clostridial neurotoxin may indicate that the target gene does not indirectly control Clostridial neurotoxin activity.

[0050] The equivalent or greater amount referred to above may be when compared to a comparable cell in which expression of the target gene is unaltered.

[0051] Similarly, in one embodiment, detecting a decreased amount of Clostridial neurotoxin receptor in a cell contacted with a) a Clostridial neurotoxin and b) an agent can indicate that the agent indirectly regulates Clostridial neurotoxin activity.

[0052] The reduced amount referred to above may be when compared to equivalent cells in the absence of the agent.

[0053] By further employing the use of a known direct inhibitor of Clostridial neurotoxin activity, it can be determined that the agent indirectly regulates Clostridial neurotoxin activity at the level of trafficking of the Clostridial neurotoxin receptor. For example, indirect regulation can be determined when the amount of receptor detected in the presence of an inhibitor is less than the amount of receptor detected in comparable cells that have also been contacted with a Clostridial neurotoxin and an inhibitor, but not with the agent (and vice versa).

[0054] In one embodiment, detecting equal or greater amounts of Clostridial neurotoxin receptors on the surface of cells contacted with a) a Clostridial neurotoxin and b) an agent can indicate that the agent does not indirectly regulate Clostridial neurotoxin activity.

[0055] The above-referenced equivalent or greater amount may be when compared to equivalent cells in the absence of the agent.

[0056] Preferably, the clostridial neurotoxin receptor is SV2.

[0057] The present invention overcomes the limitations of previous assays in that in many cell types, normal levels of cell surface SV2 are low and difficult to detect, and therefore it is impractical / difficult to determine whether cell surface SV2 levels change when expression of a target gene is altered or in the presence of an agent.

[0058] In one embodiment, the known direct inhibitor of Clostridial neurotoxin activity is an siRNA or shRNA that downregulates expression of thioredoxin reductase.

[0059] The method of the present invention is particularly suitable for high-throughput screening.In this respect, the method can preferably include the use of multiple cell samples, in each cell sample, the expression of different target genes is changed.RNAi library (or equivalent gene silencing library) is particularly suitable for use in such high-throughput method, as is drug library.

[0060] Thus, in one embodiment, the method of the present invention comprises: a. providing a plurality of samples of human neuronal cells expressing a polypeptide comprising a C-terminal detectable label, said polypeptide being cleavable by a Clostridial neurotoxin; b. altering expression of a different target gene in each of said plurality of samples, said target gene being different in each sample of cells; c. contacting the cells with a Clostridial neurotoxin; d. quantitating clostridial neurotoxin activity by measuring the amount of C-terminal detectable label; e. identifying the one or more target genes as regulators of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity differs from the quantified Clostridial neurotoxin activity when expression of the one or more target genes is unaltered; or f. identifying the one or more target genes as not regulators of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is equivalent to the quantified Clostridial neurotoxin activity when expression of the one or more target genes is unaltered; Includes.

[0061] In another embodiment, the method further comprises: a. providing a plurality of samples of human neuronal cells expressing a polypeptide comprising a C-terminal detectable label, said polypeptide being cleavable by a Clostridial neurotoxin; b. contacting the cells with a Clostridial neurotoxin and an agent, the contacting being sequential or simultaneous, and each of the samples being contacted with a different agent; c. measuring the amount of C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; d. identifying the one or more agents as regulators of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity differs from the quantified Clostridial neurotoxin activity in the absence of the one or more agents; or e. identifying the one or more agents as not being regulators of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is equivalent to the quantified Clostridial neurotoxin activity in the absence of the one or more agents; Includes.

[0062] The term "plurality," as used herein, means two or more. Preferably, the term "plurality" means more than two, for example, ≧50, ≧100, ≧150, ≧200, ≧250, or ≧300.

[0063] The method may include the use of a multi-well plate, each well containing one of a plurality of samples. The sensitivity and high signal-to-noise ratio of the method of the present invention allows the use of a multi-well plate containing ≧150 wells (preferably ≧300 wells, such as a 384-well plate). Advantageously, this allows for improved throughput when compared to methods employing <150 well plates (such as a 96-well plate).

[0064] Quantifying clostridial neurotoxin activity by measuring the amount of C-terminal detectable label according to the method of the present invention is particularly suitable for easy and rapid imaging and quantification using automated and / or high-throughput means (e.g., a microscope coupled with automated analysis software). Furthermore, the human neuronal cells of the present invention are highly sensitive to clostridial neurotoxins, which allows for shorter exposure times while still generating sufficient signal for detection. This is particularly suitable for use in automated screening using smaller multiwell plate formats (e.g., 384-well plates), e.g., containing ≧150 wells, because the shorter incubation times reduce the logistical complexity of scheduling the automated steps in the method. The severity of the bottleneck in the progression of a series of multiple plates from one step to the next (in a screening scan), where some steps are completed in minutes while others take hours to days, is reduced, fewer and shorter holding steps are required, and the equipment occupancy time is shorter.

[0065] The method for identifying genes that control Clostridial neurotoxin activity includes altering the expression of a target gene. This alteration can be up-regulation or down-regulation of expression (compared to the expression level in a comparable cell in which expression is not altered, i.e., the target gene expression is "unaltered"). Altering the expression of a target gene can be achieved by any method known in the art, for example, by gene editing, overexpression, or gene silencing.

[0066] In one embodiment, expression is altered by downregulating the expression of the target gene (e.g., by gene silencing).A preferred method for downregulating the expression of the target gene is by RNA interference (RNAi), for example by using short interfering or small hairpin RNA (siRNA or shRNA).

[0067] In embodiments where expression is altered by downregulating expression of a target gene, the target gene may be identified as a positive regulator of Clostridial neurotoxin activity if its quantified Clostridial neurotoxin activity is less than the Clostridial neurotoxin quantified when expression of the target gene is unaltered. Alternatively, the target gene may be identified as a negative regulator of Clostridial neurotoxin activity if its quantified Clostridial neurotoxin activity is greater than the Clostridial neurotoxin quantified when expression of the target gene is unaltered.

[0068] In contrast, in embodiments in which expression is altered by upregulating expression of a target gene, the target gene may be identified as a negative regulator of Clostridial neurotoxin activity if its quantified Clostridial neurotoxin activity is less than the Clostridial neurotoxin quantified when expression of the target gene is unaltered, or may be identified as a positive regulator of Clostridial neurotoxin activity if its quantified Clostridial neurotoxin activity is greater than the Clostridial neurotoxin quantified when expression of the target gene is unaltered.

[0069] A method for identifying an agent that regulates Clostridial neurotoxin activity includes contacting a cell with a Clostridial neurotoxin and an agent, either sequentially (e.g., contacting with the Clostridial neurotoxin and then with the agent, or contacting with the agent and then with the Clostridial neurotoxin) or simultaneously. Preferably, the contacting is sequential.

[0070] In one embodiment, the cell is contacted with the agent before contacting with the Clostridial neurotoxin. Such a method is particularly suitable for identifying agents that can prevent intoxication by Clostridial neurotoxins. Agents that can prevent intoxication may be suitable for use as prophylactic agents in treatment.

[0071] Alternatively, the cells may be contacted with the agent after contacting with the Clostridial neurotoxin. Such methods may be particularly suitable for identifying agents capable of inhibiting Clostridial neurotoxin activity after intoxication, and may be suitable for use as post-intoxication therapeutics in treatment.

[0072] An agent identified as a positive regulator of Clostridial neurotoxin activity may be a Clostridial neurotoxin sensitizer, which may be used in combination with a Clostridial neurotoxin in therapy to modulate the local activity of the Clostridial neurotoxin (e.g., allowing for reduced dosage and minimizing spread to other tissues).

[0073] Clostridial neurotoxin activity can be quantified by measuring the amount of the C-terminal detectable label. Preferably, Clostridial neurotoxin activity can be quantified by measuring the amount of the C-terminal detectable label and the N-terminal detectable label.

