EA048091B1 - CELLULAR ANALYSES OF CLOSTRIDIAL NEUROTOXINS - Google Patents

Description

The invention relates to cellular assays of clostridial neurotoxins.

Bacteria of the genus Clostridia produce highly potent and specific protein toxins that can poison neurons and other cells they target. Examples of such clostridial neurotoxins include those produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes AG and X (see WO 2018/009903 A2), as well as those produced by C. baratii and C. butyricum.

Clostridial neurotoxins include some of the most potent toxins known. For example, botulinum neurotoxins have median lethal dose (LD50) values in mice ranging from 0.5 to 5 ng/kg, depending on the serotype. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, particularly the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system; tetanus toxin acts in the central nervous system.

In nature, clostridial neurotoxins are synthesized as a single-chain polypeptide that undergoes post-translational modification by proteolytic cleavage to form two polypeptide chains linked by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, which is located between the cysteine residues that mediate the interchain disulfide bond. It is this double-chain form that is the active form of the toxin. The two chains are referred to as the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain contains an N-terminal translocation component (HN domain) and a C-terminal targeting component (HC domain). The cleavage site is located between the L chain and the translocation domain components. Following binding of the NS domain to its target neuron and internalization of the bound toxin into the cell via the endosome, the HN domain translocates the L chain across the endosomal membrane into the cytosol, and the L chain provides protease function (also known as non-cytotoxic protease).

Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or syntaxin) - see Gerald K. (2002) Cell and Molecular Biology (4th edition) John Wiley & Sons, Inc. The abbreviation SNARE comes from the term (Soluble NSF Attachment Receptor), where NSF stands for N-ethylmaleimide-sensitive factor. SNARE proteins are essential for the fusion of intracellular vesicles and thus the secretion of molecules via vesicular transport from the cell. The protease function is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Accordingly, after delivery to the desired target cell, the non-cytotoxic protease is able to inhibit cellular secretion from the target cell. Clostridial neurotoxin L-chain proteases are non-cytotoxic proteases that cleave SNARE proteins.

The mouse LD ₅₀ assay is currently the only FDA-approved test for the release of botulinum toxins. The assay simultaneously tests for the activity of all three domains of the botulinum neurotoxin (i.e., binding, translocation, and protease). More specifically, it determines the median lethal intraperitoneal dose of toxin at a specific time point, typically 2-4 days after dosing (the potency is expressed in mouse LD ₅₀ units). Unfortunately, however, LD 50 assays use large numbers of animals. Moreover, LD 50 units are not absolute measurements because they are not biological constants - thus, they are highly dependent on assay conditions. In particular, errors associated with this assay can be as high as 60% between different testing centers (Sesardic et al. 2003; Biologicals 31(4):265-276).

The mouse flaccid paralysis test, also known as the mouse abdominal ptosis assay, correlates the potency of botulinum toxin with the degree of abdominal distension observed following subcutaneous injection of the toxin into the left inguinal-thoracic region of the mouse - the amount of paralysis is dose dependent. This approach has been proposed as an improvement on the mouse LD50 test because it is based on a humane endpoint. The assay is approximately 10 times more sensitive than the LD50 assay, uses a sublethal dose of toxin, and is more rapid than the LD50 test, providing results within 24-48 h, compared with 72-96 h for a typical LD50 assay. Results from this assay demonstrate excellent agreement with LD50 values (Sesardic et al., 1996; Pharmacol Toxicol, 78(5):283-8). Although this assay uses 20% of the number of animals used in the LD50 assay, the use of animals is still necessary.

Assays such as the mouse/rat phrenic nerve hemidiaphragmatic assay (which is based on the use of ex vivo nerve/muscle preparations) correlate the potency of a botulinum neurotoxin with the reduction in the amplitude of the convulsive response to the drug after its application to the support medium. The usual endpoint of the assay is the time required to observe a 50% reduction in amplitude. Unfortunately, however, the hemidiaphragmatic assay (like the LD50 assay) results in the use of large numbers of animals. In addition, the

- 1 048091 analysis requires highly qualified personnel trained in the use of complex and expensive equipment.

All of the above assays have certain drawbacks, especially animal welfare issues. Furthermore, none of the above assays are suitable for high-throughput testing, such as for detecting genetic or chemical regulators of clostridial neurotoxin. Thus, there is a need in the art for alternative and/or improved clostridial neurotoxin assays.

The present invention solves one or more of the above problems.

In one aspect, the invention provides a method for identifying a gene that regulates the activity of a clostridial neurotoxin, comprising:

(a) obtaining a sample of human neuronal cells expressing a polypeptide that contains a C-terminal detectable label, wherein the polypeptide is cleavable by a clostridial neurotoxin;

b) change in the expression of the target gene of the cells;

c) contact of cells with clostridial neurotoxin;

d) measuring the amount of detectable C-terminal tag, thereby quantifying the activity of the clostridial neurotoxin; and

e) identifying the target gene as a regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity differs from the quantified Clostridial neurotoxin activity when expression of the target gene is not altered.

Alternatively, the method may include determining that the target gene is not a regulator of clostridial neurotoxin activity when the quantified clostridial neurotoxin activity is equivalent to the quantified clostridial neurotoxin activity when expression of the target gene is not altered.

In one embodiment, the cells may be contacted with a clostridial neurotoxin prior to altering the expression of the target gene.

In a related aspect, the invention provides a method for identifying an agent that regulates the activity of a clostridial neurotoxin, comprising:

(a) obtaining a sample of human neuronal cells expressing a polypeptide that contains a C-terminal detectable label, wherein the polypeptide is cleaved by a clostridial neurotoxin;

b) contacting the cells with a clostridial neurotoxin and an agent, wherein the contacting is sequential or simultaneous;

c) measuring the amount of detectable C-terminal tag, thereby quantifying the activity of the clostridial neurotoxin; and

d) identifying the agent as a regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity differs from the quantified Clostridial neurotoxin activity in the absence of the agent.

Alternatively, the method may include identifying that the agent is not a regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is equivalent to the quantified Clostridial neurotoxin activity in the absence of the agent.

An agent may be identified as a negative regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is lower than the quantified Clostridial neurotoxin activity in the absence of the agent. Alternatively, an agent may be identified as a positive regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is higher than the quantified Clostridial neurotoxin activity in the absence of the agent.

The present invention uses human neuronal cells, and this is associated with a number of advantages that are absent in conventional cell-based assays, such as assays using 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 libraries). Thus, the present invention has higher prognostic capabilities than conventional cell-based assays and has increased therapeutic relevance for humans.

Thus, in one aspect, the invention provides a human neuronal cell expressing a polypeptide that can be cleaved by a clostridial neurotoxin and comprises a C-terminal detectable label. The cell is preferably a stable cell line comprising a nucleotide sequence or vector according to the invention.

The human neural cell of the invention is preferably not a cancer cell. Preferably, the human neural cell of the invention is an immortalized cell.

- 2 048091 a human neuronal progenitor cell or a cell equivalent thereto (e.g., functionally equivalent thereto). For example, the cell may be a ReNcell® human neuronal progenitor cell (commercially available from Sigma-Aldrich) expressing the polypeptide of the invention. Preferably, the non-cancerous cells are genetically closer to natural human neurons compared to cancer cell lines (e.g., neuroblastoma cells) and thus represent an improved neuronal cell model for use in the assays described herein.

The human neuronal progenitor cell is preferably differentiated prior to use in the method of the present invention. Thus, in one embodiment, the human neuronal cell of the invention or for use in the method of the invention (e.g., a human neuron or progenitor thereof) is derived from a human neuronal progenitor cell. More preferably, the human neuronal cell of the invention or for use in the method of the invention is a human neuron or a cell equivalent thereto (e.g., functionally equivalent thereto).

A polypeptide expressed by a human neuronal cell described herein may comprise a detectable label at both the N-terminus and the C-terminus. In one embodiment, the detectable labels of the N-terminus and the C-terminus are different.

The detectable label is preferably a fluorescent label. In some embodiments, the polypeptide of the invention does not comprise an additional non-fluorescent label.

In one embodiment, the N-terminal or C-terminal detectable label is a red fluorescent protein (RFP). More preferably, one end of the protein has a detectable RFP 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). Preferably, RFP is antigenically distinct from GFP, CFP and YFP, so that the methods of the present invention allow 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 generally not suitable for use with such immunogenic detection techniques due to antibody cross-reactivity. Preferably, the polypeptide described herein comprises an N-terminal detectable RFP label and a C-terminal detectable GFP label.

Preferably, the polypeptide of the invention does not contain a tag for immobilization and/or purification; since the assay is cell-based, such tags are not necessary. In one embodiment, the polypeptide of the invention may not contain a His tag (e.g., a polyhistidine tag, such as a 6-H-tag), a FLAG tag, a protein A tag, a maltose binding protein tag, and/or a Myc tag.

Therefore, in one aspect, the invention relates to a polypeptide that is cleaved by a clostridial neurotoxin and comprises an N-terminal detectable RFP tag and a C-terminal detectable GFP tag. In a related aspect, the invention provides a nucleotide sequence encoding said 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 a human neuronal cell can be used, such as the CMV promoter.

