Cell-based neurotoxin assay

Abstract

The present invention is directed to cell-based methods, such as those for determining the clostridial neurotoxin activity of a composition or determining the presence or absence of a clostridial neurotoxin in a composition. The invention is also directed to therapeutic or cosmetic clostridial neurotoxin compositions, methods for producing the same, as well as kits, methods of treatment and uses of the same.

Description

[0001] CELL-BASED NEUROTOXIN ASSAY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to clostridial neurotoxins, including methods for determining activity of the same.

[0004] BACKGROUND

[0005] Bacteria in the genus Clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, and X (see WO 2018 / 009903 A2), as well as those produced by C. baratii and C. butyricum. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. While botulinum neurotoxins act at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system.

[0006] In nature, clostridial neurotoxins (e.g. botulinum neurotoxins [BoNTs]) are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site that is located between the cysteine residues that provide the inter-chain disulphide bond. It is this di-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises an N-terminal translocation component (HNdomain) and a C-terminal targeting component (Hcdomain). The cleavage site is located between the L-chain and the translocation domain components. Following binding of the Hcdomain to its target neuron and internalisation of the bound toxin into the cell via an endosome, the HNdomain translocates the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a non- cytotoxic protease).

[0007] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin). The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell. The protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins.

[0008] When producing and formulating clostridial neurotoxins for therapeutic and / or cosmetic purposes, there is a need to accurately assess activity of a given composition.

[0009] The mouse LD5o assay has historically been the principal assay for the assessment of clostridial neurotoxin activity. The assay simultaneously tests the action of all three domains (i.e. binding, translocation, and protease). In more detail, it defines the median lethal intraperitoneal dose of the toxin at a defined time-point usually 2-4 days after dosing (activity is expressed in mouse LD5o units). Regrettably, however, LD5o assays use large numbers of animals. Moreover, LD5o units are not absolute measurements because they are not biological constants - as such they are highly dependent on the assay conditions. In particular, errors associated with this assay can be as high as 60% between different testing facilities (Sesardic et al. 2003; Biologicals 31 (4):265-276).

[0010] The mouse flaccid paralysis assay, which is also known as the “mouse abdominal ptosis assay”, relates the activity of clostridial neurotoxins to the degree of abdominal bulging seen after the toxin is subcutaneously injected into the left inguinocrural region of a mouse - the magnitude of the paralysis is dose-dependent. This approach has been proposed as a refinement to the mouse LD5o test, because it relies on a humane endpoint. This assay is approximately 10 times more sensitive than the LD5o assay, uses a sub-lethal dose of toxin and is more rapid than the LD5o test as it provides results in 24 to 48 hours, compared to 72 to 96 hours for a typical LD5o assay. The results from this assay show excellent agreement with the LD5o values (Sesardic et al. ,1996; Pharmacol Toxicol, 78(5): 283-8). Although this assay uses 20% of the animals used in the LD50 assay it still necessitates the use of animals.

[0011] Assays such as the mouse / rat phrenic nerve hemi-diaphragm assay (which are based on the use of ex vivo nerve / muscle preparations) relate the activity of a clostridial neurotoxin to a decrease in the amplitude of a twitch response of the preparation after it is applied to a maintenance medium. The usual endpoint of the assay is the time required before a 50% decrease in amplitude is observed. Regrettably, however, the hemi-diaphragm assay (like the LD50 assay) results in the use of large numbers of animals. In addition, the assay requires highly skilled personnel trained in the use of sophisticated and expensive equipment. All of the above assays have particular failings, notably animal welfare issues. Moreover, none of the above-mentioned assays are suitable for high throughput testing. The use of clostridial neurotoxins in therapeutic and cosmetic treatment of humans and other mammals is anticipated to expand to an ever-widening range of diseases and ailments that can benefit from the properties of these toxins. In view of this, there is an increasing demand for large-scale manufacture of clostridial neurotoxins and appropriate formulation thereof. Thus, there is a need in the art for alternative and / or improved clostridial neurotoxin assays.

[0012] Cell-based assays provide an alternative to the in vivo mouse assays for assessing the activity of a given composition. Most current cell-based assays rely on the use of primary or stem cell- derived neurons. Nonetheless, use of these cells does not overcome the fallings of the in vivo assays - notably obtaining primary cells entails a laborious process that still requires animal sacrifice, while stem cell-derived neurons require differentiation that takes several weeks with variable results in cellular quality. Such cells are therefore not suitable for use in a high- thorough put clostridial neurotoxin assay. Attempts have been made to develop cell-based assays using cells that maybe more suitable to high-throughput testing such as neuronal cell lines. However, neuronal cell lines in their native state were found to have a sensitivity to clostridial neurotoxin that was too low.

[0013] To overcome the issue of low sensitivity, one approach taken has been to use differentiation of neuronal cell lines which results in increased neurotoxin sensitivity. However, there is currently no consensus on the best way to achieve the increased sensitivity in such cells, with the art showing conflicting results. For example, Hong et al., 2013 (Toxicological Sciences 134(1): 64- 72) co-cultured NG108-15 cells with S16 Schwann cells to produce differentiated cells, which resulted in improved sensitivity to BoNT / A, while Hong et al., 2016 (Journal of Biomolecular Screening, Vol 21 (1): 65-73) found that no improvement in sensitivity was achieved following addition of GT1 b to differentiated NG108-15 cells. Despite improvements in sensitivity, there are a number of draw backs to using differentiated neuronal cell lines for a high throughput clostridial neurotoxin assay. Notably, differentiation of neuronal cell lines is time consuming taking at least 5 days to achieve differentiation, and requires a number of additional reagents to be used e.g. additional cell culture supplements or additional cell lines for co-culturing with the neuronal cell line. This increases the level of complexity of the assay particularly from an analytical and quality control testing perspective, which is unfavourable for high-throughput assays. Additionally, it is difficult to control the extent of differentiation and, therefore, the sensitivity of the cells is not guaranteed due to variability that occurs with each batch of differentiated cells. Such batch-to-batch variability is undesirable as it negatively impacts the reliability and robustness of the assay results.

[0014] The present invention overcomes one or more of the above-mentioned problems.

[0015] SUMMARY OF THE INVENTION

[0016] The present inventors have developed a novel cell-based method for determining the clostridial neurotoxin activity of a composition. Advantageously, the cell-based assay provides a simple and robust clostridial neurotoxin assay which provides reliable results while achieving sensitivity levels required to determine clostridial neurotoxin activity of a composition. The assay of the present invention is therefore particularly useful in the context of high throughput analysis of clostridial neurotoxin activity. This is especially useful during production of therapeutic and / or cosmetic clostridial neurotoxin formulations. The advantageous properties of the present invention may be based, at least in part, on the expression by the population of cells of a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase.

[0017] Unlike conventional cell-based assays, the assay of the present invention may avoid the use of isolated neuronal cells (primary cells) which are difficult to isolate and require animal sacrifice, and / or complex or time-consuming cell culturing methods which require several additional supplements or co-culturing of cells. By avoiding these steps, such methods may be less complex and / or time consuming and may be associated with reduced costs, making the assay more amenable to high-throughput testing.

[0018] Additionally / alternatively, by preferably avoiding the use of differentiated cells (e.g. fully / terminally differentiated), the present methods may reduce the batch-to-batch variability in the cell population observed with known cell-assays. This may be particularly advantageous in the context of high throughput testing as it provides a more reliable and robust assay.

[0019] Additionally / alternatively, the methods of the present invention have been surprisingly found to show acceptable or improved sensitivity levels even when using cells that have not gone through the differentiation process. This further supports the use of the methods of the invention for high-throughput testing. DETAILED DESCRIPTION

[0020] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0021] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0022] (i) a first luciferase domain;

[0023] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0024] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and

[0025] (b) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity.

[0026] For example, the method may comprise:

[0027] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0028] (i) a first luciferase domain;

[0029] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0030] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0031] (b) lysing the cells, thereby providing a cell lysate; and

[0032] (c) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0033] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin in a composition, the method comprising:

[0034] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0035] (i) a first luciferase domain;

[0036] (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and

[0037] (b) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity.

[0038] For example, the method may comprise:

[0039] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0040] (i) a first luciferase domain;

[0041] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0042] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0043] (b) lysing the cells, thereby providing a cell lysate; and

[0044] (c) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0045] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0046] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0047] (i) a first luciferase domain;

[0048] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0049] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0050] (b) contacting the population of cells with the composition; and

[0051] (c) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity.

[0052] For example, the method may comprise: (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0053] (i) a first luciferase domain;

[0054] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0055] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0056] (b) contacting the population of cells with the composition;

[0057] (c) lysing the cells, thereby providing a cell lysate; and

[0058] (d) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0059] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin in a composition, the method comprising:

[0060] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0061] (i) a first luciferase domain;

[0062] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0063] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0064] (b) contacting the population of cells with the composition; and

[0065] (c) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity.

[0066] For example, the method may comprise:

[0067] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0068] (i) a first luciferase domain;

[0069] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0070] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0071] (b) contacting the population of cells with the composition;

[0072] (c) lysing the cells, thereby providing a cell lysate; and

[0073] (d) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0074] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0075] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) seeded prior to contacting comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0076] (i) a first luciferase domain;

[0077] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0078] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and

[0079] (b) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity.

[0080] Preferably, in the foregoing aspect, the method further comprises contacting the population of cells (preferably neuronal progenitor-like cells) with a ganglioside prior to contacting the population of cells with the composition.