[0074] The amount of C-terminal detectable label after contacting cells with Clostridial neurotoxin can be compared with the amount of C-terminal detectable label measured in the absence of Clostridial neurotoxin.For example, the amount of C-terminal detectable label can be measured in the same cell sample before and after contacting with Clostridial neurotoxin.Alternatively, the amount of C-terminal detectable label can be measured after contacting with Clostridial neurotoxin and compared with the amount of C-terminal detectable label present in the equivalent cell sample under equivalent conditions that is not contacted with Clostridial neurotoxin.

[0075] In one embodiment, the loss of the C-terminal detectable label (e.g., over time) when compared to equivalent cells that have not been contacted with the Clostridial neurotoxin or when compared to the same cells before contacting with the Clostridial neurotoxin indicates the presence of Clostridial neurotoxin activity. Alternatively, in one embodiment, the absence of (or detection of an equivalent amount of) the C-terminal detectable label (e.g., over time) when compared to equivalent cells that have not been contacted with the Clostridial neurotoxin or when compared to the same cells before contacting with the Clostridial neurotoxin indicates the absence of Clostridial neurotoxin activity. In one embodiment, the partial loss of the C-terminal detectable label (e.g., over time) when compared to equivalent cells that have not been contacted with the Clostridial neurotoxin or when compared to the same cells before contacting with the Clostridial neurotoxin may indicate the presence of reduced Clostridial neurotoxin activity (and vice versa).

[0076] The method may further comprise measuring the amount of N-terminal detectable label.Similar to C-terminal label, the amount of N-terminal detectable label after contacting cells with Clostridial neurotoxin can be compared with the amount of N-terminal detectable label measured in the absence of Clostridial neurotoxin.For example, the amount of N-terminal detectable label can be measured in the same cell sample before and after contacting with Clostridial neurotoxin.Alternatively, the amount of N-terminal detectable label can be measured after contacting with Clostridial neurotoxin and compared with the amount of N-terminal detectable label present in an equivalent sample of cells under equivalent conditions that are not contacted with Clostridial neurotoxin.

[0077] Preferably, clostridial neurotoxin activity is determined by comparing the amount of N-terminal label and C-terminal label, preferably a greater amount of N-terminal label relative to C-terminal label indicates that the polypeptide has been cleaved by a clostridial neurotoxin.

[0078] The detectable label of the present invention may be measured at one or more time points after contacting the cells with a Clostridial neurotoxin, such that a percentage of Clostridial neurotoxin activity may be calculated.

[0079] In one embodiment, the detectable label may be measured after contacting the cells with the Clostridial neurotoxin for at least 2, 5, 10, 15, 20, 30, 40, 50, 60, or 70 hours. In other embodiments, the detectable label may be measured after contacting the cells with the Clostridial neurotoxin for less than 100, 80, 70, 60, or 50 hours. Preferably, the detectable label may be measured after contacting the cells with the Clostridial neurotoxin for less than 72 hours, more preferably less than 50 hours. Thus, the detectable label may be measured after contacting the cells with the Clostridial neurotoxin for 20 to 60 hours, preferably 40 to 55 hours (e.g., about 48 hours). The cells may be fixed prior to measuring the amount of detectable label.

[0080] When comparing the cells of the method of the present invention to a control (e.g., equivalent cells in which expression of the target gene is unaltered or not contacted with the agent of the present invention, or another control as mentioned above), it is assumed that the measurement is performed in the same manner (e.g., at the same time point after contacting the cells with the Clostridial neurotoxin). This allows for a comparison of the quantified Clostridial neurotoxin activity of the cells of the method of the present invention to the control to identify the presence or absence of differences in Clostridial neurotoxin activity.

[0081] The measuring or detecting step of the present invention may be carried out using any suitable means known to those skilled in the art. In one embodiment, a fluorescent label present on the polypeptide (or present on an antibody that binds to a Clostridial neurotoxin receptor) is excited with light of a suitable wavelength and the resulting fluorescence is detected. For this reason, the present invention employs the use of a fluorescent microscope. In a preferred embodiment, the measuring or detecting step includes the use of a high-throughput screening system. Examples of suitable systems include the use of the Opera Phenix™ High-Content Screening System marketed by PerkinElmer and / or suitable imaging software such as Columbus™ software (marketed by PerkinElmer). In some embodiments, the measuring or detecting step of the present invention may be automated and employ the use of robotics.

[0082] The method of the present invention preferably does not employ the use of electronic coupling between detectable labels as described herein.In particular, it is preferred that these labels are not positioned so that the donor label (e.g., the N-terminal label) can transfer energy to the acceptor label (e.g., the C-terminal label) by dipole-dipole coupling mechanism or the like.Preferably, the present invention does not employ the use of fluorescence resonance energy transfer (FRET).

[0083] The method of the present invention may include a validation step of comparing the quantified Clostridial neurotoxin activity detected in the method with a positive control. Positive validation may occur when the quantified Clostridial neurotoxin activity detected is equivalent to the positive control.

[0084] The method of the present invention may further include a step of verifying that the regulator is indeed a positive regulator. In one embodiment, the method includes a step of contacting a cell with a negative regulator of a known Clostridial neurotoxin activity and showing that the effect on Clostridial neurotoxin activity can be reversed. In one embodiment, the method includes a step of up-regulating the expression of a negative regulator of a known Clostridial neurotoxin activity and showing that the effect on Clostridial neurotoxin activity can be reversed. Preferably, the method includes a step of down-regulating the expression of a positive regulator of a known Clostridial neurotoxin activity and showing that the effect on Clostridial neurotoxin activity can be reversed.

[0085] The method of the present invention may further include a step of verifying that the regulator is indeed a negative regulator. In one embodiment, the method includes a step of contacting a cell with a positive regulator of a known Clostridial neurotoxin activity and showing that the effect on Clostridial neurotoxin activity can be reversed. In one embodiment, the method includes a step of down-regulating the expression of a negative regulator of a known Clostridial neurotoxin activity and showing that the effect on Clostridial neurotoxin activity can be reversed. In one embodiment, the method includes a step of up-regulating the expression of a positive regulator of a known Clostridial neurotoxin activity and showing that the effect on Clostridial neurotoxin activity can be reversed.

[0086] A known (positive) regulator of Clostridial neurotoxin activity is thioredoxin reductase. In one embodiment, expression of thioredoxin reductase is upregulated. Preferably, expression of thioredoxin reductase is downregulated.

[0087] The term "equivalent" as used herein may mean that two or more values ​​being compared are not statistically significantly different. Preferably, the term "equivalent" as used herein means that the two or more values ​​are the same. Similarly, the term "unmodified" as used herein may mean that two or more values ​​being compared are not statistically significantly different. Preferably, the term "unmodified" as used herein means that the two or more values ​​are the same.

[0088] The difference or alteration (e.g., increase or decrease) referred to herein preferably means a statistically significant difference or alteration (e.g., a statistically significant increase or decrease). Thus, the difference in the quantified Clostridial neurotoxin activity is preferably a statistically significant difference in the quantified Clostridial neurotoxin activity. In one embodiment, the difference in the quantified Clostridial neurotoxin activity is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% difference when compared to the quantified Clostridial neurotoxin activity of a negative control (e.g., a comparable cell in which the expression of the target gene is not altered or is not contacted with the agent of the present invention or another control as mentioned above).

[0089] The method of the present invention is an in vitro method.

[0090] In one embodiment, the method of the invention is a live cell method, and optionally the method employs real-time monitoring of Clostridial neurotoxin activity when expression of a target gene is altered and / or when the cell is contacted with an agent.