The polypeptide comprises a clostridial neurotoxin substrate or a portion thereof. The substrate (or portion thereof) is selected appropriately based on the clostridial neurotoxin being analyzed.

In one embodiment, the polypeptide comprises a botulinum neurotoxin substrate (or a portion thereof), 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. Preferably, the polypeptide comprises a substrate (or a portion thereof) of BoNT/A.

The polypeptide of the invention may comprise synaptosomal protein 25 kDa (SNAP-25), synaptobrevin/vesicle-associated membrane protein (VAMP, such as VAMP1, VAMP2, VAMP3, VAMP4, or VAMP5), syntaxin (such as syntaxin 1, syntaxin 2, or syntaxin 3), Ykt6, or a portion thereof.

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

BoNT/B, BoNT/D, BoNT/F, and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/C1, BoNT/A, and BoNT/E cleave synaptosomal protein 25 kDa (SNAP-25); and BoNT/C1 cleaves syntaxin 1, syntaxin 2, and syntaxin 3. BoNT/X was found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

The term “part” of a clostridial neurotoxin substrate includes the site at which the clostridial neurotoxin is cleaved and may further include a number of amino acid residues surrounding said site, for example, if said additional amino acid residues are necessary for cleavage of the substrate by clostridial neurotoxin cleavage.

- 3 048091 stridial neurotoxin. For example, the fragment may contain <50, <25, or <15 amino acids of the clostridial neurotoxin substrate.

A polypeptide of the invention may comprise one or more polypeptides having at least 70% sequence identity to SEQ ID NO: 4, 6, 8, 10 and/or 12. In one embodiment, a polypeptide of the invention comprises one or more polypeptides having at least 80% or 90% sequence identity to SEQ ID NO: 4, 6, 8, 10 and/or 12. Preferably, a polypeptide of the invention comprises one or more polypeptides shown as SEQ ID NO: 4, 6, 8, 10 and/or 12.

The polypeptide of the invention may comprise polypeptides having at least 70% sequence identity with SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12. In one embodiment, the polypeptide of the invention comprises polypeptides having at least 80% or 90% sequence identity with SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12. Preferably, the polypeptide of the invention comprises the polypeptides shown as SEQ ID NO: 4, SEQ ID NO: 10 and SEQ ID NO: 12.

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

A nucleotide sequence of the invention (e.g., which encodes 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 shown as SEQ ID NO: 3, 5, 7, 9 and/or 11.

The nucleotide sequence of the invention may comprise one or more nucleotide sequences having at least 70% sequence identity with 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 with SEQ ID NO: 3, 9 and/or 11. Preferably, the nucleotide sequence of the invention comprises one or more nucleotide sequences shown as SEQ ID NO: 3, 9 and/or 11.

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.

As mentioned above, the invention provides methods for identifying a regulator (e.g., a gene or an agent) of clostridial neurotoxin activity. The regulation of clostridial neurotoxin activity may be an up-regulation/positive regulation (e.g., increased clostridial neurotoxin activity) or a down-regulation/negative regulation of clostridial neurotoxin activity (e.g., decreased clostridial neurotoxin activity). For example, the regulation of clostridial neurotoxin activity may be a direct regulation at the level of:

i) binding of clostridial neurotoxin to the cell;

ii) internalization of clostridial neurotoxin;

iii) translocation of the L-chain of clostridial neurotoxin from the endosome (including reduction of the disulfide bond between the L-chain and the H-chain);

iv) catalysis (e.g. inhibition or activation of SNARE cleavage); or

v) duration of effect (e.g. maintenance of L-chain activity in the cell cytoplasm).

In other embodiments, the regulation of the activity of a clostridial neurotoxin may be indirect regulation. Indirect regulation may include regulation of a pathway required for the activity of the clostridial neurotoxin. An example of indirect regulation is regulation of cellular trafficking of a clostridial neurotoxin receptor, such as synaptic vesicle glycoprotein 2A (SV2).

Preferably, regulation of clostridial neurotoxin activity is direct regulation.

The information about the indirect regulators can be used to further test any genes or agents identified using the method of the invention. In one embodiment,

- 4 048091 Clostridial neurotoxin receptor (preferably SV2) activity can be used to determine whether a gene or agent indirectly regulates Clostridial neurotoxin receptor migration, or directly regulates Clostridial neurotoxin activity (e.g., through Clostridial neurotoxin migration).

Thus, in one embodiment, the methods of the invention comprise further testing the gene or agent identified in the method of the invention, wherein the testing further comprises detecting the presence or absence of a clostridial neurotoxin receptor in a cell when the expression of the target gene has been altered in the cell or when the cell has been contacted with the agent. A suitable testing method is provided in Example 5 of the present specification.

The amount of Clostridial neurotoxin receptor detected can be compared to a negative control in which target gene expression was not altered or in which the cell was not exposed to the agent. Alternatively or additionally, the negative control may include cells that were not exposed to the Clostridial neurotoxin. When the cell was not exposed to the Clostridial neurotoxin, internalization and subsequent recycling of the Clostridial neurotoxin receptor to the cell surface occurs. In contrast, when the cell was exposed to the Clostridial neurotoxin, cleavage of SNARE proteins by the Clostridial neurotoxin prevents efficient recycling of the receptor to the cell surface.

Thus, in one embodiment, the amount of clostridial neurotoxin receptor detected is less than the amount detected on the surface of a cell that has not been exposed to the clostridial neurotoxin.

In one embodiment, detection of a reduced amount of a clostridial neurotoxin receptor on the surface of a cell: a) in which expression of a target gene has been altered; and b) which has been exposed to a clostridial neurotoxin may indicate that the target gene indirectly regulates the activity of the clostridial neurotoxin.

The above mentioned decrease may occur compared to an equivalent cell in which the expression of the target gene is not changed.

By further use of a known direct inhibitor of Clostridial neurotoxin activity, it can be confirmed that the target gene indirectly regulates Clostridial neurotoxin activity at the level of Clostridial neurotoxin receptor migration. For example, indirect regulation can be confirmed if the amount of receptor on the cell surface detected in the presence of the inhibitor is less than the amount of receptor detected in an equivalent cell (in which target gene expression is not altered) that has also been exposed to Clostridial neurotoxin and inhibitor (and vice versa).

In one embodiment, detection of an equivalent or greater amount of a clostridial neurotoxin receptor on the surface of a cell: a) in which expression of a target gene has been altered, and b) which has been exposed to a clostridial neurotoxin, may indicate that the target gene does not indirectly regulate the activity of clostridial neurotoxins.

An equivalent or greater amount as mentioned above may occur compared to an equivalent cell in which the expression of the target gene is not altered.

Similarly, in one embodiment, detection of a reduced amount of a clostridial neurotoxin receptor in a cell contacted with: a) a clostridial neurotoxin and b) an agent may indicate that the agent indirectly regulates the activity of the clostridial neurotoxin.

The reduced amount mentioned above may occur compared to an equivalent cell in the absence of the agent.

By further use of a known direct inhibitor of Clostridial neurotoxin activity, it can be confirmed that the agent indirectly regulates Clostridial neurotoxin activity at the level of Clostridial neurotoxin receptor migration. For example, indirect regulation can be confirmed if the amount of receptor detected in the presence of the inhibitor is less than the amount of receptor detected in an equivalent cell that has also been exposed to the Clostridial neurotoxin and inhibitor but not to the agent (and vice versa).

In one embodiment, detection of an equivalent or greater amount of a clostridial neurotoxin receptor on the surface of a cell in contact with a) a clostridial neurotoxin and b) an agent may indicate that the agent does not indirectly regulate the activity of the clostridial neurotoxin.

An equivalent or greater amount as mentioned above may occur compared to an equivalent cell in the absence of the agent.

Preferably, the receptor for clostridial neurotoxin is SV2.

The present invention overcomes the limitation of prior assays in that in many cell types, normal cell surface levels of SV2 are low and difficult to detect, and therefore it is impossible/difficult to determine whether these levels change when target gene expression is altered or in the presence of an agent.

- 5 048091

In one embodiment, a known direct inhibitor of clostridial neurotoxin activity is an siRNA or shRNA that inhibits the expression of thioredoxin reductase.

The methods of the present invention are particularly suitable for high-throughput screening. In this regard, the methods may involve the use of multiple cell samples, preferably wherein the expression of different target genes is altered in each of the cell samples. RNAi libraries (or equivalent gene silencing libraries) are particularly well suited for use in high-throughput methods such as drug libraries.

Thus, in one embodiment, the method of the invention comprises:

(a) providing a plurality of human neuronal cell samples expressing a polypeptide that comprises a C-terminal detectable tag, wherein the polypeptide is cleavable by a clostridial neurotoxin;

b) a change in the expression of different target genes in each of a plurality of samples, wherein the target gene is different in each cell sample;

c) contact of cells with clostridial neurotoxin;

d) measuring the amount of C-terminal detectable label, thereby quantifying the activity of the clostridial neurotoxin; and

e) identifying one or more target genes as regulators of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity differs from the quantified Clostridial neurotoxin activity when the expression of the one or more target genes is not altered; or

f) identifying that one or more target genes are not regulators of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is equivalent to the quantified Clostridial neurotoxin activity when the expression of one or more target genes is not altered.