[0081] For example, the method may comprise:

[0082] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) seeded prior to contacting comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0083] (i) a first luciferase domain;

[0084] (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0085] (b) lysing the cells, thereby providing a cell lysate; and

[0086] (c) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0087] In one aspect, the invention provides a method for determining the presence or absence of clostridial neurotoxin in a composition, the method comprising:

[0088] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) seeded prior to contacting comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0089] (i) a first luciferase domain;

[0090] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0091] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and

[0092] (b) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity.

[0093] Preferably, in the foregoing aspect, the method further comprises contacting the population of cells (preferably neuronal progenitor-like cells) with a ganglioside prior to contacting the population of cells with the composition.

[0094] For example, the method may comprise:

[0095] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) seeded prior to contacting comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0096] (i) a first luciferase domain;

[0097] (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0098] (b) lysing the cells, thereby providing a cell lysate; and

[0099] (c) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0100] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0101] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0102] (i) a first luciferase domain;

[0103] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0104] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and

[0105] (b) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity.

[0106] Preferably, in the foregoing aspect, the method further comprises contacting the population of cells (preferably neuronal progenitor-like cells) with a ganglioside prior to contacting the population of cells with the composition.

[0107] For example, the method may comprise:

[0108] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0109] (i) a first luciferase domain;

[0110] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0111] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0112] (b) lysing the cells, thereby providing a cell lysate; and

[0113] (c) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0114] In one aspect, the invention provides a method for determining the presence or absence of clostridial neurotoxin in a composition, the method comprising:

[0115] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0116] (i) a first luciferase domain;

[0117] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0118] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and

[0119] (b) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity.

[0120] Preferably, in the foregoing aspect, the method further comprises contacting the population of cells (preferably neuronal progenitor-like cells) with a ganglioside prior to contacting the population of cells with the composition.

[0121] For example, the method may comprise:

[0122] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells (preferably neuronal progenitor-like cells) comprises at least 30,000 cells, and wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0123] (i) a first luciferase domain;

[0124] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0125] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; (b) lysing the cells, thereby providing a cell lysate; and

[0126] (c) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

[0127] In a method of determining the presence or absence of a clostridial neurotoxin in a composition, the presence of a clostridial neurotoxin may be indicated by a change (e.g. a statistically significant change) in the luciferase activity (e.g. of the cell lysate), such as a change (e.g. a statistically significant change) over time. The absence of a clostridial neurotoxin may be indicated by no change (e.g. no statistically significant change) in the luciferase activity (e.g. of the cell lysate), such as no change (e.g. no statistically significant change) change over time. The absence of a clostridial neurotoxin may mean that there is no neurotoxin present in the composition or that the toxin present is below the detectable limit. Preferably, in the foregoing aspect the presence or absence of clostridial neurotoxin in the composition is determined by comparing the measured luciferase activity to a control as defined herein.

[0128] Preferably, the methods of the invention comprise contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside. Advantageously, this has been shown to improve sensitivity of the method. This may provide for an improved determination of clostridial neurotoxin activity of a composition or improved determination of the presence or absence of a clostridial neurotoxin.

[0129] Gangliosides are oligoglycosylceramides derived from lactosylceramide and containing a sialic acid residue such as N-acetylneuraminic acid (‘NANA or ‘SA or 'Neu5Ac' or 'NeuAc'). In some embodiments, the sialic acid component is N-glycolyl-neuraminic acid (Neu5Gc), or a Neu5Ac analogue in which the amine group is replaced by OH (3-deoxy-D-glycero-D-galacto- nonulosonic acid, given the abbreviation ‘KDN’). Gangliosides are defined by a nomenclature system proposed by Svennerholm in which M, D, T and Q refer to mono-, di-, tri- and tetrasialogangliosides, respectively, and the numbers 1 , 2, 3, etc. refer to the order of migration of the gangliosides on thin-layer chromatography. For example, the order of migration of monosialogangliosides is GM3 > GM2 > GM 1 . To indicate variations within the basic structures, further terms are added, e.g. GM1a, GD1 b, etc. Glycosphingolipids having 0,1 , 2, and 3 sialic acid residues linked to the inner galactose unit are termed asialo- (or 0-), a-, b- and c-series gangliosides, respectively, while gangliosides having sialic acid residues linked to the inner N- galactosamine residue are classified as a-series gangliosides. Pathways for the biosynthesis of the 0-, a-, b- and c-series of gangliosides involve sequential activities of sialyltransferases and glycosyltransferases as illustrated e.g. in Ledeen et al., 2015 (Ledeen, Robert W., and Gusheng Wu. "The multi-tasked life of GM1 ganglioside, a true factotum of nature." Trends in biochemical sciences 40.7 (2015): 407-418). Further sialization of each of the series and in different positions in the carbohydrate chain can occur to give an increasingly complex and heterogeneous range of products, such as the a-series gangliosides with sialic acid residue(s) linked to the inner N-acetylgalactosamine residue.

[0130] In the context of the cell, gangliosides are transferred to the external leaflet of the plasma membrane by a transport system involving vesicle formation. Gangliosides are present and concentrated on cell surfaces, with the two hydrocarbon chains of the ceramide moiety embedded in the plasma membrane and the oligosaccharides located on the extracellular surface, where they present points of recognition for extracellular molecules or surfaces of neighbouring cells. The sialoglycan components of gangliosides extend out from the cell surface, where they can participate in intermolecular interactions. They function by recognizing specific molecules at the cell surface and by regulating the activities of proteins in the plasma membrane. Gangliosides also bind specifically to viruses and to bacterial toxins, such as those from botulinum, tetanus and cholera. For example, the specific cell surface receptor for the cholera toxin is ganglioside GM1 (or GM1a): Neu5Aca2-3(Gal|31-3GalNAc|31-4)Gal|31- 4GlcpiCer.

[0131] BoNTs possess two independent binding regions in the Hcc domain for gangliosides and neuronal protein receptors. BoNT / A, ZB, ZE, ZF and ZG have a conserved ganglioside-binding site in the Hcc domain composed of a “E(Q) ... H(K) ... SXWY ... G” motif, whereas BoNT / C, / D and / DC display two independent ganglioside-binding sites. (Lam, Kwok-Ho, et al. "Diverse binding modes, same goal: The receptor recognition mechanism of botulinum neurotoxin." Progress in biophysics and molecular biology 117.2 (2015): 225-231.) Most BoNTs bind only to gangliosides that have an 2,3-linked N-acetylneuraminic acid residue (denoted Sia5) attached to Gal4 of the oligosaccharide core, whereas the corresponding ganglioside-binding pocket on TeNT can also bind to GM1a, a ganglioside lacking the Sia5 sugar residue. It has been shown that introducing a H1241 K mutation into a recombinant BoNT / F confers GM1 binding ability (Benson, Marc A., et al. "Unique ganglioside recognition strategies for clostridial neurotoxins." Journal of Biological Chemistry 286.39 (2011): 34015-34022). BoNT / D has been found to bind GM1a and GD1a (Kroken, Abby R., et al. "Novel ganglioside-mediated entry of botulinum neurotoxin serotype D into neurons." Journal of Biological Chemistry 286.30 (2011): 26828-26837.)

[0132] A ganglioside may be GM1 (e.g. GM1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, or GD3.

[0133] Combining the data derived from ganglioside-deficient mice and biochemical assays, BoNT / A, E, F and G display a preference for the terminal NAcGal-Gal-NAcNeu moiety being present in GD1a and GT1 b, whereas BoNT / B, C, D and TeNT require the disialyl motif found in GD1b, GT1 b and GQ1 b.

[0134] Thus, a ganglioside may comprise a terminal NAcGal-Gal-NAcNeu moiety or a disialyl motif. A ganglioside may comprise GD1a, GT1 b, GD1 b, GQ1 b, or GM1 (Neu5Aca2-3(Gaipi- 3GalNAcpi-4)Gaipi-4GlcpiCer). For example, a ganglioside may comprise GD1a, GT1 b, GD1 b, or GQI b.

[0135] Suitable gangliosides may be selected from those described in WO 2018 / 060351.

[0136] Any suitable gangliosides can be contacted with the population of cells (preferably neuronal progenitor-like cells) in the methods of the invention. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may be contacted with a ganglioside selected from GM1 (e.g. GM1a or GMI b), GM2, GM3 (e.g. NeuAc GM 3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, GD3, or a combination thereof. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may be contacted with a ganglioside selected from GT1a, GT1 b, GD1a, GM1 , or a combination thereof. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may be contacted with GT1a. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may be contacted with GD1 a. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may be contacted with GM1. Preferably, the population of cells (preferably neuronal progenitor-like cells) may be contacted with GT1 b. Advantageously, addition of a ganglioside, preferably GT1 b, improves the sensitivity of the method of the invention.

[0137] The population of cells (preferably neuronal progenitor-like cells) may be contacted with one or more ganglioside, e.g. one or more, two or more, three or more, four or more, or five or more gangliosides. In one embodiment, the population of cells may be contacted with one or more ganglioside selected from GM1 (e.g. GM1a or GM1b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1b, GalNAc-GD1a, GT1a, GT1b, GQ1b, GD2, and GD3. In one embodiment, the population of cells may be contacted with at least one ganglioside selected from GT1a, GT1b, GD1a and GM1. In one embodiment, the population of cells may be contacted with at least GT1a. In one embodiment, the population of cells may be contacted with at least GD1a. In one embodiment, the population of cells may be contacted with at least GM1. Most preferably, the population of cells may be contacted with at least GT1b, e.g. in some instances only GT 1 b.