[0091] Preferably, the term "contacting a cell with a Clostridial neurotoxin" means that the surface of the cell is contacted with the Clostridial neurotoxin. Suitably, the Clostridial neurotoxin may be added to the medium in which the cells are present, for example, cell culture medium. The term "contacting a cell with a Clostridial neurotoxin" preferably does not include contacting by expressing the Clostridial neurotoxin in the cell, which technique does not provide any information regarding the binding, internalization, and / or translocation of the Clostridial neurotoxin.

[0092] The method of the invention typically includes contacting the cell with less than 1500 nM of a Clostridial neurotoxin, hi one embodiment, the cell is contacted with less than 1000 nM of a Clostridial neurotoxin, such as less than 500 nM, 250 nM, or 100 nM.

[0093] The methods of the invention may include contacting a cell with at least 1 nM, 5 nM, 10 nM, 20 nM, or 50 nM of a Clostridial neurotoxin.

[0094] In one embodiment, the method of the invention may comprise the step of contacting a cell with 1-1000 nM of a Clostridial neurotoxin, for example 1-500 nM or 1-200 nM of a Clostridial neurotoxin.

[0095] The inventors have surprisingly found that the sensitivity of the method of the invention can be improved by contacting the cells with a buffer comprising GDNF and cell-permeable cAMP (and optionally further comprising CaCl2 and KCl), in the presence of which the cells become highly sensitive to Clostridial neurotoxins at concentrations below 100 nM, preferably below 50 nM, more preferably between 5 and 15 nM (e.g. about 10 nM).

[0096] The buffer may contain GDNF, d-cAMP, CaCl2, and KCl. GDNF may be present at 1-100 ng / ml, preferably 10 ng / ml. d-cAMP may be present at 0.1-5 mM, preferably 1 mM. CaCl2 may be present at 0.1-7 mM, preferably 2 mM. KCl may be present at 1-100 mM, preferably 56 mM.

[0097] The buffer may be a component of a kit of the invention.

[0098] Thus, in one aspect, the present invention provides a. Clostridial neurotoxins; b. a buffer containing GDNF, cell-permeable cyclic adenosine monophosphate (cAMP), CaCl2, and KCl; The present invention provides a composition comprising:

[0099] In one embodiment, the composition comprises GDNF present at 1-100 ng / ml, d-cAMP present at 0.1-5 mM, CaCl2 present at 0.1-7 mM, and KCl present at 1-100 mM.Preferably, the buffer comprises GDNF present at 10 ng / ml, d-cAMP present at 1 mM, CaCl2 present at 2 mM, and KCl present at 56 mM.

[0100] References herein to "cAMP" are preferably interchangeable with d-cAMP.

[0101] The composition may comprise less than 1500 nM of Clostridial neurotoxin. In one embodiment, the composition comprises less than 1000 nM, such as less than 500 nM, 250 nM, or 100 nM of Clostridial neurotoxin. In some embodiments, the composition may comprise at least 1 nM, 5 nM, 10 nM, 20 nM, or 50 nM of Clostridial neurotoxin. In one embodiment, the composition may comprise 1-1000 nM of Clostridial neurotoxin, such as 1-500 nM, or 1-200 nM of Clostridial neurotoxin. Preferably, the composition comprises a Clostridial neurotoxin at a concentration of less than 100 nM, preferably less than 50 nM, more preferably 5-15 nM (e.g., about 10 nM).

[0102] In some embodiments, the composition further comprises a cell, eg, a human neuronal cell as described herein.

[0103] The cells may be incubated with the Clostridial neurotoxin for any suitable time. In one embodiment, the cells may be incubated with the Clostridial neurotoxin for at least 2, 5, 10, 15, 20, 30, 40, 50, 60, or 70 hours. In another embodiment, the cells may be incubated with the Clostridial neurotoxin for less than 100, 80, 70, 60, or 50 hours. Preferably, the cells are incubated with the Clostridial neurotoxin for less than 72 hours, more preferably less than 50 hours. Thus, the cells may be incubated with the Clostridial neurotoxin for 20 to 60 hours, preferably 40 to 55 hours (e.g., about 48 hours). Advantageously, the human neuronal cells of the present invention are highly sensitive to the Clostridial neurotoxin, thereby allowing for short incubation times of less than 72 hours (about 48 hours) with the Clostridial neurotoxin, thereby reducing the time required to perform the assay.

[0104] The cells may be incubated with the Clostridial neurotoxin at any suitable temperature, hi one embodiment, the cells are incubated with the Clostridial neurotoxin at 30-40°C, preferably 37°C.

[0105] A target gene or agent identified by the method of the invention may be used in a method for treating a disorder. Thus, in one aspect, the invention provides a method for treating a disorder comprising administering to a subject an agent identified by the method of the invention. In another aspect, the invention provides a method for treating a disorder comprising altering expression of a gene identified by the method of the invention in a subject.

[0106] In one aspect, the invention provides a kit comprising a cell according to the invention, and optionally instructions for its use (e.g. in a method described herein). In a related aspect, the invention provides a kit comprising a nucleotide sequence according to the invention, and optionally instructions for its use (e.g. in a method described herein). In a related aspect, the invention provides a kit comprising a vector according to the invention, and optionally instructions for its use (e.g. in a method described herein). In one embodiment, the kit comprises a cell (preferably identical to a cell type of the invention, but which does not comprise a nucleotide sequence of the invention and does not express a polypeptide of the invention), a nucleotide sequence or vector of the invention, and optionally instructions for its use (e.g. in a method described herein). The kit may comprise one or more separate containers, each of which contains the recited components of the kit.

[0107] The present invention is suitable for application to a wide variety of Clostridial neurotoxins. Thus, in the context of the present invention, the term "Clostridial neurotoxin" includes toxins produced by Clostridium botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X), Clostridium tetani (tetanus neurotoxin), Clostridium butyricum (botulinum neurotoxin serotype E), and Clostridium baratii (botulinum neurotoxin serotype F), as well as modified Clostridial neurotoxins or derivatives derived from any of the foregoing. The term "Clostridial neurotoxin" also includes botulinum neurotoxin serotype H.

[0108] Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum in the form of large protein complexes consisting of BoNT itself and a number of accessory proteins complexed to it. Currently, there are nine different classes of botulinum neurotoxins, namely botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which share a similar structure and mode of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and such serotype classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage sequence identity of amino acids.

[0109] BoNTs are absorbed in the gastrointestinal tract and, after entering the systemic circulation, bind to the presynaptic membranes of cholinergic nerve terminals, preventing the release of their neurotransmitter, acetylcholine.

[0110] Tetanus toxin is produced in a single serotype by Clostridium tetani: Clostridium butyricum produces BoNT / E and Clostridium baratii produces BoNT / F.

[0111] The term "clostridial neurotoxin" is also intended to encompass modified clostridial neurotoxins and derivatives thereof, including but not limited to those mentioned above. A modified clostridial neurotoxin or derivative may contain one or more amino acids modified compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids not present in the native (unmodified) form of the clostridial neurotoxin. For example, a modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may alter the functional aspects of the toxin, such as biological activity or persistence. Thus, in one embodiment, the clostridial neurotoxin of the present invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.

[0112] A modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., a modified H C domain), the modified heavy chain binds to target neurons with greater or less affinity than the native (unmodified) Clostridial neurotoxin. C Such modifications in the H domain alter binding to ganglioside receptors and / or protein receptors on target neurons. C This may involve modifying residues in the ganglioside binding site or in the protein (SV2 or synaptotagmin) binding site of the domain. Examples of such modified Clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, which are incorporated herein by reference in their entireties.

[0113] A modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domains that may alter or modify the SNARE protein specificity of the modified L chain. Examples of such modified Clostridial neurotoxins are described in WO 2010 / 120766 and US 2011 / 0318385, which are incorporated by reference in their entireties.