In another embodiment, the method includes:

(a) providing a plurality of human neuronal cell samples expressing a polypeptide that comprises a C-terminal detectable tag, wherein the polypeptide is cleavable by a clostridial neurotoxin;

b) contacting the cells with a clostridial neurotoxin and an agent, wherein the contacting is sequential or simultaneous, and wherein each of the samples is contacted with a different agent;

c) measuring the amount of C-terminal detectable label, thereby quantifying the activity of the clostridial neurotoxin; and

d) identifying one or more agents as regulators of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity differs from the quantified Clostridial neurotoxin activity in the absence of one or more agents; or

e) identifying that one or more agents are not regulators of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is equivalent to the quantified Clostridial neurotoxin activity in the absence of one or more agents.

As used herein, the term plurality means two or more. Preferably, the term plurality means more than two, such as >50, >100, >150, >200, >250, or >300.

The method may include using a multi-well plate, wherein each well contains one of a plurality of samples. The sensitivity and high signal-to-noise ratio of the methods of the present invention allow the use of multi-well plates containing >150 wells (preferably >300 wells, such as a 384-well plate). Advantageously, this allows for increased throughput compared to methods that use plates with <150 wells (such as 96-well plates).

Quantitative determination of Clostridial neurotoxin activity by measuring the amount of detectable C-terminal tag according to the methods of the present invention is particularly well suited for simple and rapid imaging and quantification using automated and/or high-throughput means (e.g., microscopy coupled with automated analysis software). Moreover, the human neuronal cells of the present invention are highly sensitive to Clostridial neurotoxins, allowing for reduced exposure times to generate a signal sufficient for detection. This is particularly suitable for use in automated screening with smaller multi-well plate formats, e.g., containing >150 wells (e.g., 384-well plates), since shorter incubation times make the logistics of scheduling automated steps in the method less complex. The complexity of bottlenecks in moving a group of plates (in the screening process) from one step to the next, with some steps completed in minutes and others

- 6 048091 per hour or day; fewer and shorter holding stages are required and the equipment is occupied for less time.

Methods for identifying a gene that regulates the activity of a clostridial neurotoxin include altering the expression of a target gene. The alteration may be an up-regulation or a down-regulation of expression (compared to the level of expression in an equivalent cell in which the expression has not been altered, i.e., in which the expression of the target gene is not altered). Altering the expression of the target gene may be achieved using any method known in the art, such as gene editing, overexpression, or gene silencing.

In one embodiment, expression is altered by down-regulating the expression of a target gene (e.g., by gene silencing). A preferred method for down-regulating the expression of a target gene is RNA interference (RNAi), e.g., using short interfering or short hairpin RNAs (siRNA or shRNA).

In embodiments where expression is altered by down-regulating expression of a target gene, the target gene may be identified as a positive regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is lower than the quantified Clostridial neurotoxin activity when expression of the target gene is not altered. Alternatively, the target gene may be identified as a negative regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is higher than the quantified Clostridial neurotoxin activity when expression of the target gene is not altered.

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 when the quantified Clostridial neurotoxin activity is lower than the quantified Clostridial neurotoxin activity when expression of the target gene is not altered. Alternatively, the target gene may be identified as a positive regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is higher than the quantified Clostridial neurotoxin activity when expression of the target gene is not altered.

Methods for identifying an agent that regulates the activity of a clostridial neurotoxin include contacting cells with a clostridial neurotoxin and an agent, wherein the contacting is sequential (e.g., contacting with a clostridial neurotoxin and then with an agent, or contacting with an agent and then with a clostridial neurotoxin) or simultaneous. Preferably, the contacting is sequential.

In one embodiment, the cells are contacted with an agent prior to contact with a clostridial neurotoxin. Such methods are particularly suitable for identifying agents that can prevent clostridial neurotoxin intoxication. Agents that can prevent intoxication may be suitable for use in therapy as prophylactic agents.

Alternatively, the cells may be contacted with the agent after contact with the clostridial neurotoxin. Such methods are particularly suitable for identifying agents capable of inhibiting clostridial neurotoxin activity after intoxication and may be suitable for use in post-intoxication therapy as therapeutic agents.

An agent identified as a positive regulator of clostridial neurotoxin activity may be an agent that is sensitizing to clostridial neurotoxin. Such agents may be used in combination with clostridial neurotoxin therapy to modulate local activity of clostridial neurotoxins (e.g., to reduce dosage and minimize dissemination to other tissues).

The activity of the clostridial neurotoxin can be quantified by measuring the amount of detectable C-terminal tag. Preferably, the activity of the clostridial neurotoxin can be quantified by measuring the amount of detectable C-terminal tag and detectable N-terminal tag.

The amount of C-terminal detectable label following exposure of cells to a clostridial neurotoxin may be compared to the amount of C-terminal detectable label measured in the absence of the clostridial neurotoxin. For example, the amount of detectable C-terminal label may be measured in a sample of the same cells before and after exposure to a clostridial neurotoxin. Alternatively, the amount of C-terminal detectable label may be measured after exposure to a clostridial neurotoxin and compared to the amount of C-terminal detectable label present in an equivalent sample of cells under equivalent conditions that were not exposed to the clostridial neurotoxin.

In one embodiment, the loss of the C-terminal detectable label (e.g., over time) compared to an equivalent cell that has not been exposed to the clostridial

- 7 048 091 neurotoxin, or compared to the same cell before contact with the Clostridial neurotoxin, indicates the presence of Clostridial neurotoxin activity. Alternatively, in one embodiment, the absence of loss (or detection of an equivalent amount) of the C-terminal detectable label (e.g., over time) compared to an equivalent cell that has not been contacted with the Clostridial neurotoxin, or compared to the same cell before contact with the Clostridial neurotoxin, indicates the absence of Clostridial neurotoxin activity. In one embodiment, a partial loss of the C-terminal detectable label (e.g., over time) compared to an equivalent cell that has not been contacted with the Clostridial neurotoxin, or compared to the same cell before contact with the Clostridial neurotoxin, may indicate the presence of reduced Clostridial neurotoxin activity (and vice versa).

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

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

The detectable label of the invention can be measured at one or more time points after contact of the cells with the clostridial neurotoxin. By this means the level of activity of the clostridial neurotoxin can be calculated.

In one embodiment, the detectable label can 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 can be measured after contacting the cells with the Clostridial neurotoxin for less than 100, 80, 70, 60 or 50 hours. Preferably, the detectable label can 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 can be measured after contacting the cells with the Clostridial neurotoxin for 20-60 hours, preferably 40-55 hours (e.g., about 48 hours). The cells can be fixed before measuring the amount of detectable label.

When comparing cells according to the method of the invention with a control (e.g., an equivalent cell in which the expression of the target gene is not altered or which has not been contacted with the agent of the invention, or another control mentioned above), it is assumed that the measurements are performed by the same method (e.g., at the same time after contacting the cells with the clostridial neurotoxin). This allows comparison of the quantitatively determined clostridial neurotoxin activity of the cells according to the method and the control, so as to identify the presence or absence of a difference in the clostridial neurotoxin activity.

The measuring or detecting step of the invention can be performed using any suitable means known to the skilled person. 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. Thus, fluorescence microscopy can be used in the invention. In a preferred embodiment, the measuring or detecting step includes using a high-throughput screening system. An example of a suitable system is the Opera Phenix™ High-Content Screening System, which is commercially available from PerkinElmer, and/or using suitable image processing software, such as Columbus™ software (commercially available from PerkinElmer). In some embodiments, the measuring or detecting step of the invention is automated and can include the use of robotics.

The method of the invention preferably does not use electronic coupling between the detectable labels described herein. In particular, it is preferred that the labels are not arranged in such a way that a donor label (e.g. an N-terminal label) can transfer energy to an acceptor label (e.g. a C-terminal label), for example by a dipole-dipole coupling mechanism. Preferably, the invention does not involve the use of fluorescence resonance energy transfer (FRET).

The methods of the invention may include a verification step in which the quantitatively determined clostridial neurotoxin activity detected in the method is compared with a positive

- 8 048091 control. A positive test may occur when the quantitatively determined activity of the clostridial neurotoxin is equivalent to the activity of the positive control.

The method of the invention may further include verifying that the regulator is indeed a positive regulator. In one embodiment, the method includes contacting the cells with a known negative regulator of Clostridial neurotoxin activity and demonstrating that the effect on Clostridial neurotoxin activity is reversible. In one embodiment, the method includes upregulating the expression of a known negative regulator of Clostridial neurotoxin activity and demonstrating that the effect on Clostridial neurotoxin activity is reversible. Preferably, the method includes downregulating the expression of a known positive regulator of Clostridial neurotoxin activity and demonstrating that the effect on Clostridial neurotoxin activity is reversible.

The method of the invention may further include verifying that the regulator is indeed a negative regulator. In one embodiment, the method includes contacting the cells with a known positive regulator of Clostridial neurotoxin activity and demonstrating that the effect on Clostridial neurotoxin activity is reversible. In one embodiment, the method includes down-regulating the expression of a known negative regulator of Clostridial neurotoxin activity and demonstrating that the effect on Clostridial neurotoxin activity is reversible. In one embodiment, the method includes up-regulating the expression of a known positive regulator of Clostridial neurotoxin activity and demonstrating that the effect on Clostridial neurotoxin activity is reversible.