[0138] In one embodiment, the population of cells may be contacted with two or more gangliosides selected from GM1 (e.g. GM1a or GM 1b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1b, GalNAc-GD1a, GT1a, GT1b, GQ1b, GD2, and GD3. In one embodiment, the population of cells may be contacted with two or more gangliosides selected from GT 1 a, GT1 b, GD1a and GM1. In one embodiment, the population of cells may be contacted by GT1a, and at least one other ganglioside selected from GT1b, GD1a and GM1. In one embodiment, the population of cells may be contacted with GD1a and at least one other ganglioside selected from GT1a, GT1b, and GM1. In one embodiment, the population of cells may be contacted with GM1, and at least one other ganglioside selected from GT1a, GT1b, and GD1a. Preferably, the population of cells may be contacted with at least GT 1 b and at least one other ganglioside selected from GT1a, GD1a and GM1.

[0139] In one embodiment, the population of cells may be contacted with three or more gangliosides selected from GM1 (e.g. GM1a or GM 1b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1b, GalNAc-GD1a, GT1a, GT1b, GQ1b, GD2, and GD3. In one embodiment, the population of cells may be contacted with three or more gangliosides selected from GT1a, GT1b, GD1a and GM1. In one embodiment, the population of cells may be contacted with GT1a, and at least two other gangliosides selected from GT1b, GD1a and GM1. In one embodiment, the population of cells may be contacted with GD1a and at least two other gangliosides selected from GT1a, GT1b, and GM1. In one embodiment, the population of cells may be contacted with GM1, and at least two other gangliosides selected from GT1a, GT1b, and GD1a. Preferably, the population of cells may be contacted with at least GT1b and two other gangliosides selected from GT1a, GD1a and GM1.

[0140] In one embodiment, the population of cells may be contacted with four or more gangliosides selected from GM1 (e.g. GM1a or GM 1b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, and GD3. Preferably, the population of cells may be contacted with at least GT1 b, GT1a, GD1a and GM1.

[0141] A ganglioside for use in the present invention may be an isolated ganglioside. Said ganglioside may have been isolated from a cell and / or purified. A ganglioside for use in the present invention may be a natural or synthetic ganglioside. The synthetic ganglioside may be otherwise identical to its natural counterpart.

[0142] The term “contacting a population of cells with a ganglioside” means that the ganglioside and cell (e.g. a component thereof, such a clostridial neurotoxin receptor) are allowed to physically and / or chemically interact. Contacting may comprise a surface of the cell (e.g. a component thereof) being contacted with a ganglioside. For example, a ganglioside may interact with a clostridial neurotoxin receptor, such as SV2, SYT-I, and / or SYT-II. The ganglioside may form a complex with the clostridial neurotoxin receptor. Thus, “contacting a population of cells with a ganglioside” may mean that the ganglioside and cell (preferably a neuronal progenitor-like cell) are brought into physical proximity so as to allow interaction between the ganglioside and a clostridial neurotoxin receptor. The term “contacting a population of cells with a ganglioside” is differentiated from a cell simply expressing or over expressing a ganglioside.

[0143] The term “contacting a population of neuronal progenitor-like cells with a ganglioside” means that the ganglioside and cell (e.g. a component thereof, such a clostridial neurotoxin receptor) are allowed to physically and / or chemically interact. Contacting may comprise a surface of the cell (e.g. a component thereof) being contacted with a ganglioside. For example, a ganglioside may interact with a clostridial neurotoxin receptor, such as SV2, SYT-I, and / or SYT-II. The ganglioside may form a complex with the clostridial neurotoxin receptor. Thus, “contacting a population of neuronal progenitor-like cells with a ganglioside” may mean that the ganglioside and cell are brought into physical proximity so as to allow interaction between the ganglioside and a clostridial neurotoxin receptor. The term “contacting a population of neuronal progenitorlike cells with a ganglioside” is differentiated from a cell simply expressing or over expressing a ganglioside.

[0144] A ganglioside, preferably GT1 b, may be added to a medium in which the cell is present, for example a cell culture medium. The ganglioside may be added to fresh medium (e.g. cell culture medium). Said medium may replace existing medium (e.g. cell culture medium). In other words, the existing medium may be removed (e.g. aspirated) and fresh medium may be added which contains the ganglioside diluted to the appropriate concentration. Alternatively, or additionally, the ganglioside may be added to the existing cell culture medium. The ganglioside, preferably GT 1 b, may be present at a concentration of at least 10, 20, 30 40 or 50 pg / ml. The ganglioside, preferably GT1 b, may be present at a concentration of <200, <150, <100, or <75 pg / ml. The ganglioside, preferably GT1 b, may be present at a concentration of 10-100 pg / ml, 10-80 pg / ml, 25-100 pg / ml, 25-80 pg / ml, 30-90 pg / ml, 40-100 pg / ml, 40-80 pg / ml or 50-70 pg / ml. The ganglioside, preferably GT1 b, concentration may be selected from 10 pg / ml, 25 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, or 100 pg / ml. Preferably, the concentration of the ganglioside is 60 pg / ml. In some instances, the above concentrations may be concentration per ganglioside type, preferably the above concentrations are the total ganglioside concentration with which the cells (preferably neuronal progenitor-like cells) are contacted.

[0145] The ganglioside, preferably GT 1 b, may be contacted with the cell for a period of up to 48 hours, up to 35 hours, 24 hours, up to 22 hours, up to 20 hours, up to 18 hours or up to 16 hours, preferably up to 24 hours, more preferably up to 22 hours. The ganglioside, preferably GT1 b, may be contacted with the cell for a period of at least 10, 15, 18 or 20 hours. The ganglioside, preferably GT 1 b, may be contacted with the cell (preferably neuronal progenitor-like cell) for a period of 16 to 24 hours, 17 to 23 hours, or 18 to 22 hours. Preferably, the ganglioside, preferably GT1 b, may be contacted with the cell (preferably neuronal progenitor-like cell) for 18 to 22 hours.

[0146] The ganglioside, preferably GT1 b, may be contacted with the population of cells (preferably neuronal progenitor-like cells) at any suitable temperature. In one embodiment, the ganglioside may be contacted with the population of cells at 15-45°C, 15-40°C, 20°C-40°C, 25-40°C, 30- 40°C or 35-40°C. Preferably, the ganglioside (preferably GT1 b) may be contacted with the population of cells at a 37°C ±2°C.

[0147] The ganglioside may be present or absent (e.g. in the cell culture medium) when the population of cells are contacted with the composition. When the ganglioside is absent from the medium (e.g. cell culture medium), the medium (e.g. cell culture medium) containing the ganglioside may be replaced with fresh medium (e.g. cell culture medium) which does not contain the ganglioside prior to contacting the cell with the composition to be tested. In one embodiment, the ganglioside is present in the medium (e.g. cell culture medium) during contacting with the composition, (e.g. the ganglioside may not be removed from the medium (e.g. cell culture medium) prior to contacting with the composition or the medium (e.g. cell culture medium) may be replaced with fresh medium (e.g. cell culture medium) comprising the ganglioside). Preferably, the ganglioside (e.g. GT1 b), is not present in the medium (e.g. the cell culture medium may be replaced with medium without the ganglioside) when the cells are contacted with the composition.

[0148] Thus, following contact of the population of cells with the ganglioside(s), preferably GT 1 b, said ganglioside(s) may be removed from the population of cells prior to the population of cells being contacted with the composition. For example, the medium (e.g. cell culture medium) comprising the ganglioside may be removed and replaced with fresh medium (e.g. cell culture medium). In this context the term “removed” may mean that the medium (e.g. cell culture medium) comprising the ganglioside(s) is taken out (e.g. aspirated) from the cell culture vessel in which the cells are grown. However, it is not necessary for all of the medium (e.g. cell culture medium) comprising the ganglioside(s) to be removed or replaced. Some medium (e.g. cell culture medium) comprising the ganglioside(s) may still be present in the cell culture vessel. The medium (e.g. cell culture medium) comprising the ganglioside(s) may be partially removed e.g. at least 20%, 30%, 35%, 40%, 45%, or 50% of the medium (e.g. cell culture medium) comprising the ganglioside may be removed. For example, substantially all of the medium (e.g. cell culture medium) comprising the ganglioside(s) may be removed or replaced. “Substantially all” means that at least 85%, 90%, 95%, 97%, 98% or 99% of the medium (e.g. cell culture medium) comprising the ganglioside(s) has been removed. In some instances, 100% of the medium (e.g. cell culture medium) comprising the ganglioside(s) may be removed or replaced. By removing the medium (e.g. cell culture medium) comprising the ganglioside(s) from the cell culture vessel, the ganglioside(s) may no longer be in contact with the population of cells. In one embodiment, most or all of the ganglioside(s) may no longer be in contact with the population of cells. The ganglioside(s) may be partially removed from the population of cells, e.g. at least 20%, 30%, 35%, 40%, 45%, or 50% of the ganglioside(s) may no longer be in contact with the population of cells. In one embodiment, substantially all of the ganglioside(s) may no longer be in contact with the population of cells, e.g. at least 85%, 90%, 95%, 97%, 98% or 99% may no longer be in contact with the population of cells. In some instances, 100% of the gangliosides may no longer be in contact with the population of cells.

[0149] Methods of the invention make use of a population of cells (preferably neuronal progenitor-like cells).

[0150] The population of cells used in the methods of the invention preferably expresses a clostridial neurotoxin receptor and / or (preferably and) a ganglioside and / or (preferably and) is sensitive to intoxication by a clostridial neurotoxin (e.g. when contacted with a clostridial neurotoxin in accordance with a method of the invention). In other words, the population of cells when contacted with a clostridial neurotoxin may be suitable to enable the binding and internalisation of the clostridial neurotoxin. The population of cells may also be a population of cells that have the ability to differentiate into a neuron-like cell. Preferably, said cells may share some characteristics of a neuron-like cell and further be capable of differentiation (e.g. into a neuronlike cell). Preferably, the population of cells may be a population of neuronal progenitor-like cells.