[0114] The modified Clostridial neurotoxin may contain one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified Clostridial neurotoxin. For example, the modified Clostridial neurotoxin may contain a leucine-based or tyrosine-based motif that increases or decreases the biological activity and / or biological persistence of the modified Clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL (SEQ ID NO: 22), xExxxLL (SEQ ID NO: 23), xExxxIL (SEQ ID NO: 24), and xExxxLM (SEQ ID NO: 25), where x is any amino acid. Suitable tyrosine-based motifs include Yxx-Hy (SEQ ID NO: 26), where Hy is a hydrophobic amino acid. Examples of modified Clostridial neurotoxins containing leucine-based and tyrosine-based motifs are described in WO 2002 / 08268, which is incorporated herein by reference in its entirety.

[0115] The term "Clostridial neurotoxin" is intended to encompass hybrid and chimeric Clostridial neurotoxins. A hybrid Clostridial neurotoxin comprises at least a portion of a light chain from one Clostridial neurotoxin or subtype and at least a portion of a heavy chain from another Clostridial neurotoxin or Clostridial neurotoxin subtype. In one embodiment, the hybrid Clostridial neurotoxin may contain the entire light chain from one Clostridial neurotoxin subtype and a heavy chain from another Clostridial neurotoxin subtype. In another embodiment, a chimeric Clostridial neurotoxin may contain the heavy chain protein (e.g., binding domain) of one Clostridial neurotoxin subtype and another portion of the heavy chain may be from another Clostridial neurotoxin subtype. Similarly or alternatively, the therapeutic element may contain light chain proteins from different Clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of such clostridial neurotoxins to patients who are immunologically resistant to a given clostridial neurotoxin subtype, who may have a lower than average concentration of receptors for a given clostridial neurotoxin heavy chain binding domain, or who may have protease-resistant mutants of membrane or vesicle toxin substrates (e.g., SNAP-25, VAMP, and syntaxin).Hybrid and chimeric clostridial neurotoxins are described in US 8,071,110, which is incorporated herein by reference in its entirety.Thus, in one embodiment, the clostridial neurotoxin of the present invention is a hybrid clostridial neurotoxin or a chimeric clostridial neurotoxin.

[0116] The term "clostridial neurotoxin" may also encompass newly discovered botulinum neurotoxin protein family members expressed by non-clostridial microorganisms, such as the Enterococcus-encoded toxin with the closest sequence identity to BoNT / X, the Weissella oryzae-encoded toxin called BoNT / Wo that cleaves VAMP2 at W89-W90 (NCBI Reference Sequence, WP_027699549.1), the Enterococcus faecium-encoded toxin that cleaves VAMP2 and SNAP25 (GenBank, OTO22244.1), and the Chryseobacterium pipero-encoded toxin (NCBI Reference Sequence, WP_034687872.1).

[0117] In a preferred embodiment, the Clostridial neurotoxin is a botulinum neurotoxin, more preferably BoNT / A.

[0118] In one embodiment, the Clostridial neurotoxin can be BoNT / A. A reference BoNT / A sequence is provided as SEQ ID NO:13.

[0119] In another embodiment, the Clostridial neurotoxin can be BoNT / B. A reference BoNT / B sequence is provided as SEQ ID NO:14.

[0120] In another embodiment, the Clostridial neurotoxin can be BoNT / C. The reference BoNT / C1 sequence is provided as SEQ ID NO:15.

[0121] In another embodiment, the Clostridial neurotoxin can be BoNT / D. A reference BoNT / D sequence is provided as SEQ ID NO:16.

[0122] In another embodiment, the Clostridial neurotoxin can be BoNT / E. A reference BoNT / E sequence is provided as SEQ ID NO:17.

[0123] In another embodiment, the Clostridial neurotoxin can be BoNT / F. A reference BoNT / F sequence is provided as SEQ ID NO:18.

[0124] In another embodiment, the Clostridial neurotoxin can be BoNT / G. A reference BoNT / G sequence is provided as SEQ ID NO:19.

[0125] In one embodiment, the Clostridial neurotoxin can be BoNT / X. A reference BoNT / X sequence is provided as SEQ ID NO:20.

[0126] In another embodiment, the Clostridial neurotoxin can be TeNT. A reference TeNT sequence is provided as SEQ ID NO:21.

[0127] It is intended that embodiments relating to the various methods of the invention apply equally to other methods, cells, polypeptides, nucleotide sequences, kits, and compositions of the invention, and vice versa.

[0128] Sequence identity

[0129] To determine percent identity, any of a variety of sequence alignment methods can be used, including but not limited to global, local, and combined methods, such as segment approach methods. Protocols for determining percent identity are routine procedures within the scope of those skilled in the art. Global methods perform sequence alignment from the beginning to the end of the molecule and determine the best alignment by adding up the scores of individual residue pairs and applying gap penalties. Non-limiting methods include, for example, CLUSTAL W (see, e.g., Julie D, Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994)) and iterative refinement methods (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838(1996)). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509(1992)), Gibbs Sampling (see, e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214(1993)), and Align-M (see, e.g., Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435(2004)).

[0130] Thus, percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned and the alignment score is optimized using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.), as shown below (amino acids are identified by the standard one-letter code):

[0131] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of the percent sequence identity can also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The alignment of two or more sequences and the determination of the percent identity can be performed using specialized mathematical algorithms such as BLAST, with which the skilled person will be familiar.

[0132] Alignment scores for determining sequence identity

number

[0133] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, typically small deletions of from one to about 30 amino acids, and small amino- or carboxy-terminal extensions such as an amino-terminal methionine residue, small linker peptides of up to about 20-25 residues, or affinity tags.

[0134] Conservative Amino Acid Substitutions

[0135] Basicity: Arginine lysine Histidine Acidic: Glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Ballin Aromatic: Phenylalanine Tryptophan Tyrosine small: glycine Alanine Serine Threonine Methionine

[0136] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the invention can also include non-naturally occurring amino acid residues.

[0137] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for introducing non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system including E. coli S30 extracts and commercially available enzymes, as well as other reagents. Proteins are purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993. In the second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In a third method, E. coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine) that is introduced into the polypeptide in place of its natural counterpart.See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0138] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may substitute for amino acid residues in the polypeptides of the invention.

[0139] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of amino acids at putative contact sites. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identification of essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0140] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods in which two or more positions in a polypeptide are randomized simultaneously, selection is performed for functional polypeptides, and then the mutagenized polypeptides are sequenced to determine the spectrum of substitutions allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., US Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0141] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary for many of the terms used in this disclosure.

[0142] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation.