A known (positive) regulator of clostridial neurotoxin activity is thioredoxin reductase. In one embodiment, the expression of thioredoxin reductase is up-regulated. Preferably, the expression of thioredoxin reductase is down-regulated.

As used in this description, the term equivalent may mean that two or more values being compared do not differ at a statistically significant level. As used in this description, the term equivalent preferably means that two or more values are identical. Similarly, the term unchanged as used in this description may mean that two or more values being compared do not differ at a statistically significant level. Preferably, the term unchanged as used in this description means that two or more values are identical.

The difference or change (e.g., increase or decrease) referred to herein preferably means a statistically significant difference or change (e.g., a statistically significant increase or decrease). Thus, the difference in the quantitatively determined activity of the Clostridial neurotoxin is preferably a statistically significant difference in the quantitatively determined activity of the Clostridial neurotoxin. In one embodiment, the difference in the quantitatively determined activity of the Clostridial neurotoxin is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50% compared to the quantitatively determined activity of the Clostridial neurotoxin for a negative control (e.g., an equivalent cell in which the expression of the target gene is not altered or which has not been contacted with the agent of the invention or another control mentioned above).

The methods of the present invention are in vitro methods.

In one embodiment, the methods of the invention are methods using live cells and, optionally, the methods employ real-time monitoring of clostridial neurotoxin activity when target gene expression changes and/or when cells are contacted with the agent.

Preferably, the term contacting a cell with a clostridial neurotoxin means that the cell surface is contacted with a clostridial neurotoxin. Accordingly, the clostridial neurotoxin can be added to a medium in which the cell is present, such as a cell culture medium. The term contacting a cell with a clostridial neurotoxin preferably excludes contact by expressing a clostridial neurotoxin in a cell, since this technique does not provide information regarding binding, internalization and/or translocation of the clostridial neurotoxin.

The method of the invention typically comprises contacting the cells with a Clostridial neurotoxin at a concentration of less than 1500 nM. In one embodiment, the cells are contacted with a Clostridial neurotoxin at a concentration of less than 1000 nM, such as less than 500 nM, 250 nM, or 100 nM.

The method of the invention may comprise contacting the cells with a clostridial neurotoxin at a concentration of at least 1 nM, 5 nM, 10 nM, 20 nM, or 50 nM.

In one embodiment, the method of the invention may comprise contacting the cells with a Clostridial neurotoxin at a concentration of 1-1000 nM, such as a Clostridial neurotoxin at a concentration of 1-500 nM or 1-200 nM.

- 9 048091

The inventors have surprisingly found that by contacting cells with a buffer containing GDNF and cell-permeable cAMP (and optionally further containing CaCl2 and KCl), the sensitivity of the methods of the present invention can be increased. In the presence of the buffer, the cells become highly sensitive to the clostridial neurotoxin at a concentration of less than 100 nM, preferably less than 50 nM, more preferably 5-15 nM (e.g., ~10 nM).

The buffer may contain GDNF, d-cAMP, CaCl2 and KCl. GDNF may be present in an amount of 1100 ng/ml, preferably 10 ng/ml. d-cAMP may be present in a concentration of 0.1-5 mM, preferably 1 mM. CaCl2 may be present in a concentration of 0.1-7 mM, preferably 2 mM. KCl may be present in an amount of 1-100 mM, preferably 56 mM.

The buffer may be a component of the kit of the present invention.

Thus, in one aspect, the invention provides a composition comprising:

a) clostridial neurotoxin and

b) a buffer containing GDNF, cell-permeable cyclic adenosine monophosphate (cAMP), CaCl2 and KCl.

In one embodiment, the composition comprises GDNF present at a concentration of 1-100 ng/ml, d-cAMP present at a concentration of 0.1-5 mM, _CaCl2 present at a concentration of 0.1-7 mM, and KCl present at a concentration of 1-100 mM. Preferably, the buffer comprises GDNF at a concentration of 10 ng/ml, d-cAMP at a concentration of 1 mM, _CaCl2 at a concentration of 2 mM, and KCl at a concentration of 56 mM.

Reference in the present description to cAMP is preferably interchangeable with d-cAMP.

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

In some embodiments, the composition further comprises cells, such as human neural cells, as described herein.

The cells can be incubated with the clostridial neurotoxin for any suitable time. In one embodiment, the cells can be incubated with the clostridial neurotoxin for at least 2, 5, 10, 15, 20, 30, 40, 50, 60 or 70 hours. In other embodiments, the cells can 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 can be incubated with the clostridial neurotoxin for 20-60 hours, preferably 40-55 hours (e.g., about 48 hours). Preferably, the human neuronal cells of the present invention are highly sensitive to clostridial neurotoxins, allowing for short incubation periods with the clostridial neurotoxin of less than 72 h (~48 h), thereby reducing the time required to perform the assay.

The cells may be incubated with the clostridial neurotoxin at any suitable temperature. In one embodiment, the cells are incubated with the clostridial neurotoxin at 30-40°C, preferably 37°C.

A target gene or agent identified by the method of the present invention can be used in a method of treating a disorder. Thus, in one aspect, the invention provides a method of 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 of treating a disorder, comprising altering the expression of a gene in a subject, wherein the gene has been identified by the method of the invention.

In one aspect, the invention provides a kit comprising: a cell of the invention; and, optionally, instructions for use thereof (e.g., in a method described herein). In a related aspect, the invention provides a kit comprising a nucleotide sequence of the invention; and, optionally, instructions for use thereof (e.g., in a method described herein). In a related aspect, the invention provides a kit comprising a vector of the invention; and, optionally, instructions for use thereof (e.g., in a method described herein). In one embodiment, the kit comprises a cell (preferably a cell identical to a cell type of the invention, but lacking a nucleotide sequence of the invention and not expressing a polypeptide of the invention), a nucleotide sequence or a vector of the invention, and, optionally, instructions for use thereof (e.g., in a method described herein). Said kits may comprise one or more separate containers, each of which contains said component of the kit.

- 10 048091

The present invention is suitable for application to many different varieties of clostridial neurotoxin. Thus, in the context of the present invention, the term clostridial neurotoxin encompasses toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, Cl, D, E, F, G, H and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E) and C. baratii (botulinum neurotoxin serotype F), as well as modified clostridial neurotoxins or derivatives obtained from any of the foregoing neurotoxins. The term clostridial neurotoxin also encompasses botulinum neurotoxin serotype H.

Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex consisting of BoNT itself linked to a number of accessory proteins. There are currently nine different classes of botulinum neurotoxins, namely botulinum neurotoxin serotypes A, B, Cl, D, E, F, G, H, and X, which have similar structures and mechanisms of action. The different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and this serotype classification correlates with percent sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on percent amino acid sequence identity.

BoNTs are absorbed in the gastrointestinal tract and, entering the general bloodstream, bind to the presynaptic membrane of cholinergic nerve endings and prevent the release of their neurotransmitter acetylcholine.

Tetanus toxin is produced in a single serotype of C. tetani. C. butyricum produces BoNT/E, and C. baratii produces BoNT/F.

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

A modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., a modified _HC domain), wherein said modified heavy chain binds to target neural cells with a higher or lower affinity than a native (unmodified) Clostridial neurotoxin. Such modifications in the _HC domain may include modifying residues in the ganglioside binding site of the _HC domain or in the protein binding site (SV2 or synaptotagmin) that alter binding to a ganglioside receptor and/or a target neural cell protein receptor. 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.

The modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the binding substrate or catalytic domain, which 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 herein by reference in their entireties.

The modified Clostridial neurotoxin may comprise 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 comprise a leucine-based or tyrosine-based motif, wherein said motif 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 an arbitrary amino acid). Suitable tyrosine-based motifs include YxxHy (SEQ ID NO: 26) (where Hy is a hydrophobic amino acid). Examples of modified clostridial neurotoxins containing leucine and tyrosine-based motifs are described in WO 2002/08268, which is incorporated herein by reference in its entirety.

The term clostridial neurotoxin encompasses hybrid and chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin contains at least part of the light chain

- 11 048091 one Clostridial neurotoxin or subtype thereof and at least a portion of the heavy chain of another Clostridial neurotoxin or a Clostridial neurotoxin of another subtype. In one embodiment, the hybrid Clostridial neurotoxin may comprise the entire light chain of the light chain of one subtype of Clostridial neurotoxin and the heavy chain of another subtype of Clostridial neurotoxin. In another embodiment, the chimeric Clostridial neurotoxin may comprise a portion (e.g., a binding domain) of the heavy chain of one subtype of Clostridial neurotoxin, wherein the other portion of the heavy chain belongs to another subtype of Clostridial neurotoxin. Similarly or alternatively, the therapeutic element may comprise portions of the light chain 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, patients who may have a below average concentration of receptors for a given clostridial neurotoxin heavy chain binding domain, or patients who may have a protease-resistant variant of a membrane or vesicular toxin substrate (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in U.S. Patent 8,071,110, the publication of which is incorporated herein by reference in its entirety. Thus, in one embodiment, a clostridial neurotoxin of the invention is a hybrid clostridial neurotoxin or a chimeric clostridial neurotoxin.