[0151] A population of cells used in a method of the invention may be a population of neuronal progenitor-like cells (or may comprise neuronal progenitor cells) at the time of seeding said cells. During the course of the method, the cells may progress in differentiation (e.g. to or towards a neuron-like cell). Thus, when contacted with a clostridial neurotoxin, said cells may be differentiated towards being neuron-like cells or may be neuron-like cells.

[0152] The term “neuronal progenitor-like cell” may refer (preferably refers) to any cell that has the ability to differentiate into a neuron-like cell. The neuronal progenitor-like cell preferably expresses a clostridial neurotoxin receptor and / or (preferably and) a ganglioside and / or (preferably and) is sensitive to intoxication by a clostridial neurotoxin. Preferably, said neuronal progenitor-like cell may share some characteristics of a neuron-like cell and further be capable of differentiation (e.g. into a neuron-like cell).

[0153] A neuronal progenitor-like cell may be a cell that retains plasticity (e.g. dependent on cell culture or media conditions). For example, a neuronal progenitor-like cell may be a cell that can be differentiated and dedifferentiated. A neuronal progenitor-like cell may be a cell that is capable of differentiation (e.g. into a neuron-like cell) when exposed to appropriate media (e.g. neural differentiation media) but that reverts to an undifferentiated state when said media is removed. For example, a neuronal progenitor-like cell may be differentiated into a neuron-like cell by exposure to appropriate neural differentiation media containing one or more neural differentiation supplements for a suitable period of time, however, removal of said neural differentiation media (e.g. by growing the cells in a standard growth medium lacking one or more neural differentiation supplements) may cause the cell to revert to an undifferentiated state.

[0154] A “neuronal progenitor-like cell” may be a neuronal progenitor cell (e.g. a natural neuronal progenitor cell, such as a normal neuronal progenitor cell) or any equivalent cell. A “neuronal progenitor-like cell” may be a non-natural cell (e.g. a non-natural cell having one or more properties of a natural neuronal progenitor cell, such as a normal neuronal progenitor cell) and / or an abnormal cell, such as a cancer cell. A non-natural cell may be a hybrid cell, such as a hybrid between one or more abnormal cells (e.g. a hybrid between a neuroblastoma cell and a glioma cell). The hybrid may be a fusion of one or more cell types. Said cell may be immortalized as described herein.

[0155] A cell (preferably a neuronal progenitor-like cell) may still be capable of cell division even when in a differentiated state. Alternatively, a cell (preferably a neuronal progenitor-like cell) may be a postmitotic cell that does not divide further. Said postmitotic cell preferably has the ability to differentiate into a neuron-like cell.

[0156] A cell (preferably a neuronal progenitor-like cell) may be a neuroblast or have one or more properties of a neuroblast. A neuroblast may be an example of a natural neuronal progenitor cell, such as a normal neuronal progenitor cell.

[0157] Thus, the cells (preferably neuronal progenitor-like cells), in one embodiment, do not express all (or in some instances any) markers of terminally-differentiated neuronal cells and / or extend processes resembling axons and dendrites. Preferably, the cells (preferably neuronal progenitor-like cells) do not comprise axonal and / or dendritic processes / outgrowth and / or do not express Beta Tubulin III. Preferably, the cells (preferably neuronal progenitor-like cells) do not express Beta Tubulin III. Differentiation of cells (preferably neuronal progenitor-like cells) into neuron-like cells may involve culturing the cells in a neural differentiation media comprising varying combinations of neural differentiation supplements such as B18, N2 and GS21 or coculturing with suitable cells of a different type, e.g. Schwann cells. During differentiation, the cells (preferably neuronal progenitor-like cells) may be cultured in a neural differentiation media or co-cultured with other suitable cells of a different type for several days or weeks e.g. at least 2 days, at least 5 days, at least 7 days, at least 14 days, and up to 30 days, to achieve full differentiation of the cells into neuron-like cells. The term “neural differentiation supplement” may refer to a neuronal culture supplement which is a mixture of nutrients and vitamins that, when added to the cell culture medium and incubated for a suitable period of time, initiates differentiation of a cell. Examples of such neural differentiation supplements include retinoic acid, purmorphamine, B18 (Brewer and Cotman 1989, Brain Res. 494 65-74), N2 (commercially available from Thermo Fisher Scientific, formulation based on Bottenstein and Sato 1979, PNAS, 76, 514-517), N21 (Chen et al., 2008, J. Neurosci. Methods, 171 (2), 239- 247), and GS21 (commercially available from MTI-GlobalStem; formulation based on N21) supplements. Preferably, the population of cells (preferably neuronal progenitor-like cells) are not incubated in a neural differentiation media containing at least one neural differentiation supplement selected from the group consisting of retinoic acid, purmorphamine, B18, N2, N21 and GS21.

[0158] A “neuron-like cell” may be a cell having one or more properties of a terminally-differentiated neuron e.g. in terms of morphology such as axonal and / or dendritic processes / outgrowth, and / or markers such as Beta Tubulin III and / or receptors which neurons typically express. For example, a neuron-like cell may express one or more (e.g. all) markers of a terminally- differentiated neuron cell and / or extend processes resembling axons and dendrites. A “neuronlike cell” is preferably incapable of further differentiation. The term “neuron-like cell” may encompass a neuron (e.g. a natural neuron). Preferably a neuron-like cell expresses Beta Tubulin III.

[0159] A neuron-like cell may be a terminally-differentiated neuron, for example, a natural terminally- differentiated neuron, such as a normal terminally-differentiated neuron.

[0160] A “neuron-like cell” may be a non-natural cell (e.g. a non-natural cell having one or more properties of a natural neuron, such as a normal neuron) and / or an abnormal cell, such as a cancer cell. The neuron-like cell may be a cell that has one or more properties of a natural (e.g. normal) neuron and that has been differentiated from a cell (preferably a neuronal progenitorlike cell), such as a non-natural cell and / or an abnormal cell, such as a cancer cell. A “neuronlike cell” may be a cell that has one or more properties of a natural (e.g. normal) neuron and that has been differentiated from a hybrid cell, such as a hybrid between one more abnormal cells (e.g. a hybrid between a neuroblastoma cell and a glioma cell). The hybrid may be a fusion of one or more cell types. Said cell may be immortalized as described herein. A natural (e.g. normal) neuron may be a terminally-differentiated neuron.

[0161] A ”neuron-like cell” may be a motor neuron-like cell. A neuron-like cell may have one or more properties of a terminally-differentiated motor neuron.

[0162] In some embodiments, the population of cells (preferably neuronal progenitor-like cells) may not be differentiated during any step of the methods of the invention. In some embodiments, the population of cells (preferably neuronal progenitor-like cells) may not be differentiated when the population of cells are contacted with a ganglioside. Preferably, the population of cells (preferably neuronal progenitor-like cells) may not be differentiated when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition. In some embodiments, the population of cells (preferably neuronal progenitor-like cells) may not be terminally-differentiated during any step of the methods of the invention. In some embodiments, the population of cells (preferably neuronal progenitor-like cells) may not be terminally-differentiated when the population of cells are contacted with a ganglioside. Preferably, the population of cells (preferably neuronal progenitor-like cells) may not be terminally-differentiated when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition.

[0163] In some embodiments, the population of cells (preferably neuronal progenitor-like cells) may remain undifferentiated throughout the entire method of the invention. In other words, the population of cells (preferably neuronal progenitor-like cells) may remain undifferentiated during all steps of the methods of the invention. In some embodiments, the population of cells (preferably neuronal progenitor-like cells) may remain undifferentiated at least when the population of cells are contacted with a ganglioside. Preferably, the population of cells (preferably neuronal progenitor-like cells) may remain undifferentiated when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition.

[0164] The term “undifferentiated” may mean that the population of cells (preferably neuronal progenitor-like cells) have not undergone one or more active steps to induce cellular changes into neuron-like cells. In other words, in one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not have undergone one or more active steps to differentiate (e.g. partially or fully differentiate, preferably fully differentiate) the cells so that the population of cells (preferably neuronal progenitor-like cells) forms a population of neuron-like cells.

[0165] An “active step” may include a differentiation step as described above, e.g. culturing in neural differentiation media containing neural differentiation supplements and / or growing in co-culture with other cell types e.g. Schwan cells for a suitable period of time, such as at least 2 days, at least 5 days, at least 7 days, at least 14 days, and up to 30 days, e.g. between 5-30 days. Cells (preferably neuronal progenitor-like cells) may be considered to be undifferentiated prior to one or more active differentiation steps being taken. Thus, preferably, a method of the invention does not comprise an active step, e.g. to promote differentiation of the cells (preferably neuronal progenitor-like cells). For example, the population of cells (preferably neuronal progenitor-like cells) may be undifferentiated prior to culturing in neural differentiation media comprising neural differentiation supplements or co-culturing with other cell types for a suitable time period (e.g. at least 2 days, at least 5 days, at least 7 days, at least 14 days, and up to 30 days, e.g. between 5-30 days) to induce cellular changes into neuron-like cells. In other words, the population of cells (preferably neuronal progenitor-like cells) may be undifferentiated prior to the occurrence of full differentiation into a population of neuron-like cells.