[0143] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0144] Amino acids are referred to herein using their amino acid name, three-letter abbreviation, or one-letter abbreviation. The term "protein" as used herein encompasses proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0145] Other definitions of terms may be provided throughout this specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, as such may vary. It should also be understood that the terminology used herein is intended to describe only specific embodiments, and is not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0146] Where a range of values ​​is provided, each intervening value between the upper and lower limit of that range is also understood to be specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either or both of the upper and lower limits are included in the smaller range, or neither, is also encompassed within the disclosure, provided that any specifically excluded limit in the stated range applies. Where the stated range includes one or both of its limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0147] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents, and reference to "the clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0148] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0149] Embodiments of the invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0150] [Figure 1] FIG. 1 shows the method for generating ReNcell VM stably expressing TagRFPT-SNAP25-TagGFP. Images (bottom) show red fluorescence (right) and green fluorescence (right). [Diagram 2] BoNT / A-induced degradation of the C-terminal fragment of the construct is shown. ReD SNAPR cells were differentiated for 14 days without growth factors, after which 100 nM BoNT / A was added to the cells and intoxicated for 48 h. (A) Cells were imaged using a fluorescent microscope. Corresponding GFP, RFP, and mixed GFP / RFP channels are indicated. (B) Lysates were subjected to SDS-PAGE for Western blot analysis. Rabbit antibodies against tagRFPT and tagGFP were used for Western blot. [Diagram 3] A diagram summarizing the degradation of constructs by BoNT / A is shown. [Figure 4] Figure 1 shows that differentiation and stimulation enhanced buffer sensitized ReD SNAPR cells to BoNT / A. (A) ReD SNAPR cells were subjected to ReNcell differentiation medium supplemented with and without GDNF and d-cAMP during differentiation and to high potassium buffer during intoxication. The resulting modified medium is known as ReDS (ReNcell enhanced differentiation and stimulation) medium. Loss of tagGFP fluorescence upon cleavage of the dual-tagged SNAP25 construct was observed in a dose-dependent manner. ReD SNAPR cells in ReDS medium showed enhanced sensitivity to BoNT / A compared to normal ReNcell medium as detected using confocal microscopy. (B) Quantification of BoNT / A-mediated cleavage in both conditions showed an improvement in EC50 from 43 nM to 6 nM in ReD SNAPR exposed to ReDS medium. (C) Western blot detection of BoNT / A-mediated SNAP25 construct cleavage in ReD SNAPR cells in normal and ReDS medium. The upper blot shows cell lysates probed with tRFP antibody, and the lower blot shows cell lysates probed with tGFP antibody. [Diagram 5]13 shows that siRNA against TrxR rescues BoNT / A-mediated construct cleavage. A graph of TrxR1 knockdown level and construct cleavage is 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). [Figure 6] Figure 1: BoNT / A intoxication prevents SV2 transport to the cell surface. A) ReNcell VM cells were treated with siNT3, siVAMP2, and siTrxR1 for 72 hours before BoNT / A was added. Cells were fixed and stained with a primary antibody against SV2A (without permeabilization). An Alexa-488 secondary antibody was used against the primary antibody. (B) Schematic of BoNT / A-mediated decreased transport of SV2 to the cell surface. [Figure 7] FIG. 1 shows a schematic diagram for performing genome-wide siRNA screening using the cell line of the present invention.

[0151] Array List

[0152] Where the first Met amino acid residue or the corresponding first codon is shown in any of the SEQ ID NOs below, said residue / codon can be optional.

[0153] SEQ ID NO:1 (nucleic acid sequence, construct TagRFPT-SNAP25-TagGFP)

[0154] SEQ ID NO:2 (polypeptide sequence, construct TagRFPT-SNAP25-TagGFP) *

[0155] SEQ ID NO:3 (nucleic acid sequence, TagRFPT) ATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAGAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCAACACCGAGATGCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAACCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTCAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACCACAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATGGCATGGACGAGCTGTACAAG

[0156] SEQ ID NO:4 (polypeptide sequence, TagRFPT) MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGMDELYK

[0157] SEQ ID NO:5 (nucleic acid sequence, Glycine-Serine rich linker 1) GGCTCGGGCTCGGGCTCGGGC

[0158] SEQ ID NO:6 (polypeptide sequence, Glycine-Serine rich linker 1) GSGSGSG

[0159] SEQ ID NO: 7 (nucleic acid sequence, Glycine-Serine rich linker 2) GGCGGCTCGGGCTCGGGGC

[0160] SEQ ID NO:8 (polypeptide sequence, Glycine-Serine rich linker 2) GGSGSG

[0161] SEQ ID NO:9 (nucleic acid sequence, SNAP25) GTGGCCGAAGACGCAGACATGCGCAATGAGCTGGAGGAGATGCAGCGAAGGGCTGACCAGTTGGCTGATGAGTCGCTGGAAAGCACCCGTCGTATGCTGCAACTGGTTGAAGAGAGTAAAGATGCTGGTATCAGGACTTTGGTTATGTTGGATGAACAAGGAGAACAACTCGATCGTGTCGAAGAAGGCATGAACCATATCAACCAAGACATGAAGGAGGCTGAGAAAAATTTAAAAGATTTAGGGAAATGCTGTGGCCTTTTCATATGTCCTTGTAACAAGCTTAAATCAAGTGATGCTTACAAAAAAGCCTGGGGCAATAATCAGGACGGAGTGGTGGCCAGCCAGCCTGCTCGTGTAGTGGACGAACGGGAGCAGATGGCCATCAGTGGCGGCTTCATCCGCAGGGTAACAAATGATGCCCGAGAAAATGAAATGGATGAAAACCTAGAGCAGGTGAGCGGCATCATCGGGAACCTCCGTCACATGGCCCTGGATATGGGCAATGAGATCGATACACAGAATCGCCAGATCGACAGGATCATGGAGAAGGCTGATTCCAACAAAACCAGAATTGATGAGGCCAACCAACGTGCAACAAAGATGCTGGGAAGTGGTTAC

[0162] SEQ ID NO:10 (polypeptide sequence, SNAP25) VAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLDRVEEGMNHINQDMKEAEKNLKDLGKCCGLFICPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSGY

[0163] SEQ ID NO:11 (nucleic acid sequence, TagGFP) GTGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACGGCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACGGCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCACATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCGCACCATCCAGTTCCAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCCAACAACGGCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCGACCACTACCAGACCAACGTGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGACCAAGATCAGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAA

[0164] SEQ ID NO:12 (polypeptide sequence, TagGFP) VSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICCTTGKLPVPWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVKFEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLESFSACCHTHGMDELYR*

[0165] SEQ ID NO:13 (BoNT / A - UniProt P10845)

[0166] SEQ ID NO:14 (BoNT / B - UniProt P10844)

[0167] SEQ ID NO:15 (BoNT / C - UniProt P18640)

[0168] SEQ ID NO:16 (BoNT / D - UniProt P19321)

[0169] SEQ ID NO:17 (BoNT / E - UniProt Q00496)

[0170] SEQ ID NO:18 (BoNT / F - UniProt A7GBG3)

[0171] SEQ ID NO:19 (BoNT / G - UniProt Q60393)

[0172] SEQ ID NO:20 (polypeptide sequence, BoNT / X)

[0173] SEQ ID NO:21 (TeNT - UniProt P04958) EXAMPLES

[0174] Example 1 – Generation of stable cell lines

[0175] Gene synthesis and subcloning

[0176] The nucleotide sequences of tagRFPT and tagGFP were obtained from Evrogen and synthesized by GeneArt (Thermo Fisher Scientific). The gene product, tRFPT-SNAP25-tGFP, was flanked by attB sequences for Gateway® cloning. The synthesized gene product was then subcloned into the lentiviral vector, pLenti6.3 / V5-dest, using the BP Clonase Enzyme Kit (Thermo Fisher) according to the manufacturer's protocol. The resulting vector, pLenti6.3-tRFPT-SNAP25-tGFP, was transformed into E. coli BL21 cells and selected with ampicillin antibiotic. Positive bacterial clones were subjected to maxiprep using Machery-Nagel endotoxin-free maxiprep kit according to the manufacturer's protocol.

[0177] Generation of lentivirus from HEK293FT cells

[0178] To prepare for lentivirus production, HEK293FT cells were cultured in high glucose Dulbecco's modified Eagle's medium with 4500 mg / L glucose supplemented with 10% fetal bovine serum (FBS) (Gibco) and then transfected into T75cm 2The cells were seeded at 80% confluence in flasks and incubated overnight at 37°C, 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 the supplier, and the flasks were incubated for 6 hours at 37°C, 5% CO2. Six hours after transfection, the medium containing the lipid-DNA complex was carefully removed from the flask, discarded, and replaced with 10 ml of pre-warmed medium. The cells were incubated overnight at 37°C, 5% CO2. 24 hours after transfection, 10 ml of cell supernatant (first batch of virus) was collected and stored at 4°C in a 15 ml conical tube. The harvested medium was replaced with 10 ml of pre-warmed medium and the flasks were incubated overnight at 37°C, 5% CO2. A second batch of virus was harvested 48 hours post-transfection. Supernatants from both batches were centrifuged at 2000 rpm for 10 minutes at room temperature to remove cell debris. After centrifugation, the cleared lentiviral supernatant was collected and filtered through a 0.45 μm pore filter to remove remaining cell debris. Virus was aliquoted into 1 ml and stored at -80°C.