The term clostridial neurotoxin may also encompass recently discovered members of the botulinum neurotoxin protein family expressed by non-clostridial microorganisms, such as the enterococcal-encoded toxin, which has the highest sequence identity to BoNT/X, the Weissella oryzae-encoded toxin called BoNT/Wo (NCBI Ref Seq: WP_027699549.1) that cleaves VAMP2 at W89-W90, the Enterococcus faecium-encoded toxin (GenBank: OTO22244.1) that cleaves VAMP2, and the SNAP-25-encoded Chryseobacterium pipero toxin (NCBI Ref Seq: WP_034687872.1).

In a preferred embodiment, the clostridial neurotoxin is a botulinum neurotoxin, more preferably BoNT/A.

In one embodiment, the clostridial neurotoxin may be BoNT/A. The reference sequence of BoNT/A is provided as SEQ ID NO: 13.

In another embodiment, the clostridial neurotoxin may be BoNT/B. The reference sequence of BoNT/B is provided as SEQ ID NO: 14.

In another embodiment, the clostridial neurotoxin may be BoNT/C. The reference sequence of BoNT/C is provided as SEQ ID NO: 15.

In another embodiment, the clostridial neurotoxin may be BoNT/D. The reference sequence of BoNT/D is provided as SEQ ID NO: 16.

In another embodiment, the clostridial neurotoxin may be BoNT/E. The reference sequence of BoNT/E is provided as SEQ ID NO: 17.

In another embodiment, the clostridial neurotoxin may be BoNT/F. The reference sequence of BoNT/F is provided as SEQ ID NO: 18.

In another embodiment, the clostridial neurotoxin may be BoNT/G. The reference sequence of BoNT/G is provided as SEQ ID NO: 19.

In one embodiment, the clostridial neurotoxin may be BoNT/X. The reference sequence of BoNT/X is provided as SEQ ID NO: 20.

In another embodiment, the clostridial neurotoxin may be TeNT. The reference sequence of TeNT is provided as SEQ ID NO: 21.

Embodiments relating to the various methods of the invention are equally intended to be applied to other methods, cells, polypeptides, nucleotide sequences, kits and compositions of the invention, and vice versa.

Sequence homology

Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global alignment methods, local alignment methods, and hybrid methods such as, for example, segment approach methods. Protocols for determining percent identity are routine procedures within the skill of the art. Global alignment methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up individual residue pair scores and applying gap penalties. Non-limiting methods include, for example, Clustal W., see, for example, 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 46734680 (1994); and iterative refinement, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823–838 (1996). Local alignment methods align sequences by

- 12 048091 identification of one or more conserved motifs that are common to all input sequences. Non-limiting methods include, for example, Match-box, see, for example, 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, for example, C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262 (5131) Science 208–214 (1993); Align-M., see, for example, Ivo Van Walle et al., Align-M. - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

Thus, the percentage of sequence identity is determined by conventional methods; see, for example, 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 to optimize alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the blosum 62 substitution matrix of Henikoff and Henikoff (ibid.), as shown below (amino acids are designated by standard single-letter codes).

The percentage of 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, % identity can be calculated as the number of identical nucleotides/amino acids divided by the total number of nucleotides/amino acids, and then multiplied by 100. When calculating % sequence identity, the number of gaps and the length of each gap that must be introduced to optimize the alignment of two or more sequences can also be taken into account. Sequence comparison and determination of the percentage of identity between two or more sequences can be performed using certain mathematical algorithms, such as BLAST, which will be familiar to those skilled in the art.

Alignment scores for determining sequence identity ARNDCQEGHI LKMFPSTWYV

A 4

R-15

N -2 06

D -2 -2 16

CO -3-3-3 9

Q -1 1 0 0 -35

E -1 0 0 2 -4 25

G0 -2 0 -1-3 -2 -2 6

H -2 0 1 -1 -3 0 0 -28

I -1 -3 -3 -3 -1 -3 -3 -4 -34

L -1 -2 -3 -4 -1 -2 -3 -4-3 24

K -1 2 0 -1 -3 1 1 -2-1 -3 -25

Μ -1 -1 -2 -3 -1 0 -2-3-2 1 2-15

F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 06

R -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -47

S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -14

T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2-11 5 w -3 -3 -4 -4 -2 -2 -3 -2 -2 - 3 -2 -3 -1 1 -4 -3 -211

Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 27

V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3-1 4

The percent identity is then calculated as:

Total number of matching characters ------z----------------------------------x 100 length of the longer sequence plus the number of gaps introduced into the longer sequence to align the two sequences

Substantially homologous polypeptides are characterized by the presence of one or more amino acid substitutions, deletions, or insertions. These changes are preferably minor, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically from one to about 30 amino acids; and small extensions at the amino- or carboxyl terminus, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues in length, or an affinity tag.

- 13 048091

Conservative amino acid substitutions

Main:

arginine, lysine, histidine. Acidic: glutamic acid, aspartic acid.

Polar:

glutamine, asparagine.

Hydrophobic:

leucine, isoleucine, valine.

Aromatic:

phenylalanine, tryptophan, tyrosine.

Small:

glycine, alanine, serine, threonine, methionine.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can replace amino acid residues in the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids can replace amino acid residues of the polypeptide. The polypeptides of the present invention can also contain non-naturally occurring amino acid residues.

Unnatural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating unnatural amino acid residues into proteins are known in the art. For example, an in vitro system can be used 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 accomplished in a cell-free system containing E. coli S30 extract and commercially available enzymes and 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 accomplished in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991–8, 1996). In the third method, E. coli cells are grown in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired unnatural amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnatural amino acid is inserted 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 unnatural species by chemical modification in vitro. 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).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, unnatural amino acids, and unnatural amino acids may replace amino acid residues of the polypeptides of the present invention.

Essential amino acids in the polypeptides of the present 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). Biological interaction sites can also be determined by physical analysis of the structure, as determined by techniques such as nuclear

- 14 048091 magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutation of putative contact site amino acids; 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 identity of essential amino acids can also be inferred from homology analysis with related components (e.g., translocation or protease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods 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 for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions 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., U.S. 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).

Unless defined otherwise, 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 pertains. 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 a general dictionary of many of the terms used in this disclosure for those skilled in the art.

The present disclosure is not limited to 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. Numerical ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in a 5' to 3' orientation; amino acid sequences are written left to right in an amino to carboxy orientation, respectively.

The headings provided in this description are not intended to limit the various aspects or embodiments of the present disclosure.

In the present specification, amino acids are referred to using the amino acid name, a three-letter abbreviation, or a one-letter abbreviation. As used herein, the term protein includes 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 cases, the term amino acid sequence is synonymous with the term peptide. In some cases, the term amino acid sequence is synonymous with the term enzyme. The terms protein and polypeptide are used interchangeably in the present specification. Conventional one-letter and three-letter codes for amino acid residues may be used in the present disclosure and claims. The three-letter code for amino acids is defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It should also be understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear in the description. Before exemplary embodiments are described in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and therefore may vary. It should also be understood that the terminology used in the present description is for the purpose of describing specific embodiments only and is not intended to be limiting, since the scope of the present disclosure will be determined only by the appended claims.

Where a range of values is provided, it is intended that each sub-range, down to one-tenth of a unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of the range is also specifically disclosed. Each minor range between any stated value or sub-range and any other stated value or sub-range within the stated range is encompassed by this disclosure. The upper and lower limits of these minor ranges may independently be included or excluded from the range, and each range in which any, none, or both limits are included in the minor ranges is also encompassed by this disclosure, subject to any specifically excluded limit in the stated range. If a stated range includes one or both limits, ranges excluding one or both of these included limits are also included in this disclosure.

It should be noted that, as used in the present specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a certain clostridial neurotoxin includes the plural.

- 15 048091 the number of such candidate agents, and a reference to said clostridial neurotoxin includes a reference to one or more clostridial neurotoxins and their equivalents known to those skilled in the art, and so on.

The publications discussed herein are intended solely for their disclosure prior to the filing date of this application. Nothing in this specification shall be construed as an admission that such publications constitute prior art with respect to the appended claims.

Brief description of the drawings

Embodiments of the invention will now be described by way of example only with reference to the following figures and examples.

Fig. 1 shows the method for generating ReNcell VM stably expressing TagRFPT-SNAP25TagGFP. The images (bottom) show red fluorescence (right) and green fluorescence (right).

Figure 2 shows BoNT/A-induced degradation of the C-terminal fragment of the construct. ReD SNAPR cells were differentiated for 14 days without growth factors, then 100 nM BoNT/A was added to the cells and they were intoxicated for 48 h. (A) The cells were visualized using fluorescence microscopy and the corresponding combined GFP, RFP and GFP/RFP channels are shown in the figure. (B) The lysates were subjected to SDS-PAGE for Western blotting. Rabbit antibodies against tagRFPT and tagGFP were used for Western blotting.

Fig. 3 shows a diagram summarizing the degradation of the structure under the influence of BoNT/A.