[0166] In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not undergo (or may not have undergone) one or more active steps to differentiate (preferably to fully differentiate) the cells so that the population of cells (preferably neuronal progenitor-like cells) forms a population of neuron-like cells. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not undergo (or may not have undergone) one or more active differentiation steps (e.g. culturing in neural differentiation media comprising neural differentiation supplements) for a suitable time period (e.g. at least 2 days, at least 5 days, at least 7 days, at least 14 days, and up to 30 days, e.g. between 5-30 days). In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not undergo (or may not have undergone) one or more active differentiation steps (e.g. culturing in neural differentiation media comprising neural differentiation supplements) for more than 24 hours. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not undergo (or may not have undergone) one or more active differentiation steps (e.g. culturing in neural differentiation media comprising neural differentiation supplements) fora suitable time period (e.g. at least 2 days, at least 5 days, at least 7 days, at least 14 days, and up to 30 days, e.g. between 5-30 days) prior to contacting with a ganglioside and / or (preferably and) the composition. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not undergo (or may not have undergone) one or more active differentiation steps (e.g. culturing in neural differentiation media comprising neural differentiation supplements) for more than 115 minutes (preferably more than 95 minutes) prior to contacting with a ganglioside. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not undergo (or may not have undergone) one or more active differentiation steps (e.g. culturing in neural differentiation media comprising neural differentiation supplements) for more than 24 hours prior to contacting with a composition.

[0167] Growth of cells in a neural differentiation media containing neural differentiation supplements for less than 24 hours (preferably no more than 18-24 hours) prior to any contacting step preferably may not be considered an active differentiation step for the purposes of this invention. In other words, the population of cells (preferably neuronal progenitor-like cells) may be cultured in a neural differentiation media comprising neural differentiation supplements for no more than 24 hours (preferably no more than 18-24 hours) prior to any contacting step and be considered undifferentiated.

[0168] The population of cells (preferably neuronal progenitor-like cells) may be cultured (or incubated) (e.g. in a neural differentiation media comprising neural differentiation supplements) for no more than 24 hours (preferably no more than 18-24 hours) prior to any contacting step. The population of cells (preferably neuronal progenitor-like cells) may be cultured (or incubated) (e.g. in a neural differentiation media comprising neural differentiation supplements) for no more than 24 hours (preferably no more than 18-24 hours) prior to contacting with a composition. The population of cells (preferably neuronal progenitor-like cells) may be incubated (e.g. in a neural differentiation media comprising neural differentiation supplements) for no more than 115 minutes (preferably no more than 95 minutes, more preferably 60-90 minutes) prior to contacting with a ganglioside. The population of cells (preferably neuronal progenitor-like cells) may be incubated (e.g. in a neural differentiation media comprising neural differentiation supplements) for no more than 95 minutes (preferably 60-90 minutes) prior to contacting with a ganglioside. Preferably, the population of cells (preferably neuronal progenitor-like cells) may be incubated in a neural differentiation media comprising neural differentiation supplements for no more than 24 hours (preferably no more than 18-24 hours) prior to any contacting step. Preferably, the population of cells (preferably neuronal progenitorlike cells) may be incubated in a neural differentiation media comprising neural differentiation supplements for no more than 24 hours (preferably no more than 18-24 hours) prior to contacting with the composition. Preferably, the population of cells (preferably neuronal progenitor-like cells) may be incubated in a neural differentiation media comprising neural differentiation supplements for no more than 115 minutes (preferably no more than 95 minutes, more preferably 60-90 minutes) prior to contacting with a ganglioside. The neural differentiation media may comprise the neural differentiation supplement B27. Preferably, the neural differentiation media does not comprise a neural differentiation supplement selected from the group consisting of retinoic acid, purmorphamine, B18, N2, N21 or GS21.

[0169] In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may not be co-cultured with cells of a different type, e.g. Schwann cells prior to contacting with a ganglioside and / or composition. Preferably, the population of cells (preferably neuronal progenitor-like cells) may (i) be incubated (e.g. in a neural differentiation media comprising neural differentiation supplements) for no more than 24 hours (preferably no more than 18-24 hours) and (ii) not be co-cultured with cells of a different type, e.g. Schwann cells, prior to contacting with a composition. Preferably, the population of cells (preferably neuronal progenitor-like cells) may (i) be incubated (e.g. in a neural differentiation media comprising neural differentiation supplements) for no more than 115 minutes (preferably no more than 95 minutes, more preferably for 60-90 minutes) and (ii) not be co-cultured with suitable cells of a different type, e.g. Schwann cells, prior to contacting with a ganglioside.

[0170] A population of undifferentiated cells (preferably neuronal progenitor-like cells) may encompass some cells which have undergone differentiation without one or more active steps being taken. The skilled person will appreciate that there may be some differentiation of the cells (preferably neuronal progenitor-like cells) when carrying out a method of the invention. Said differentiation may be induced by standard / normal cell culture conditions which do not involve one or more active steps. For example, differentiation may be induced without one or more active steps due to direct cell to cell contact of two or more cells (preferably neuronal progenitor-like cells) and / or cell to cell communication of two or more cells (preferably neuronal progenitor-like cells) through the secretion of soluble factors with paracrine effects. In one embodiment, differentiation of cells may not involve the use of a neural differentiation media comprising neural differentiation supplements or co-culturing with other cell types and may be considered to be a natural differentiation. The cells (preferably neuronal progenitor-like cells) may be cultured or incubated in a neural differentiation media comprising neural differentiation supplements for less than a suitable period of time required for differentiation (e.g. less than 5 days, preferably less than 24 hours (e.g. prior to any contacting step), more preferably 18-24 hours) and may be considered to be a natural differentiation. A population of cells comprising cells which have undergone a natural differentiation process may be considered to be undifferentiated for the purposes of the present invention. Accordingly, an undifferentiated population of cells (preferably neuronal progenitor-like cells) may include some differentiated neuron-like cells. In other words, the population of cells (preferably neuronal progenitor-like cells) may be considered to be undifferentiated even when the population includes neuron-like cells. Thus, an “undifferentiated” population may encompass a “partially undifferentiated” population. The term “partially undifferentiated” means that a small proportion of the cells of the population may be differentiated, for example, the population of cells may include up to 30%, 20%, 10%, 5% or 1% (preferably up to 1%) of cells which may have been differentiated without one or more active steps having been taken. In some instances, substantially all of the population of cells may be undifferentiated. In this context, the term “substantially all” means that most of the cells of the population may be undifferentiated but a small proportion of cells may be differentiated (e.g. partially or fully differentiated) without any active steps being taken, for example, up to 15%, 10%, 5%, or 1% of the cells may be differentiated. A differentiated cell may be a cell that exhibits properties that are more similar to a neuron-like cell than an undifferentiated cell (preferably neuronal progenitor-like cell). A cell may be considered “fully differentiated” (e.g. terminally differentiated) when it develops all characteristics of a neuron-like cell as described herein. A cell may be considered “partially differentiated” when it has developed some but not all of the characteristics of a neuron-like cell as described herein. A partially differentiated cell (preferably neuronal progenitor-like cell) may comprise axonal and / or dendritic processes / outgrowth. A partially differentiated cell (preferably neuronal progenitor-like cell) may retain plasticity (e.g. dependent on cell culture or media conditions). For example, a partially differentiated cell may be a cell that can be further differentiated and also dedifferentiated. A partially differentiated cell may be a cell that is capable of further differentiation (e.g. into a neuron-like cell) when exposed to appropriate media (e.g. neural differentiation media) but that reverts to an undifferentiated state when said media is removed. For example, a partially differentiated cell may be further differentiated into a neuron-like cell by exposure to appropriate neural differentiation media containing one or more neural differentiation supplements for a suitable period of time, however, removal of said neural differentiation media (e.g. by growing the cells in a standard growth medium lacking one or more neural differentiation supplements) may cause the cell to revert to an undifferentiated state. For example, a population of cells comprising cells which have undergone a differentiation process without any active differentiation steps for a suitable period of time may be considered to be partially differentiated forthe purposes of the present invention. A partially differentiated cell (preferably neuronal progenitor-like cell) may maintain the ability to undergo cell division.

[0171] A cell (preferably neuronal progenitor-like cell) may be undifferentiated (e.g. when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition).

[0172] A cell (preferably neuronal progenitor-like cell) may be undifferentiated or partially differentiated (e.g. when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition).

[0173] The population of cells (preferably neuronal progenitor-like cells) may be partially differentiated throughout the entire method of the invention. In otherwords, the population of cells (preferably neuronal progenitor-like cells) may be partially differentiated during all steps of the methods of the invention. The population of cells (preferably neuronal progenitor-like cells) may be partially differentiated at least when the population of cells are contacted with a ganglioside. Preferably, the population of cells (preferably neuronal progenitor-like cells) may be partially differentiated when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition.

[0174] Preferably, a cell may be a neuronal progenitor-like cell.

[0175] A cell (preferably neuronal progenitor-like cell) may be a cancer cell or a hybrid (e.g. fusion) thereof. A cell (preferably neuronal progenitor-like cell) is preferably a neuroblastoma cell or a hybrid (e.g. fusion) thereof (e.g. a neuroblastoma-glioma hybrid cell). A cell (preferably neuronal progenitor-like cell) is preferably immortalized. In a particularly preferred embodiment, a cell (preferably neuronal progenitor-like cell) is a neuroblastoma cell hybrid (e.g. fusion) (preferably a neuroblastoma-glioma hybrid cell), is immortalized, and is undifferentiated (e.g. when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition).

[0176] The cells (preferably neuronal progenitor-like cells) may be immortalized cell lines (preferably immortalized neuronal progenitor-like cell lines), for example neuroblastoma cells or neuroblastoma-glioma hybrid cells.