[0179] Measuring lentiviral titers by GFP selection

[0180] HEK293FT cells were seeded in 96-well plates (Nunc) at a density of 10,000 cells / well in 100 μl of culture medium. 10% of the virus was added using fresh culture medium containing 8 mg / ml (final concentration) of polybrene reagent (Sigma catalog number H9268). -1 ~10 -4A serial dilution of 100 μl of the prepared dilution was performed. The cells were transduced by removing the existing medium and replacing it with 100 μl of the prepared dilution for the corresponding well. The plate was incubated overnight at 37° C., 5% CO2. The next day, the culture medium was changed to fresh medium without polybrene. The cells were incubated for another 3 days, after which the virus titer was calculated. The titer was calculated in transduction units (TU) per ml based on the percentage of GFP-positive cells using the appropriate dilution factor. The desired transduction range was 1-20%. The virus titer was then determined using the following formula: Titer=(F×C / V)×D, where F=frequency of GFP-positive cells (percent GFP-positive cells / 100), C=number of cells per well at the time of transduction, V=volume of inoculum in ml (0.1 ml), and D=lentivirus dilution factor.

[0181] Generation of ReNcell VM stable cell lines from lentivirus

[0182] ReNcell VM (Millipore) cells were seeded at 80% confluence onto 24-well plates coated with laminin (final concentration 20 μg / ml) and incubated overnight at 37°C, 5% CO2. The medium was removed and 500 μl of lentivirus per well was added with polybrene reagent at a final concentration of 8 mg / ml. The cells were incubated overnight at 37°C, 5% CO2. The next day, the medium was replaced with fresh medium without polybrene. After transduction, the cells were expanded and subjected to FAC sorting using GFP wavelength.

[0183] result

[0184] The assay construct consisting of full-length SNAP25 flanked by tagRFPT and tagGFP was cloned into a lentiviral vector backbone. Generation of stable cell lines was achieved using a modified lentiviral production protocol consisting of lipofection of the construct with lentiviral packaging plasmids into the HEK293T cell line. The resulting lentivirus was purified and added to ReNcell VM cells, which were finally sorted using FACS (see FIG. 1) to confirm the generation of stable cell lines. This v-myc immortalized cell line was derived from human neural progenitor cells (NPCs), which are genetically closer to native human neurons compared to cancer cell lines and therefore a better neuronal model for use in the assays described herein.

[0185] Example 2 - The construct is susceptible to BoNT / A cleavage

[0186] Materials and Methods

[0187] Perkin Elmer CellCarrier 384 Ultra™ imaging plates and Nunc 24-well tissue culture dishes were incubated overnight at 4° C. with 20 μg / mL laminin (Invitrogen).

[0188] Imaging

[0189] The stable cell line of the present invention (referred to as ReD SNAPR cell line) was differentiated according to the ReNcell VM cell manufacturer's protocol. Briefly, cells were seeded at 3000 cells per well on pre-coated Perkin Elmer CellCarrier 384 Ultra™ imaging plates. Cells were maintained in ReNcell NSC maintenance medium (differentiation medium) without growth factors (EGF and FGF2) for 14 days with medium changes every 3 days. Cells were incubated with 100 nM BoNT / A in differentiation medium for 48 hours. Cells were fixed using fixative (4% paraformaldehyde and 2% sucrose). Fixed cells were imaged using Opera™ Phenix.

[0190] Western blot

[0191] ReD SNAPR cells were differentiated according to the ReNcell VM cell manufacturer's protocol. Briefly, cells were seeded at 30,000 cells per well in Nunc 24-well tissue culture dishes. Cells were maintained in ReNcell NSC maintenance medium (differentiation medium) without growth factors (EGF and FGF2) for 14 days with medium changes every 3 days. Cells were incubated with 100 nM BoNT / A in differentiation medium for 48 h. Medium was aspirated and cells were lysed in NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 nM Tris-Cl, pH 8.0). To prepare samples for loading on SDS-PAGE gels, 10% DTT and 6X loading solution (BioRad) were added to the samples and boiled for 5 min. 20 μL of sample was added to each lane of a NuPAGE Bis Tris 4-12% gel (Thermo Fisher) and scanned at 120 V until the dye front had run out. The gel was transferred to a nitrocellulose membrane and probed overnight with anti-tRFP and anti-tag(CGY)FP (Evrogen).

[0192] result

[0193] FIG. 2 shows that the assay construct is susceptible to degradation by BoNT / A. Conventional cell-based assays focus on FRET interactions or direct Western blot detection of cleaved SNAP25 in cell lysates, which are not suitable for high-throughput screening (HTS) applications. Under normal circumstances, the BoNT / A-cleaved C-terminal fragment of full-length SNAP25 is barely detectable due to its small molecular weight, and therefore its fate is usually not elucidated. This previously unrecognized observation indicates that the C-terminal fragment is degraded together with the fluorophore attached to it. The relative simplicity of the methodology makes it highly suitable for low-throughput to high-throughput applications.

[0194] Figure 3 provides a schematic showing BoNT / A-mediated degradation of the C-terminus of the SNAP25 construct. Upon internalization 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 degradation of the C-terminal fragment while retaining the N-terminal construct. This degradation can be detected using fluorescence microscopy and Western blot.

[0195] Example 3 - Improved sensitivity of ReD SNAPR cells to BoNT / A

[0196] ReD SNAPR cells were seeded onto 384-well plates as described above. To enhance differentiation, ReD SNAPR cells were cultured in standard ReNcell medium containing 10 ng / mL GDNF and 1 mM d-cAMP (cell-permeable cAMP). Various concentrations (0–1 μM) of BoNT / A were added to standard medium and to ReDS medium containing 10 ng / mL GDNF, 1 mM d-cAMP, 2 mM CaCl2, and 56 mM KCl. Differentiated ReD SNAPR cells were intoxicated with BoNT / A-containing medium, fixed, and imaged as described above.

[0197] result

[0198] Figure 4 shows that the addition of GDNF and d-cAMP during differentiation and high potassium conditions during intoxication improved the sensitivity of the cell lines to BoNT / A. Dose-response curves and EC 50 Values ​​were determined using low nM values ​​when cells were exposed to ReDS medium (see FIG. 4B).

[0199] Example 4 - Thioredoxin reductase (TrxR1) as an assay control

[0200] ReDS SNAPR cells were seeded into 384-well plates and differentiated as described above. Differentiated cells were treated with 25 nmol of either siRNA non-targeting control NT3 or siRNA against TrxR1 using Lipofectamine RNAimax according to the manufacturer's protocol, and cells were left for 72 hours. ReDS medium containing 10 nM BoNT / A was added to cells 48 hours later, they were fixed and imaged as described above. Briefly, cells were fixed and TrxR1 was detected using an antibody against TrxR1, and fluorescence was imaged using Opera Phenix. Mean fluorescence intensity levels in the GFP, RFP, and near-infrared channels were captured and measured.

[0201] result

[0202] Figure 5 shows that siTrxR-treated cells are more resistant to BoNT / A-mediated cleavage compared to control. Therefore, TrxR1 can be used as a suitable positive control to identify genes related to intracellular transport of BoNT / A. In particular, TrXR1 knockdown can be used to show that BoNT / A intoxication can be rescued, so that the genes identified in the assay can be further validated.

[0203] Example 5 – Use of BoNT receptor SV2

[0204] ReNcell VM cells were seeded and differentiated in 384-well plates as described above. Differentiated cells were treated with 25 nmol of either siNT3, siVAMP2, or siTrxR using Lipofectamine RNAimax according to the manufacturer's protocol, and cells were left for 72 hours. ReDS medium containing 10 nM BoNT / A was added to cells for 48 hours before fixation. For immunostaining, 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. Alexa-488 conjugated secondary antibody was added to cells for 1 hour before cells were imaged using Opera Phenix. Cells were then imaged using Opera Phenix with GFP and DAPI channels shown.