Figure 4 shows that enhanced differentiation and stimulation buffer sensitized ReD SNAPR cells to BoNT/A. (A) ReD SNAPR cells were exposed to ReNcell differentiation medium with and without GDNF and d-cAMP during differentiation and to high potassium buffer during intoxication. The resulting modified medium is known as ReNcell enhanced differentiation and stimulation (ReDS) 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 exhibited increased sensitivity to BoNT/A compared to normal ReNcell medium, as detected by confocal microscopy. (B) Quantification of BoNT/A-mediated cleavage under both conditions showed that the EC50 improved from 43 to 6 nM in ReD SNAPR exposed to ReDS medium. (C) Western blot detection of BoNT/A-mediated cleavage of the SNAP25 construct in ReD SNAPR cells in normal and ReDS medium. The upper blot shows cell lysates probed with tRFP antibody and the lower blot probed with tGFP antibody.

Figure 5 shows siRNA against TrxR preventing BoNT/A-mediated cleavage of the construct. Graphs of TrxR1 knockdown levels and construct cleavage are shown. The top 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 both green and red fluorescence (siTrxR, -BoNT/A). The bottom panel shows TrxR1 staining in the presence of siNT3 (for both -BoNT/A and +BoNT/A conditions) and no TrxR1 staining in the presence of siTrxR (for both -BoNT/A and +BoNT/A conditions).

Figure 6 shows that BoNT/A intoxication prevents SV2 cell surface trafficking. A) ReNcell VM cells were treated with siNT3, siVAMP2, and siTrxR1 for 72 h prior to addition of BoNT/A. Cells were fixed and stained with anti-SV2A primary antibody (without permeabilization). Alexa-488 secondary antibody was used against the primary antibody. (B) Illustration of BoNT/A-mediated reduction in SV2 cell surface trafficking.

Fig. 7 shows a scheme for conducting a genome-wide screening of miRNAs using a cell line according to the present invention.

List of sequences

If the initial Met amino acid residue or the corresponding start codon is indicated in any of the following SEQ ID NOs, then the indicated residue/codon may be optional.

SEQ ID NO: 1 (nucleotide sequence of construct TagRFPT-SNAP25TagGFP)

ATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGT ACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGC AAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCT CCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAGAACCTTCAT CAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCAC ATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACA CCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTC CCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCAACACCGA GATGCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAACCGACATGGCCCTGAAGC

- 16 048091

TCGTGGGCGGGGGCCACCTGATCTGCAACTTCAAGACCACATACAGATCCAAGAAA CCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACCACAGACTGGAAAG AATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCC AGATACTGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATGGCATGGACGAGCT GTACAAGGGCTCGGGCTCGGGCTCGGGCGTGGCCGAAGACGCAGACATGCGCAATG AGCTGGAGGAGATGCAGCGAAGGGCTGACCAGTTGGCTGATGAGTCGCTGGAAAGC ACCCGTCGTATGCTGCAACTGGTTGAAGAGGTAAAGATGCTGGTATCAGGACTTTG GTTATGTTGGATGAACAAGGAGAACAACTCGATCGTGTCGAAGAAGGCATGAACCA TATCAACCAAGACATGAAGGAGGCTGAGAAAAATTTAAAAGATTTAGGGAAATGCT GTGGCCTTTTCATATGTCCTTGTAACAAGCTTAAATCAAGTGATGCTTACAAAAAAG CCTGGGGCAATAATCAGGACGGAGTGGTGGCCAGCCAGCCTGCTCGTGTAGTGGAC GAACGGGAGCAGATGGCCATCAGTGGCGGCTTCATCCGCAGGGTAACAAATGATGC CCGAGAAAATGAAATGGATGAAAACCTAGAGCAGGTGAGCGGCATCATCGGGAAC CTCCGTCACATGGCCCTGGATATGGGCAATGAGATCGATACACAGAATCGCCAGAT CGACAGGATCATGGAGAAGGCTGATTCCAACAAAACCAGAATTGATGAGGCCAACC AACGTGCAACAAAGATGCTGGGAAGTGGTTACGGCGGCTCGGGCTCGGGCGTGAGC GGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACGGCGA CGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACG GCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCC ACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCAC ATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCG CACCATCCAGTTCCAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCG AGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGA CGGCAACATCCTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCACAACGTGTACA TCCGCCCCGACAAGGCCAACAACGGCCTGGAGGCTAACTTCAAGACCCCGCCACAAC ATCGAGGGCGGCGGCGTGCAGCTGGCCGACCACTACCAGACCAACGTGCCCCTGGG CGACGGCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGACCAAGATCA GCAAGGACCGCAACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTTCAGCGCC TGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAA

SEQ ID NO: 2 (polypeptide sequence of the construct TagRFPT-SNAP25TagGFP)

MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEG GPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTS LQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMALKLVG GGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYCDL PSKLGHKLNGMDELYKGSGSGSGVAEDADMRNELEEMQRRADQLADESLESTRRMLQ LVEESKDAGIRTLVMLDEQGEQLDRVEEGMNHINQDMKEAEKNLKDLGKCCGLFICPC NKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDEN LEQVSGIIGNLRHMALMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSGYG GSGSGVSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICTTGKLPV

- 17 048091

PWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVK FEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEANFKTRHNIE GGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLESFSACCHTH GMDELYR*

SEQ ID NO: 3 (nucleotide sequence TagRFPT)

ATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGT ACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGC AAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCT CCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAGAACCTTCAT CAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCAC ATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACA CCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTC CCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCAACACCGA GATGCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAACCGACATGGCCCTGAAGC TCGTGGGCGGGGGCCACCTGATCTGCAACTTCAAGACCACATACAGATCCAAGAAA CCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACCACAGACTGGAAAG AATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCC AGATACTGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATGGCATGGACGAGCT GTACAAG

SEQ ID NO: 4 (TagRFPT polypeptide sequence)

MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEG GPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTS LQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMALKLVG GGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYCDL PSKLGHKLNGMDELYK

SEQ ID NO: 5 (nucleotide sequence of Glycine-Serine rich linker 1)

GGCTCGGGCTCGGGCTCGGGC

SEQ ID NO: 6 (glycine-serine rich linker η polypeptide sequence

GSGSGSG

SEQ ID NO: 7 (nucleotide sequence of glycine-serine rich linker 2)

GGCGGCTCGGGCTCGGGC

SEQ ID NO: 8 (glycine-serine rich linker polypeptide sequence 21

GGSGSG

SEQ ID NO: 9 (nucleotide sequence of SNAP25)

GTGGCCGAAGACGCAGACATGCGCAATGAGCTGGAGGAGATGCAGCGAAGG GCTGACCAGTTGGCTGATGAGTCGCTGGAAAGCACCCGTCGTATGCTGCAACTGGTT GAAGAGAGTAAAGATGCTGGTATCAGGACTTTGGTTATGTTGGATGAACAAGGAGA

- 18 048091

ACAACTCGATCGTGTCGAAGAAGGCATGAACCATATCAACCAAGACATGAAGGAGG CTGAGAAAAATTTAAAAGATTTAGGGAAATGCTGTGGCCTTTTCATATGTCCTTGTA ACAAGCTTAAATCAAGTGATGCTTACAAAAAAGCCTGGGGCAATAATCAGGACGGA GTGGTGGCCAGCCAGCCTGCTCGTGTAGTGGACGAACGGGAGCAGATGGCCATCAG TGGCGGCTTCATCCGCAGGGTAACAAATGATGCCCGAGAAAATGAAATGGATGAAA ACCTAGAGCAGGTGAGCGGCATCATCGGGAACCTCCGTCACATGGCCCTGGATATG GGCAATGAGATCGATACACAGAATCGCCAGATCGACAGGATCATGGAGAAGGCTGA TTCCAACAAAACCAGAATTGATGAGGCCAACCAACGTGCAACAAAGATGCTGGGAA GTGGTTAC

SEQ ID NO: 10 (SNAP25 polypeptide sequence)

VAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQ GEQLDRVEEGMNHINQDMKEAEKNLKDLGKCCGLFICPCNKLKSSDAYKKAWGNNQD GVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMG NEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSGY

SEQ ID NO: 11 (nucleotide sequence of TagGFP)

GTGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGC TGGACGGCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGA CGCCGACTACGGCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCG TGCCCTGGCCCACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCT ACCCCGAGCACATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTAC ATCCAGGAGCGCACCATCCAGTTCCAGGACGACGGCAAGTACAAGACCCGCGGCGA GGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAAGGACT TCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCAC AACGTGTACATCCGCCCCGACAAGGCCAACAACGGCCTGGAGGCTAACTTCAAGAC CCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCGACCACTACCAGACCAACG TGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGA CCAAGATCAGCAAGGACCGCAACGAGGCCCGACCACATGGTGCTCCTGGAGTCC TTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAA

SEQ ID NO: 12 (TagGFP polypeptide sequence)

VSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICTTGKLPV PWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVK FEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEANFKTRHNIE GGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLESFSACCHTH GMDELYR*

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

MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDL N

PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

- 19 048091

RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNK A

KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFK V

LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNF T

GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKAT EA

AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININ KFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKD K

VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELN EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLK GPR

GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQ A

GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIA K

LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEO ID NO: 14 (BoNT/B - UniProt P10844)

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGL Y

GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGM

- 20 048091 γ

KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFD L

SIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSY SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRD LY

IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

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

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNP A

M

WANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILL EAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQF NNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNI N

KFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSF QNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQ LNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIID SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGN M

KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKEL DGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRR NNN

DFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIY A

- 21 048091

IGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYR HNYLVPTVKQGNYASLLESTSTHWGFVPVSE

SEO ID NO: 16 (BoNT/D - UniProt P19321)

MTWPVKDFNYSDPVNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDS STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSE VVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA LQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYL NSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWAN E

R

ECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTM PFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTI YTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS GWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYI N

GELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQI LRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSV SDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNY GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLS TSSFWKFISRDPGWVE

SEO ID NO: 17 (BoNT/E - UniProt 000496)

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGS IAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKL SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK

- 22 048091

NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTII E

I

SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTUNCMRDNNSGWKVSLNHNEII WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTM NF

KNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK SEO ID NO: 18 (BoNT/F - UniProt A7GBG3)

MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFD PPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGN EHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVY F

KVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKG LVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLN NNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFF YLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIR DFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELL IPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIER F

ITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNN SIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIY STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTimC IRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGN SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETL YSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSN TRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKL IRTSNSNNSLGQUVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTS SNGCFWSFISKEHGWQEN

SEO Yu NO: 19 (BoNT/G - UniProt 060393)

MPVNIKXFNYNDPINNDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFN

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ASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYL G

NASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGH SPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLY GIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIA NRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETN L

AGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAY EEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYN TQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGD SLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWV K

GVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFI PELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTI KERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFI NQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKD S

IPFDLSLYTKDTILIQVFNNYISNISSNAILLSSYRGGRLIDSSGYGATMNVGSDVIFND IGGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTII SCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSrriTNDRLG NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVS SLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNA AI

NYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQ LFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTY D

NYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

SEQ ID NO: 20 (BoNT/X polypeptide sequence)

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIP LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEG TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRD

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QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDD HOW

IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNL GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREY WS

SFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMG ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

SEO ID NO: 21 (TeNT - UniProt P04958)

MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFN PPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGN SYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDN KNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLH GLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYK AIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTE IELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNT NAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAE KNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAP EYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVI SKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGN FIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYK LVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLK N

KLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIG ITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDIS GFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVP K

VSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLP DKFNAYLANKWVFIITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNN NQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDV QLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYV SYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLY DD

KNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND Examples

Example 1. Creation of a stable cell line.

Gene synthesis and subcloning.

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 pLenti6.3-tRFPT-SNAP25-tGFP vector was transformed into E. coli BL21 cells and selected using the antibiotic ampicillin. Positive bacterial clones were pretreated using the endotoxin-free Machery-Nagel Maxiprep kit according to the manufacturer's protocol.

Generation of lentivirus from HEK293FT cells.

To prepare for lentivirus generation, HEK293FT cells were cultured in Dulbecco's modified Eagle's high glucose medium with 4500 mg/L glucose supplemented with 10% fetal bovine serum (FBS) (Gibco) and then plated in a T75 ^cm2 flask at 80% confluence and incubated overnight at 37°C with 5% CO2. Cells were then co-transfected with plenti6.3-tRFPT-SNAP25-tGFP plasmid and ViraPower lentivirus packaging mix (Invitrogen Cat No. K497000) using Lipofactamine 3000 reagent (Invitrogen) according to the supplier's manual and incubated in the flask for 6 h at 37°C with 5% CO2. After 6 h of transfection, the medium containing the lipid-DNA complexes was carefully removed and unloaded from the flask and

- 25 048091 was replaced with 10 ml of pre-warmed medium. The cells were incubated overnight at 37°C with 5% CO2. 10 ml of cell supernatant (first batch of virus) was collected 24 h after transfection and stored in 15 ml conical tubes at 4°C. The collected medium was replaced with 10 ml of pre-warmed medium, and the flask was incubated overnight at 37°C with 5% _CO2 . The second batch of virus was collected 48 h after transfection. Both batches of supernatant were centrifuged at 2000 rpm for 10 min at room temperature to remove cellular debris. The clarified lentiviral supernatant was collected after centrifugation and filtered using a 0.45 μm filter to remove any remaining cellular debris. The virus was divided into 1 ml aliquots and stored at -80°C.

Measurement of lentiviral titer by GFP selection.

HEK293FT cells were seeded in a 96-well plate (Nunc) at a density of 10,000 cells/well in 100 μl of culture medium. Serial dilutions of virus from 10 ^-1 to 10 ^-4 were made using fresh culture medium with 8 mg/ml (final concentration) Polybrene reagent (Sigma catalog number H9268). Cells were transduced by removing the existing medium and replacing it with 100 μl of the prepared dilutions in the appropriate well. Plates were incubated overnight at 37°C with 5% CO ₂ . The following day, the culture medium was changed to fresh medium without Polybrene. Cells were incubated for an additional 3 days before calculating the virus titer. The appropriate dilution factor was used to calculate the titer in transduction units (TU) per ml based on the percentage of GFP-positive cells. The desired transduction range was 1-20%. Therefore, the viral titer was determined using the following formula: Titer = (FXC/V) X D, where F=frequency of GFP-positive cells (percentage of GFP-positive cells/100), C=number of cells per well at the time of transduction, V=inoculation volume in ml (0.1 ml) and D=lentivirus dilution factor.

Generation of a stable cell line ReNcell VM from lentiviruses.

ReNcell VM cells (Millipore) were seeded in 24 wells coated with laminin (final concentration 20 μg/ml) at 80% confluency and incubated overnight at 37°C with 5% _CO2 . The medium was removed and 500 μl lentivirus per well with Polybrene reagent was added to a final concentration of 8 mg/ml. Cells were incubated overnight at 37°C with 5% _CO2 . The following day, the medium was replaced with fresh medium without Polybrene. Transduced cells were expanded and sorted by FAC using the GFP wavelength.

Results.

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

Example 2. The construct is sensitive to BoNT/A cleavage.

Materials and methods.

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

Visualization.

The stable cell line of the invention (referred to as the ReD SNAPR cell line) was differentiated according to the manufacturer’s protocol for ReNcell VM cells. Briefly, cells were seeded onto pre-coated Perkin Elmer CellCarrier 384 Ultra™ imaging plates at 3,000 cells per well. Cells were maintained in ReNcell NSC maintenance medium without growth factors (EGF and FGF2) (differentiation medium) for 14 days, with medium changed every 3 days. Cells were incubated with 100 nM BoNT/A in differentiation medium for 48 h. Cells were fixed with fixative (4% paraformaldehyde and 2% sucrose). Fixed cells were imaged using Opera™ Phenix.

Western blotting.

ReD SNAPR cells were differentiated according to the manufacturer’s protocol for ReNcell VM cells. Briefly, cells were seeded in a 24-well Nunc tissue culture dish at 30,000 cells per well. Cells were maintained in ReNcell NSC maintenance medium without growth factors (EGF and FGF2) (differentiation medium) for 14 days, with medium changed every 3 days. Cells were incubated with 100 nM BoNT/A in differentiation medium for 48 h. The medium was aspirated and cells were lysed with NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 nM Tris-Cl, pH 8.0). To prepare samples for loading onto SDS-PAGE gels, 10% DTT and 6X loading buffer (Bio

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Rad) and boiled for 5 min. 20 µl of samples were added to each lane of a NuPAGE Bis-tris 4-12% gel (Thermo Fisher) and run at 120 V until the dye front was gone. The gel was transferred to a nitrocellulose membrane and probed with anti-tRFP and anti-tag (CGY) FP (Evrogen) overnight.

Results.

Figure 2 demonstrates that the assay construct is sensitive to degradation by BoNT/A. Conventional cell-based assays focus on FRET interactions or direct Western blotting of cleaved SNAP25 in cell lysate, which is not suitable for high-throughput screening (HTS) applications. Under normal conditions, the C-terminal fragment of full-length SNAP25 cleaved by BoNT/A is difficult to detect due to its low molecular weight, so its fate is usually unknown. This previously unreported observation demonstrates that the C-terminal fragment is degraded along with the fluorophore that is attached to it. The relative simplicity of this methodology is well suited for a range of low- and high-throughput applications.

Figure 3 is a schematic showing the BoNT/A-mediated degradation of the C-terminus of the SNAP25 construct. Following internalization of BoNT/A in ReD SNAPR cells, the BoNT/A light chain enters the cytoplasm and cleaves tagRFPT-SNAP25-tagGFP (stably expressed in ReD SNAPR cells). This results in the degradation of the C-terminal fragment while preserving the N-terminal construct. The degradation can be detected by fluorescence microscopy and Western blotting.

Example 3. Increased sensitivity of ReD SNAPR cells to BoNT/A.

ReD SNAPR cells were seeded in 384-well plates as described above. For enhanced differentiation, ReD SNAPR cells were cultured in normal ReNcell medium with 10 ng/ml GDNF and 1 mM d-cAMP (cell-permeable cAMP). Different concentrations (0-1 μM) of BoNT/A were added to normal and ReDS medium, where ReDS medium contained 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.

Results.

Figure 4 shows that addition of GDNF and d-cAMP during differentiation and high potassium conditions during intoxication increased the sensitivity of the cell line to BoNT/A. Dose response curves and _EC50 values were determined for BoNT/A in the assay (see Figure 4b) with low nM values when cells were exposed to ReDS medium.