[0177] The cells (preferably neuronal progenitor-like cells) may be selected from BE(2)-M17 (ATCC no. CRL-2267), C6-BU-1 (UK Health Security Agency culture collections catalogue no. 08062563), Kelly (NB-19; UK Health Security Agency culture collections catalogue No 92110411), LA1-55n (UK Health Security Agency culture collections catalogue no. 06041203), LA-N-2 (UK Health Security Agency culture collections catalogue no.06041202), IMR-32 (ATCC no. CCL-127; UK Health Security Agency culture collections catalogue no. 86041809), N1 E-115 (ATCC no. CRL-2263; UK Health Security Agency culture collections catalogue no. 88112303), NB41A3 (ATCC no. CCL-147; UK Health Security Agency culture collections catalogue no. 89121405), N4TG3 (UK Health Security Agency culture collections catalogue no. 08062515), N18 (UK Health Security Agency culture collections catalogue no. 88112301); N18TG2 (UK Health Security Agency culture collections catalogue no. 08062523), Neuro-2a (N2a) (ATCC no. CCL-131 ; UK Health Security Agency culture collections catalogue no. 89121404), NG108-15 (ATCC no. HB-12317; UK Health Security Agency culture collections catalogue no.88112302), PC12 (ATCC no. CRL-1721), SH-SY5Y (ATCC no. CRL-2266), SiMa(ATCC no. CRL-3265), and / or SK-N-BE(2)-C (ATCC no.CRL-2271). The cells (preferably neuronal progenitor-like cells) may be neuroblastoma cells e.g. BE(2)-M17, Kelly (NB-19), LA1-55n, N1 E-115, N4TG3, N18, Neuro2a (N2a), IMR-32, N18, SH-SY5Y, SiMa, SK-N-BE(2)- C, SKNSH, NB41A3 and LA-N-2 cells, or neuroblastoma-glioma hybrid cells e.g. NG108-15. Preferably, the cells (preferably neuronal-progenitor-like cells) may be NG108-15 cells. The neuronal progenitor-like cells may be neuroblastoma cells e.g. BE(2)-M17, Kelly (NB-19), LA1- 55n, N1 E-115, N4TG3, N18, Neuro2a (N2a), IMR-32, N18, SH-SY5Y, SiMa, SK-N-BE(2)-C, SKNSH, NB41A3 and LA-N-2 cells, or neuroblastoma-glioma hybrid cells e.g. NG108-15. Preferably, the neuronal-progenitor-like cells may be NG 108- 15 cells. Preferably, the NG IOS- 15 cells may be undifferentiated when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition. Preferably, the NG 108- 15 cells may be partially differentiated when the population of cells are contacted with a ganglioside and / or (more preferably and) when the population of cells are contacted with the composition.

[0178] A cell population of the invention may comprise or consist of neuronal progenitor-like cells.

[0179] Advantageously, the present inventors found that the number or concentration of cells seeded may have an impact on the sensitivity of the method. In other words, the number of cells when first contacted with the ganglioside or composition may positively influence the sensitivity of the method.

[0180] At least 30,000 cells may be seeded (e.g. plated). Preferably, greater than 30,000 cells may be seeded (e.g. plated). Advantageously, the sensitivity of a method of the invention may be further improved in such instances. At least 35,000 cells, at least 40,000 cells, at least 45,000 cells, or at least 50,000 cells may be seeded (e.g. plated). Greater than 30,000 cells, greater than 35,000 cells, greater than 40,000 cells, greater than 45,000 cells or greater than 50,000 cells may be seeded (e.g. plated). Preferably, at least 45,000 cells may be seeded (e.g. plated). <100,000 cells, or <60,000 cells may be seeded (e.g. plated). Preferably, <55,000 cells may be seeded (e.g. plated). 30,000-100,000 cells, 30,000-60,000 cells, or 35,000-55,000 cells may be seeded (e.g. plated). Preferably, 45,000-55,000 cells (more preferably 50,000 cells) may be seeded (e.g. plated).

[0181] At least 30,000 cells / well may be seeded (e.g. plated). Preferably, greater than 30,000 cells / well may be seeded (e.g. plated). Advantageously, the sensitivity of a method of the invention may be further improved in such instances. At least 35,000 cells / well, at least 40,000 cells / well, at least 45,000 cells / well, or at least 50,000 cells / well may be seeded (e.g. plated). Greater than 30,000 cells / well, greater than 35,000 cells / well, greater than 40,000 cells / well, greater than 45,000 cells / well or greater than 50,000 cells / well may be seeded (e.g. plated). Preferably, at least 45,000 cells / well may be seeded (e.g. plated). <100,000 cells / well, or <60,000 cells / well may be seeded (e.g. plated). Preferably, <55,000 cells / well may be seeded (e.g. plated). 30,000-100,000 cells / well, 30,000-60,000 cells / well, or 35,000-55,000 cells / well may be seeded (e.g. plated). Preferably, 45,000-55,000 cells / well (more preferably 50,000 cells / well) may be seeded (e.g. plated).

[0182] The cells may be seeded at a concentration of at least 300,000 cells / ml. Preferably, the cells may be seeded at a concentration of greater than 300,000 cells / ml. Advantageously, the sensitivity of a method of the invention may be further improved in such instances. The cells may be seeded at a concentration of at least 350,000 cells / ml, at least 400,000 cells / ml, at least 450,000 cells / ml, or at least 500,000 cells / ml. The cells may be seeded at a concentration of greater than 300,000 cells / ml, greater than 350,000 cells / ml, greater than 400,000 cells / ml, greater than 450,000 cells / ml or greater than 500,000 cells / ml. Preferably, the cells may be seeded at a concentration of at least 450,000 cells / ml. The cells may be seeded at a concentration of <1 ,000,000 cells / ml, or <600,000 cells / ml. Preferably, the cells may be seeded at a concentration of <550,000 cells / ml. The cells may be seeded at a concentration of 300,000-1 ,000,000 cells / ml, 300,000-600,000 cells / ml, or 350,000-550,000 cells / ml. Preferably, the cells may be seeded at a concentration of 450,000-550,000 cells / ml (more preferably 500,000 cells / ml).

[0183] The population of cells when contacted with a ganglioside may comprise at least 30,000 cells. Preferably, greater than 30,000 cells. Advantageously, the sensitivity of a method of the invention may be further improved in such instances. The population of cells when contacted with a ganglioside may comprise at least 35,000 cells, at least 40,000 cells, at least 45,000 cells, or at least 50,000 cells. The population of cells when contacted with a ganglioside may comprise greater than 30,000 cells, greater than 35,000 cells, greater than 40,000 cells, greater than 45,000 cells or greater than 50,000 cells. Preferably, the population of cells when contacted with a ganglioside may comprise at least 45,000 cells. The population of cells when contacted with a ganglioside may comprise <100,000 cells, or <60,000 cells. Preferably, the population of cells when contacted with a ganglioside may comprise <55,000 cells. The population of cells when contacted with a ganglioside may comprise 30,000-100,000 cells, 30,000-60,000 cells, or 35,000-55,000 cells. Preferably, the population of cells when contacted with a ganglioside may comprise 45,000-55,000 cells (more preferably 50,000 cells).

[0184] The population of cells when contacted with a ganglioside may comprise at least 30,000 cells / well. Preferably, greater than 30,000 cells / well. Advantageously, the sensitivity of a method of the invention may be further improved in such instances. The population of cells when contacted with a ganglioside may comprise at least 35,000 cells / well, at least 40,000 cells / well, at least 45,000 cells / well, or at least 50,000 cells / well. The population of cells when contacted with a ganglioside may comprise greater than 30,000 cells, greater than 35,000 cells / well, greater than 40,000 cells / well, greater than 45,000 cells / well or greater than 50,000 cells / well. Preferably, the population of cells when contacted with a ganglioside may comprise at least 45,000 cells / well. The population of cells when contacted with a ganglioside may comprise <100,000 cells / well, or <60,000 cells / well. Preferably, the population of cells when contacted with a ganglioside may comprise <55,000 cells / well. The population of cells when contacted with a ganglioside may comprise 30,000-100,000 cells / well, 30,000-60,000 cells / well, or 35,000-55,000 cells / well. Preferably, the population of cells when contacted with a ganglioside may comprise 45,000-55,000 cells / well (more preferably 50,000 cells / well).

[0185] The population of cells when contacted with a ganglioside may be at a concentration of at least 300,000 cells / ml. Preferably, at a concentration of greater than 300,000 cells / ml. Advantageously, the sensitivity of a method of the invention may be further improved in such instances. The population of cells when contacted with a ganglioside may be at a concentration of at least 350,000 cells / ml, at least 400,000 cells / ml, at least 450,000 cells / ml, or at least 500,000 cells / ml. The population of cells when contacted with a ganglioside may be at a concentration of greater than 300,000 cells / ml, greater than 350,000 cells / ml, greater than 400,000 cells / ml, greater than 450,000 cells / ml or greater than 500,000 cells / ml. Preferably, the population of cells when contacted with a ganglioside may be at a concentration of at least 450,000 cells / ml. The population of cells when contacted with a ganglioside may be at a concentration of <1 ,000,000 cells / ml, or <600,000 cells / ml. Preferably, the population of cells when contacted with a ganglioside may be at a concentration of <550,000 cells / ml. The population of cells when contacted with a ganglioside may be at a concentration of 300,000- 1 ,000,000 cells / ml, 300,000-600,000 cells / ml, or 350,000-550,000 cells / ml. Preferably, the population of cells when contacted with a ganglioside may be at a concentration of 450,000- 550,000 cells / ml (more preferably 500,000 cells / ml).