[0205] result

[0206] Although SV2 is the main receptor for BoNT / A, its post-intoxication trajectory in cells has not been further studied. Figure 6 shows that BoNT / A intoxication leads to a decrease in SV2 at the cell surface, which may be a typical consequence of BoNT / A-mediated defective transport to the cell surface. In this case, the circulation of SV2 back to the cell surface is blocked. Therefore, SV2 can be used as a readout for BoNT / A intoxication.

[0207] Many genes can control BoNT / A activity in cells. An example of indirect control can be at the level of BoNT receptor SV2 transport (rather than modifying the toxin activity itself). To select candidates involved in SV2 transport, surface SV2 staining can be an ideal selection criterion. One example shown here is cells depleted of VAMP2, which resulted in reduced surface SV2 staining upon BoNT / A intoxication. This can be attributed to the synergistic effect of blocking vesicle exocytosis at the cell surface by reducing VAMP2 and BoNT / A intoxication.

[0208] Surface SV2 can be rescued through depletion of TrxR, indicating that TrxR itself does not affect exocytosis of SV2 at the cell surface, but directly modifies BoNT / A activity through the release of its light chain (LC), which inadvertently results in restoration of surface SV2 due to the reduction of BoNT / A LC in the cytoplasm.

[0209] Thus, SV2 is useful in the assays of the present invention because it can be used to select candidate genes directly involved in BoNT / A transport from among genes that alter the transport of the BoNT / A receptor SV2.

[0210] Example 6 – Genome-wide siRNA screening

[0211] Figure 7 provides a diagram showing a method for performing genome-wide siRNA screening using the cell lines of the present invention. ReD SNAPR cells are plated and differentiated as described above. siRNA libraries are prepared and complexed with Lipofectamine™ RNAiMAX using standard protocols, and siRNAs are transfected into ReD SNAPR cells. Cells are spiked with BoNT / A in stimulation buffer, then fixed, imaged using Opera™ Phenix, and quantified using Columbus™ software.

[0212] Positive hits may be subjected to further validation by assessing restoration in the presence of siRNA against TrxR1.Confirmation that the gene directly regulates BoNT activity is performed by SV2 cell surface staining as described above.

[0213] Example 7 –Identification of prophylactic antibotulism therapeutics

[0214] ReD SNAPR cells are plated and differentiated as described above and exposed to agents (e.g., small molecule drugs). Cells are treated with BoNT / A in stimulation buffer, then fixed, imaged using Opera™ Phenix, and quantified using Columbus™ software.

[0215] If cleavage of the construct is inhibited, the agent is identified as a prophylactic antibotulism therapeutic.

[0216] Example 8 –Identification of post-intoxication anti-botulism therapeutic agents

[0217] ReD SNAPR cells are plated and differentiated as described above, and cells are stimulated with BoNT / A in stimulation buffer to observe cleavage of the construct (loss of GFP). Cells are then exposed to an agent (e.g., a small molecule drug). Finally, cells are fixed and imaged using Opera™ Phenix and quantified using Columbus™ software.

[0218] If restoration of GFP is observed, the agent is identified as a post-intoxication anti-botulism therapeutic.

[0219] Example 9 –Identification of BoNT sensitizers

[0220] ReD SNAPR cells are plated and differentiated as described above and exposed to agents (e.g., small molecule drugs). Cells are treated with BoNT / A in stimulation buffer, then fixed, imaged using Opera™ Phenix, and quantified using Columbus™ software.

[0221] If cleavage of the construct is improved (e.g., faster or more cleavage is evident), the agent is identified as a BoNT sensitizer, which is pursued for further investigation for use as a combination product to modulate the local activity of the clostridial neurotoxin (e.g., allowing for reduced doses and minimizing spread to other tissues).

[0222] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain 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 bioengineering or related fields are intended to be within the scope of the following claims. [Explanation of symbols]

[0223] [Figure 1] promoter truncated: Truncated type Linker: Linker Modified Lentivirus generation protocol: Modified Lentivirus generation protocol Fluorescence activated cell sorting: Fluorescence activated cell sorting [Figure 2] No Toxin: Merged: Overlapped Percentage of GFP / RFP relative to control (no toxin) Percentage of remaining constructs [Figure 3] Degradation: Decomposition [Figure 4] Normal media: Standard medium ReDS media: ReDS medium Merged: Overlapped % GFP / RFP normalized to ctrl: GFP / RFP percentage normalized to control Normal: Standard [Figure 5] siRNA control: siRNA control % TrxR1 knockdown relative to NT3: Percentage of TrxR1 knockdown relative to NT3 % GFP / RFP relative to ctrl (no tox): GFP / RFP percentage relative to control (no toxin) [Figure 6] Surface SV2A: Surface SV2A Merged: Overlapped Nornal: Standard Surface SV2: Surface SV2 Internalized SV2: Internalized SV2 Low surface SV2: Low surface SV2 Lower surface SV2: Lower surface SV2 Rescued surface SV2: Rescued surface SV2 [Figure 7] Seed cells in laminin-coated Perkin Elmer CellCarrier 384 Ultra TM Imaging plate: Cells are seeded onto laminin-coated Perkin Elmer CellCarrier 384 Ultra™ imaging plates. Enhanced differentiation with withdrawal of EGF and FGF2, addition of GDNF and d-CAMP: Enhanced differentiation with withdrawal of EGF and FGF2, addition of GDNF and d-CAMP Prepare siRNA assay library plates from stock plate using Agilent Bravo TM Liquid handling platform: Prepare siRNA assay library plates from stock plates using the Agilent Bravo™ liquid handling platform. Complex siRNA with Lipofectamine TMRNAiMAX: Complexing siRNA with Lipofectamine™ RNAiMAX siRNA forward transfection: transfer siRNA complexes into cell plate using Integra VIAFLO TM 384: siRNA forward transfection: Transfer siRNA complexes to cell plates using Integra VIAFLO™ 384 Addition of BoNT / A prepared in stimulation buffer (+KCL, +CaCl2): Addition of BoNT / A prepared in stimulation buffer (+KCL, +CaCl2) Plates are fixed and imaged using Opera TM Phenix High content screen microscope: Plates are fixed and imaged using an Opera™ Phenix high content screening microscope. Images are quantified using Columbus TM Software: Images are quantified using Columbus™ software.

Claims

1. 1. A method for identifying a gene that controls Clostridial neurotoxin activity, comprising the steps of: a. altering expression of a target gene in human neuronal cells in a sample, wherein the cells express a polypeptide that includes a C-terminal detectable label and is cleavable by a Clostridial neurotoxin; b. contacting said cell with said Clostridial neurotoxin; c. measuring the amount of C-terminal detectable label, thereby quantifying Clostridial neurotoxin activity; and d. identifying the target gene as a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity differs from the Clostridial neurotoxin activity quantified when expression of the target gene is unaltered; or e. identifying the target gene as not being a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is equivalent to the Clostridial neurotoxin activity quantified when expression of the target gene is unaltered.

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

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

4. The method of claim 2, wherein the target gene is identified as a negative regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is greater than the quantified Clostridial neurotoxin activity when expression of the target gene is unaltered.

5. 5. The method of any one of claims 1 to 4, wherein expression of a different target gene is altered in each sample of a plurality of samples of human neuronal cells.

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

7. The method of any one of claims 1 to 6, 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 of claim 7, wherein the direct regulator regulates Clostridial neurotoxin activity at the level of: i. Binding of clostridial neurotoxins to cells; ii. internalization of clostridial neurotoxins; iii. translocation of the light chains of clostridial neurotoxins out of endosomes; iv. Catalysis; and / or v. Persistence of light chain activity within the cytoplasm of the cell.