Example 4. Thioredoxin reductase (TrxR1) as an assay control.

ReD SNAPR cells were seeded in 384-well plates and differentiated as described above. Differentiated cells were treated with 25 nM of either non-targeting control siRNA, NT3, or anti-TrxR1 siRNA using Lipofectamine RNAimax according to the manufacturer’s protocol and left on the cells for 72 h. ReDS medium containing 10 nM BoNT/A was added to the cells for 48 h and then fixed and imaged as described above. Briefly, cells were fixed and anti-TrxR1 antibody was used to detect TrxR1 and visualize fluorescence using Opera Phenix. Mean fluorescence intensities of GFP, RFP, and far-red channels were recorded and measured.

Results.

Figure 5 shows that siTrxR-treated cells are more resistant to BoNT/A-mediated cleavage compared to controls. Thus, TrxR1 can be used as a suitable positive control to identify genes involved in the intracellular trafficking of BoNT/A. In particular, knockdown of TrXR1 can be used to show that BoNT/A intoxication can be rescued, thereby further validating the genes identified in the assay.

Example 5. Use of the BoNT SV2 receptor.

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

Results.

Although SV2 is the major receptor for BoNT/A, it has not been previously studied for its post-intoxication pathway in the cell. Figure 6 shows that BoNT/A intoxication results in a decrease in SV2 at the cell surface, which may be a typical consequence of BoNT/A-mediated defective trafficking to the cell surface. In this case, the recirculation of SV2 back to the cell surface is blocked. Therefore, SV2 can be used as an indicator of BoNT/A intoxication.

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Many genes can regulate BoNT/A activity in the cell. An example of indirect regulation would be the level of BoNT receptor SV2 migration (instead of modulating the toxin activity itself). In order to screen out candidates involved in SV2 trafficking, SV2 surface staining could be an ideal selection criterion. An example shown here is VAMP2-depleted cells, which after BoNT/A intoxication resulted in decreased SV2 surface staining. This could be due to the synergistic effect of blocking cell surface vesicle exocytosis by reducing VAMP2 and BoNT/A intoxication.

Surface SV2 can be restored by depleting TrxR, indicating that TrxR itself does not affect SV2 exocytosis at the cell surface, but directly modulates BoNT/A activity through the release of its light chain (LC). This inadvertently results in surface SV2 restoration due to decreased BoNT/A LC in the cytoplasm.

Thus, SV2 is useful in the assay of the invention because it can be used to separate candidate genes directly involved in BoNT/A transmission from those that modulate BoNT/A receptor SV2 transmission.

Example 6. Genome-wide screening of miRNAs.

Fig. 7 is a schematic diagram showing a method for performing a genome-wide miRNA screen with a cell line of the invention. ReD SNAPR cells are seeded and differentiated as described above. The miRNA library is prepared and combined with Lipofectamine™ RNAiMAX using standard protocols, and ReD SNAPR cells are transfected with miRNA. BoNT/A in stimulation buffer is added to the cells prior to fixation and imaging using Opera™ Phenix and quantification using Columbus™ software.

Positive results can be further verified by assessing recovery in the presence of siRNA against TrxR1. Confirmation that genes directly regulate BoNT activity is confirmed by SV2 cell surface staining as described above.

Example 7. Determination of prophylactic therapeutic agents against botulism.

ReD SNAPR cells are seeded and differentiated as described above and exposed to an agent (e.g., small molecule drug). BoNT/A in stimulation buffer is added to the cells prior to fixation and imaging using Opera™ Phenix and quantification using Columbus™ software.

The agent is identified as a prophylactic against botulism if cleavage of the construct is inhibited.

Example 8. Determination of therapeutic agents against botulism after intoxication.

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

An agent is identified as an antibotulism agent after intoxication if GFP recovery is observed.

Example 9. Identification of agents that increase sensitivity to BoNT.

ReD SNAPR cells are seeded and differentiated as described above and exposed to an agent (e.g., small molecule drug). BoNT/A in stimulation buffer is added to the cells prior to fixation and imaging using Opera™ Phenix and quantification using Columbus™ software.

An agent is identified as a BoNT sensitizer if the cleavage of the construct is improved (e.g., occurs faster or is apparently more cleaved). The sensitizer is proposed for further study for use as a co-product to modulate the local activity of clostridial neurotoxins (e.g., to allow for lower dosage and minimize spread to other tissues).

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

-

Claims (5)

CLAUSE OF INVENTION 1. A method for identifying a gene that regulates the activity of a clostridial neurotoxin, comprising: (a) obtaining a sample of human neuronal cells modified to express a polypeptide that contains a C-terminal detectable tag, wherein the polypeptide is cleavable by a clostridial neurotoxin; b) change in the expression of the target gene of the cells; c) contact of cells with clostridial neurotoxin; d) measuring the amount of C-terminal detectable label, thereby quantifying the activity of the clostridial neurotoxin; and e) identifying a target gene as a regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity differs from the quantified Clostridial neurotoxin activity when expression of the target gene is not altered; or f) identifying that the target gene is not a regulator of Clostridial neurotoxin activity when the quantified Clostridial neurotoxin activity is equivalent to the quantified Clostridial neurotoxin activity when expression of the target gene is not altered. 2. The method according to claim 1, wherein the expression is changed by suppressing the expression of the target gene. 3. The method according to claim 2, wherein the target gene is identified as a positive regulator of the activity of the clostridial neurotoxin when the quantitatively determined activity of the clostridial neurotoxin is lower than the quantitatively determined activity of the clostridial neurotoxin in the case where the expression of the target gene is not altered. 4. The method according to claim 2 or 3, wherein the target gene is identified as a negative regulator of the activity of the clostridial neurotoxin when the quantitatively determined activity of the clostridial neurotoxin is higher than the quantitatively determined activity of the clostridial neurotoxin in the case where the expression of the target gene is not altered. 5. The method of any one of the preceding claims, wherein a plurality of human neuronal cell samples are provided and each of the human neuronal cell samples has altered expression of a different target gene. 6. The method of claim 5, wherein the expression of a distinct target gene is altered in each of the samples using a human RNAi library. 7. The method of any one of the preceding claims, further comprising determining whether the target gene is a direct regulator of Clostridial neurotoxin activity or an indirect regulator of Clostridial neurotoxin activity, wherein: (i) the direct regulator regulates Clostridial neurotoxin activity at a level of: 1) binding of clostridial neurotoxin to the cell; 2) internalization of clostridial neurotoxin; 3) translocation of the L-chain of clostridial neurotoxin from the endosome; 4) catalysis and/or 5) maintaining the activity of the L-chain in the cell cytoplasm; and/or (ii) the indirect regulator regulates the process of cellular migration of the clostridial neurotoxin receptor. 8. The method of claim 7, wherein the determining comprises detecting the presence or absence of a clostridial neurotoxin receptor in a cell when the expression of the target gene has been altered. 9. The method according to claim 7 or 8, wherein the detection of a smaller amount of the Clostridial neurotoxin receptor on the surface of a cell when the expression of the target gene is altered (preferably reduced) compared to an equivalent cell in contact with the Clostridial neurotoxin in which the expression of the target gene is not altered indicates that the target gene indirectly regulates the activity of the Clostridial neurotoxin; or the detection of an equivalent or greater (preferably greater) amount of the Clostridial neurotoxin receptor on the surface of a cell when the expression of the target gene has been altered (preferably down-regulated) compared to an equivalent cell in contact with the Clostridial neurotoxin in which the expression of the target gene is not altered indicates that the target gene directly regulates the activity of the Clostridial neurotoxin. 10. The method according to any one of claims 7 to 9, wherein the clostridial neurotoxin receptor is synaptic vesicle glycoprotein 2A (SV2). 11. The method of any one of the preceding claims, wherein the human neuronal cell is a non-cancerous cell. 12. The method according to any of the preceding claims, wherein the human neural cell is an immortalized human neural progenitor cell or, preferably, the human neural cell has been derived (e.g. differentiated) from an immortalized cell - 29 048091 human neuronal precursors. 13. The method according to any of the preceding claims, wherein the polypeptide further comprises an N-terminal detectable label and the N-terminal detectable label is different from the C-terminal detectable label. 14. The method according to claim 13, 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). 15. The method according to any one of the preceding claims, wherein the polypeptide comprises an N-terminal RFP and a C-terminal GFP. 16. The method according to any of the preceding claims, wherein the polypeptide: a) is encoded by a nucleotide sequence containing: i) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 3; ii) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 9; and/or iii) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 11; or b) is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1; or c) contains a polypeptide sequence comprising: i) a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4; ii) a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 10; and/or iii) a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 12; or d) comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2. 17. The method according to any of the preceding claims, wherein the polypeptide: a) is encoded by a nucleotide sequence containing: i) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3; ii) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9; and/or iii) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 11; or b) is encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1; or c) contains a polypeptide sequence comprising: i) a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 4; ii) a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 10; and/or iii) a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 12; or d) comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2. 18. The method according to any of the preceding claims, wherein the polypeptide: a) is encoded by a nucleotide sequence containing: i) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3; ii) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9; and/or iii) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11; or b) is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1; or c) contains 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 - 30 048091 activity with SEQ ID NO: 12; or d) contains a polypeptide