[0186] The “population of cells when contacted with a ganglioside” may refer to the point in time when the contacting first occurs.

[0187] The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise at least 30,000 cells. Preferably, greater than 30,000 cells. Advantageously, the sensitivity of a method of the invention may be further improved in such instances. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise at least 35,000 cells, at least 40,000 cells, at least 45,000 cells, or at least 50,000 cells. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise greater than 30,000 cells, greater than 35,000 cells, greater than 40,000 cells, greater than 45,000 cells or greater than 50,000 cells. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise at least 45,000 cells. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise <100,000 cells, or <60,000 cells. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise <55,000 cells. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise 30,000-100,000 cells, 30,000-60,000 cells, or 35,000-55,000 cells. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise 45,000-55,000 cells (more preferably 50,000 cells).

[0188] The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise at least 30,000 cells / well. Preferably, greater than 30,000 cells / well. Advantageously, the sensitivity of a method of the invention may be further improved in such instances. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise at least 35,000 cells / well, at least 40,000 cells / well, at least 45,000 cells / well, or at least 50,000 cells / well. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise greater than 30,000 cells, greater than 35,000 cells / well, greater than 40,000 cells / well, greater than 45,000 cells / well or greater than 50,000 cells / well. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise at least 45,000 cells / well. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise <100,000 cells / well, or <60,000 cells / well. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise <55,000 cells / well. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise 30,000-100,000 cells / well, 30,000- 60,000 cells / well, or 35,000-55,000 cells / well. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may comprise 45, DOO- 55, 000 cells / well (more preferably 50,000 cells / well).

[0189] The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of at least 300,000 cells / ml. Preferably, at a concentration of greater than 300,000 cells / ml. Advantageously, the sensitivity of a method of the invention may be further improved in such instances. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of at least 350,000 cells / ml, at least 400,000 cells / ml, at least 450,000 cells / ml, or at least 500,000 cells / ml. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of greater than 300,000 cells, greater than 350,000 cells / ml, greater than 400,000 cells / ml, greater than 450,000 cells / ml or greater than 500,000 cells / ml. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of at least 450,000 cells / ml . The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of <1 ,000,000 cells / ml, or <600,000 cells / ml. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of <550,000 cells / ml. The population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of 300,000-1 ,000,000 cells / ml, 300,000-600,000 cells / ml, or 350,000-550,000 cells / ml. Preferably, the population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin) may be at a concentration of 450,000-550,000 cells / ml (more preferably 500,000 cells / ml).

[0190] The “population of cells when contacted with a composition (e.g. comprising a clostridial neurotoxin)” may refer to the point in time when the contacting first occurs.

[0191] The foregoing paragraphs preferably define the number or concentration of cells when contacted with a ganglioside and when contacted with a composition (e.g. comprising a clostridial neurotoxin). The number or concentration of cells may be the same or similar during both contacting steps.

[0192] The number of cells and / or concentration of cells can be calculated using any standard method in the art, for example, using Vi-CELL or manually counting using a hemocytometer. Once the cells are counted, they may be diluted to the desired number or concentration (where necessary) and then allowed to settle in the cell culture vessel (e.g. a cell culture flask or well within a multi-well plate). The cells can then be incubated prior to any contacting steps (e.g. contacting with a ganglioside and / or composition). The cells may be incubated for at least 20 minutes, 40 minutes, 50 minutes, 60 minutes, 65 minutes, 70 minutes, 80 minutes, 85 minutes, 90 minutes, 2 hours, 4 hours, 6 hours, 8 hours or 10 hours prior to a contacting step (e.g. contacting with a ganglioside and / or composition). The incubation step may be 20 minutes to 10 hours, 30 minutes to 5 hours, or 40 minutes to 2 hours, preferably 50 minutes to 100 minutes, more preferably 60-90 minutes. In one embodiment, the cells may not be incubated for more than 24 hours prior to a contacting step (e.g. contacting with a ganglioside and / or composition). In one embodiment, the cells may not be incubated for more than 95 minutes prior to contacting with a ganglioside. In one embodiment, the cells may not be incubated for more than 24 hours prior to contacting with a composition. The cells may be incubated in a neural differentiation media for no more than 24 hours prior to a contacting step. In one embodiment, the cells may not be incubated in a neural differentiation media for more than 95 minutes prior to contacting with a ganglioside. In one embodiment, the cells may not be incubated in a neural differentiation media for more than 24 hours prior to contacting with a composition. The cells may be incubated at any suitable temperature. In one embodiment, the population of cells are incubated at 15-40°C, 20°C-40°C, 25-40°C, 30-40°C or 35-40°C prior to any contacting steps. Preferably, the population of cells are incubated at 37°C ±2°C prior to any contacting steps.

[0193] In the context of cell number or concentration, the specific value or range provided may vary to a certain extent to encompass dilution variations when preparing and plating cells. For example, at least 300,000 cells / ml may encompass 295,000 cells / ml or 305,000 cells / ml. In other words, the values recited may encompass somewhat more or somewhat less that the stated numerical amount or range to a deviation of up to 5%, 4%, 3%, 2%, or 1%. The deviation may be less than 1%.

[0194] The population of cells may be grown in any suitable cell culture vessel e.g. a cell culture flask or a multi-well plate. To aid use in high-throughput testing a multi-well plate may be used. Any suitable multi-well plate may be used in the methods of the invention. Preferably, clear bottomed multi-well plates may be used.

[0195] The population of cells used in the methods of the invention express a clostridial neurotoxin receptor and / or a ganglioside. Preferably, said cells express at least a clostridial neurotoxin receptor. The term “cells express a clostridial neurotoxin receptor and / or a ganglioside” may encompass cells which are expressing or over expressing a clostridial neurotoxin receptor and / or a ganglioside, as well as cells which have previously expressed or over expressed a clostridial neurotoxin receptor and / or a ganglioside. The cell may normally (e.g. naturally) express a clostridial neurotoxin receptor and / or a ganglioside. In other words, the clostridial neurotoxin and / or ganglioside may be under the expression of a normal (wild type) promoter and may be expressed at normal (e.g. wild type or physiological levels) for the specified cell type. In one embodiment, the cells may not be genetically modified to express or over express (e.g. express above / higher than wild type levels) a clostridial neurotoxin receptor and / or ganglioside. For example, the cells may not be genetically modified to express or over express (e.g. express above / higher than wild type levels) a SV2a, SV2c and / or SYT-II clostridial neurotoxin receptor and / or GD3 ganglioside. Preferably, the cells may not be genetically modified to over express (e.g. express above / higher than wild type levels) a clostridial neurotoxin receptor and / or ganglioside. For example, when the cell is NG108-15, the clostridial neurotoxin receptor and / or ganglioside may be expressed at the levels which are considered normal (e.g. wild type or physiological levels) for unmodified NG108-15 cells (preferably expressed at the levels which are considered normal for undifferentiated NG 108-15 cells). Preferably, the NG 108-15 cells may not be genetically modified to over express (e.g. express above / higher than wild type levels) a clostridial neurotoxin receptor and / or ganglioside. Alternatively, the cell may be genetically engineered to express a clostridial neurotoxin receptor and / or a ganglioside, for example a nucleic acid may be introduced into the cell which encodes for a clostridial neurotoxin and / or a ganglioside. The nucleic acid may be introduced into the cell by any means known in the art. For example, it may be present in a vector, such as a plasmid, which is used to introduce the nucleic acid into a cell.

[0196] In one embodiment, a cell that expresses a clostridial neurotoxin receptor and / or a ganglioside as defined herein may not continuously express said clostridial neurotoxin receptor and / or ganglioside, e.g. throughout an entire method of the invention. However, the cell preferably expresses said clostridial neurotoxin receptor and / or ganglioside at least when the cell is contacted with a composition (e.g. comprising a clostridial neurotoxin) as described herein. The cell preferably expresses said clostridial neurotoxin receptor and / or ganglioside at least when the cell is contacted with a ganglioside as described herein. Preferably, the cell continuously expresses said clostridial neurotoxin receptor and / or ganglioside.

[0197] The term “clostridial neurotoxin receptor” may encompass a full-length clostridial neurotoxin receptor or a portion thereof. The clostridial neurotoxin receptor may be a neuronal clostridial neurotoxin receptor polypeptide, such as a full-length neuronal clostridial neurotoxin receptor polypeptide or a portion thereof. The clostridial neurotoxin receptor may comprise (or consist of) an extracellular portion of a neuronal clostridial neurotoxin receptor. The clostridial neurotoxin receptor may comprise (or consist of) an extracellular portion of a neuronal botulinum neurotoxin receptor. Thus, when the population of cells expresses a clostridial neurotoxin receptor, the receptor may be a full-length clostridial neurotoxin receptor, or a fragment thereof to which a clostridial neurotoxin binds. Preferably, the clostridial neurotoxin receptor comprises a full-length clostridial neurotoxin receptor, more preferably comprises a full-length botulinum neurotoxin receptor.

[0198] A clostridial neurotoxin receptor may be a human neuronal clostridial neurotoxin receptor (e.g. an extracellular portion thereof). A neuronal clostridial neurotoxin receptor may be synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), or synaptotagmin II (SYT-II). A neuronal clostridial neurotoxin receptor may comprise (or consists of) an extracellular portion of SV2a, SV2b, SV2c, SYT-I, or SYT-II. Preferably, a neuronal clostridial neurotoxin receptor comprises (or consists of) the full length SV2a, SV2b, SV2c, SYT-I, or SYT-II. The polypeptide sequence of the neuronal clostridial neurotoxin receptor (e.g. full length receptor) may be the same as the wild-type (e.g. human) polypeptide sequence. In another embodiment, the polypeptide of the neuronal clostridial neurotoxin receptor (e.g. full length receptor) may comprise one or more modifications when compared to the wild-type (e.g. human) polypeptide sequence.