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

10. The method of any one of claims 7 to 9, wherein the determining step comprises detecting the presence or absence of a Clostridial neurotoxin receptor in the cell when expression of the target gene is altered.

11. detecting a decreased amount of Clostridial neurotoxin receptor on the surface of the cell when expression of the target gene is altered, compared to a comparable cell contacted with a Clostridial neurotoxin in which expression of the target gene is unaltered, indicates that the target gene indirectly regulates Clostridial neurotoxin activity; or detecting an equal or greater amount of Clostridial neurotoxin receptor on the surface of the cells when expression of the target gene is altered, compared to comparable cells contacted with a Clostridial neurotoxin in which expression of the target gene is unaltered, indicating that the target gene directly regulates Clostridial neurotoxin activity. The method according to any one of claims 7 to 10, Here, the term "equivalent cells" refers to human neuronal cells that express a polypeptide that includes a C-terminal detectable label and is cleavable by a Clostridial neurotoxin.

12. The method of any one of claims 9 to 11, wherein the Clostridial neurotoxin receptor is synaptic vesicle glycoprotein 2A (SV2).

13. 1. A method for identifying an agent that regulates Clostridial neurotoxin activity, comprising the steps of: a. contacting human neuronal cells in a sample with a Clostridial neurotoxin and an agent, wherein the contacting is sequential or simultaneous, and the cells express a polypeptide that includes a C-terminal detectable label and is cleavable by the Clostridial neurotoxin; b. measuring the amount of C-terminal detectable label, thereby quantifying clostridial neurotoxin activity; and c. identifying the agent as a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity differs from the Clostridial neurotoxin activity quantified in the absence of the agent; or d. identifying the agent as not being a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is equivalent to the Clostridial neurotoxin activity quantified in the absence of the agent; wherein the method comprises treating the cells with glial cell line-derived neurotrophic factor (GDNF), cell-permeable cyclic adenosine monophosphate (cAMP), CaCl 2 and contacting with KCl.

14. 1. A method for identifying an agent that regulates Clostridial neurotoxin activity, comprising the steps of: a. contacting human neuronal cells in a sample with a Clostridial neurotoxin and an agent, wherein the contacting is sequential or simultaneous, and the cells express a polypeptide that includes a C-terminal detectable label and is cleavable by the Clostridial neurotoxin; b. measuring the amount of C-terminal detectable label, thereby quantifying Clostridial neurotoxin activity; c. identifying the agent as a regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity differs from the Clostridial neurotoxin activity quantified in the absence of the agent; and d. Determining whether the agent is a direct regulator of Clostridial neurotoxin activity or an indirect regulator of Clostridial neurotoxin activity.

15. The method of claim 13 or 14, wherein the agent is identified as a negative regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is less than the Clostridial neurotoxin activity quantified in the absence of the agent.

16. The method of claim 13 or 14, wherein the agent is identified as a positive regulator of Clostridial neurotoxin activity if the quantified Clostridial neurotoxin activity is greater than the quantified Clostridial neurotoxin activity in the absence of the agent.

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

18. 18. The method of claim 14 or 17, wherein the direct regulator regulates Clostridial neurotoxin activity at the level of: i. Binding of clostridial neurotoxins to cells; ii. internalization of clostridial neurotoxins; iii. translocation of the light chains of clostridial neurotoxins out of endosomes; iv. Catalysis; and / or v. Persistence of light chain activity within the cytoplasm of the cell.

19. The method according to any one of claims 14 or 17 to 18, wherein the indirect regulator regulates cellular trafficking of a Clostridial neurotoxin receptor.

20. 20. The method of any one of claims 14 or 17-19, wherein the determining step comprises detecting the presence or absence of a Clostridial neurotoxin receptor in the cell when the cell is targeted by the agent.

21. detecting a decreased amount of Clostridial neurotoxin receptor on the surface of the cell when the cell is contacted with the agent, compared to a comparable cell not contacted with the agent but contacted with a Clostridial neurotoxin, indicates that the agent indirectly regulates Clostridial neurotoxin activity; or detecting an equal or greater amount of Clostridial neurotoxin receptor on the surface of the cells when the cells are contacted with the agent, compared to comparable cells not contacted with the agent but contacted with a Clostridial neurotoxin, indicating that the agent directly regulates Clostridial neurotoxin activity. The method according to any one of claims 14 or 17 to 20, Here, the term "equivalent cells" refers to human neuronal cells that express a polypeptide that includes a C-terminal detectable label and is cleavable by a Clostridial neurotoxin.

22. The method of any one of claims 19 to 21, wherein the Clostridial neurotoxin receptor is synaptic vesicle glycoprotein 2A (SV2).

23. The method of any one of claims 1 to 22, wherein the human neuronal cells are non-cancerous cells.

24. 24. The method of any one of claims 1 to 23, wherein the human neuronal cell is an immortalized human neural progenitor cell or the human neuronal cell is derived from an immortalized human neural progenitor cell.

25. 25. The method of claim 24, wherein the human neural cells are differentiated from immortalized human neural progenitor cells.

26. The method of any one of claims 1 to 25, wherein the polypeptide further comprises an N-terminal detectable label that is different from the C-terminal detectable label.

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

28. The method according to any one of claims 1 to 27, wherein the polypeptide comprises an N-terminal RFP and a C-terminal GFP.

29. The method according to any one of claims 1 to 28, wherein a. the polypeptide 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. the polypeptide is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:1; or c. the polypeptide 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. the polypeptide comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:

2.

30. The method according to any one of claims 1 to 29, wherein a. the polypeptide 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. the polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO:1; or c. the polypeptide comprises the polypeptide sequence: i. SEQ ID NO:4, ii. SEQ ID NO: 10, and iii. SEQ ID NO: 12; or d. the polypeptide comprises the polypeptide sequence of SEQ ID NO:

2.

31. The method according to any one of claims 1 to 30, wherein the polypeptide consists of the polypeptide sequence of SEQ ID NO:

2.

32. The method further comprises injecting the cells with GDNF, cAMP, CaCl 2 32. The method of any one of claims 1 to 12 or 14 to 31, comprising contacting with KCl.

33. The GDNF is present at a concentration of 1-100 ng / ml, cAMP is present at a concentration of 0.1-5 mM, and CaCl 2 The method of claim 32, wherein is present at a concentration of 0.1 to 7 mM and / or KCl is present at a concentration of 1 to 100 mM.

34. A kit comprising: (a) Glial cell line-derived neurotrophic factor (GDNF), cell-permeable cyclic adenosine monophosphate (cAMP), CaCl 2 , and KCl, and (b-1) a human neuronal cell capable of expressing a polypeptide, wherein the polypeptide is cleavable by a Clostridial neurotoxin and comprises a C-terminal detectable label; or (b-2) a nucleic acid comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide is cleavable by a Clostridial neurotoxin and comprises an N-terminal red fluorescent protein (RFP) and a C-terminal green fluorescent protein (GFP), the 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 iii. a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:11; Contains, or (b-3) A vector comprising 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, the 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 iii. a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:11; Including, and (c) optionally, instructions for their use; Including the kit.

35. The GDNF is present at a concentration of 1-100 ng / ml, cAMP is present at a concentration of 0.1-5 mM, and CaCl 2 35. The kit of claim 34, wherein is present at a concentration of 0.1 to 7 mM and KCl is present at a concentration of 1 to 100 mM.

36. 1. A composition comprising: a. Clostridial neurotoxins and b. Glial cell line-derived neurotrophic factor (GDNF), cell-permeable cyclic adenosine monophosphate (cAMP), CaCl 2 , and KCl, A composition comprising:

37. The GDNF is present at a concentration of 1-100 ng / ml, cAMP is present at a concentration of 0.1-5 mM, and CaCl 2 37. The composition of claim 36, wherein is present at a concentration of 0.1 to 7 mM and KCl is present at a concentration of 1 to 100 mM.

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