[0199] A clostridial neurotoxin receptor may comprise a post-translational modification, such as a glycosylation. A clostridial neurotoxin receptor may be glycosylated. The glycosylation may be N-linked glycosylation.

[0200] A full-length human SYT-II may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 18. In one embodiment, a full-length human SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 18. Preferably, a full-length human SYT-II may comprise SEQ ID NO: 18. A full-length human SYT-II may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 18. In one embodiment, a full-length human SYT- II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 18. Preferably, a full-length human SYT-II may consist of SEQ ID NO: 18.

[0201] A full-length mouse SYT-II may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 19. In one embodiment, a full-length mouse SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 19. Preferably, a full-length mouse SYT-II may comprise SEQ ID NO: 19. A full-length mouse SYT-II may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 19. In one embodiment, a full-length mouse SYT- II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 19. Preferably, a full-length mouse SYT-II may consist of SEQ ID NO: 19.

[0202] A full-length rat SYT-II may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 74. In one embodiment, a full-length rat SYT-II may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 74. Preferably, a full-length rat SYT-II may comprise SEQ ID NO: 74. A full- length rat SYT-II may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 74. In one embodiment, a full-length rat SYT-II may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 74. Preferably, a full-length rat SYT-II may consist of SEQ ID NO: 74.

[0203] The SYT-II is preferably rodent SYT-II, such as mouse or rat SYT-II.

[0204] A full-length mouse SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 69. In one embodiment, a full-length mouse SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 69. Preferably, a full-length mouse SYT-I may comprise SEQ ID NO: 69. A full-length mouse SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 69. In one embodiment, a full-length mouse SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 69. Preferably, a full-length mouse SYT-I may consist of SEQ ID NO: 69.

[0205] A full-length rat SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 75. In one embodiment, a full-length rat SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 75. Preferably, a full-length rat SYT-I may comprise SEQ ID NO: 75. A full- length rat SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 75. In one embodiment, a full-length rat SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 75. Preferably, a full-length rat SYT-I may consist of SEQ ID NO: 75.

[0206] The SYT-I is preferably rodent SYT-I, such as mouse or rat. A full-length human SYT-I may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 23. In one embodiment, a full-length human SYT-I may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 23. Preferably, a full-length human SYT-I may comprise SEQ ID NO: 23. A full-length human SYT-I may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 23. In one embodiment, a full-length human SYT-I may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 23. Preferably, a full-length human SYT-I may consist of SEQ ID NO: 23.

[0207] A full-length human SV2a may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 20. In one embodiment, a full-length human SV2a may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 20. Preferably, a full-length human SV2a may comprise SEQ ID NO: 20. A full-length human SV2a may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 20. In one embodiment, a full-length human SV2a may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 20. Preferably, a full-length human SV2a may consist of SEQ ID NO: 20.

[0208] A full-length human SV2b may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 21. In one embodiment, a full-length human SV2b may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 21 . Preferably, a full-length human SV2b may comprise SEQ ID NO: 21. A full-length human SV2b may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 21 . In one embodiment, a full-length human SV2b may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 21. Preferably, a full-length human SV2b may consist of SEQ ID NO: 21.

[0209] A full-length human SV2c may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 22. In one embodiment, a full-length human SV2c may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22. Preferably, a full-length human SV2c may comprise SEQ ID NO: 22. A full-length human SV2c may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 22. In one embodiment, a full-length human SV2c may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22. Preferably, a full-length human SV2c may consist of SEQ ID NO: 22.

[0210] The SV2c is preferably rodent SV2c, such as mouse or rat.

[0211] A full-length mouse SV2c may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 78. In one embodiment, a full-length mouse SV2c may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 78. Preferably, a full-length mouse SV2c may comprise SEQ ID NO: 78. A full-length mouse SV2c may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 78. In one embodiment, a full-length mouse SV2c may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 78. Preferably, a full-length mouse SV2c may consist of SEQ ID NO: 78.

[0212] A full-length rat SV2c may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 79. In one embodiment, a full-length rat SV2c may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 79. Preferably, a full-length rat SV2c may comprise SEQ ID NO: 79. A full- length rat SV2c may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 79. In one embodiment, a full-length rat SV2c may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 79. Preferably, a full-length rat SV2c may consist of SEQ ID NO: 79.

[0213] In one embodiment, the population of cells (preferably neuronal progenitor-like cells), preferably NG108-15 cells, may express a clostridial neurotoxin receptor (e.g. SYT-I, SYT-II, and / or SV2c). In another embodiment, the population of cells (preferably neuronal progenitorlike cells), preferably NG 108-15 cells, may express a ganglioside. In another embodiment, the cells may express a clostridial neurotoxin receptor and a ganglioside. The cells are preferably not modified, e.g. genetically engineered, to express a clostridial neurotoxin receptor, more preferably SV2a, SV2c and / or SYT-II.

[0214] The population of cells may express a ganglioside selected from GM1 (e.g. GM 1 a or GM 1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1 a, GD1 b, GalNAc-GD1 a, GT1 a, GT 1 b, GQ1 b, GD2, GD3 or a combination thereof. The population of cells may express at least one ganglioside selected from GM1 (e.g. GM1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, and GD3. The population of cells may express one or more of the following gangliosides GT1 b, GM3, GM2, GT1a, GD1 a and GM1. Preferably the population of cells express at least GT1 b, e.g. in some instances only GT 1 b. Most preferably the population of cells express at least GM3, GM2, GM1 and GD1a (e.g. at natural / wild type levels).

[0215] When the population of cells (preferably neuronal progenitor-like cells) express a ganglioside, the ganglioside contacted with the population of cells may be the same as the ganglioside expressed by the cells. In another embodiment, the ganglioside contacted with the population of cells may be different to the ganglioside expressed by the cells. Preferably, the population of cells express at least GT 1 b and the population of cells may also be contacted with at least GT1 b. Most preferably, the population of cells express at least GM3, GM2, GM1 and GD1a (e.g. at natural / wild type levels) and the population of cells may also be contacted with at least GT1 b.

[0216] A cell (preferably neuronal progenitor-like cell) according to the invention preferably does not express GT1 b.

[0217] The population of cells (preferably neuronal progenitor-like cells) may express a clostridial neurotoxin receptor selected from synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), synaptotagmin II (SYT-II) or a combination thereof. The population of cells may express at least one clostridial neurotoxin receptor selected from synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), or synaptotagmin II (SYT-II). The population of cells may express at least two clostridial neurotoxin receptor selected from synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), or synaptotagmin II (SYT-II). Preferably, the clostridial neurotoxin receptor may be SV2a, SV2c and / or SYT-II. When the cells express SV2c or SYT- II, the cells may not be genetically engineered to express the receptors e.g. the receptors may be expressed at normal (e.g. wild type or physiological levels) for the cell type.

[0218] The population of cells (preferably neuronal progenitor-like cells) may express a clostridial neurotoxin receptor selected from synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), synaptotagmin II (SYT-II) or a combination thereof; and may also express a ganglioside selected from GM1 (e.g. GM 1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, GD3 or a combination thereof. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may express at least one clostridial neurotoxin receptor selected from synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), or synaptotagmin II (SYT- II); and may also express at least one ganglioside selected from GM1 (e.g. GM1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, or GD3. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may express at least two clostridial neurotoxin receptors selected from synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), orsynaptotagmin II (SYT-II); and may also express at least one ganglioside selected from GM1 (e.g. GM1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, or GD3. Preferably, the population of cells (preferably neuronal progenitor-like cells) may express SV2c and / or SYT-II at normal (e.g. wild type or physiological) levels for the cell type and may also express (e.g., either at normal or overexpressed levels) at least GT1 b. Most preferably, the population of cells (preferably neuronal progenitor-like cells) may express SV2c and / or SYT-II at normal (e.g. wild type or physiological) levels for the cell type, GM3, GM2, GM1 and GD1a at normal (e.g. wild type or physiological) levels and may also express (e.g., either at normal or overexpressed levels) at least GT1 b.

[0219] Where the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / A Hcc (preferably Hc) domain, the population of cells may express a clostridial neurotoxin receptor selected from SV2c, SV2a, or SV2b, preferably SV2c. There are three SV2 isoforms found in humans, SV2a, SV2b, and SV2c however BoNT / A has the greatest affinity for SV2c. BoNT / A may bind specifically to the luminal domain 4 of SV2 (SV2-LD4) via direct backbone-backbone interactions between a p-strand of SV2-LD4 and a p-strand of BoNT Hc, and also through interactions with an N559-linked glycan.

[0220] Where the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / B Hcc (preferably Hc) domain, the population of cells may express a clostridial neurotoxin receptor selected from SYT-II and / or SYT-I, preferably SYT-II. While BoNT / B binds to SYT-I and SYT-II, SYT-II is believed to be more abundant on human neuronal cells. Therefore, SYT-II may be a preferred neuronal clostridial neurotoxin receptor polypeptide for use in the present invention. Where the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / D Hcc (preferably Hc) domain, the population of cells may express a clostridial neurotoxin receptor selected from SV2c, SV2a, or SV2b.

[0221] Where the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / E Hcc (preferably Hc) domain, the population of cells may express a clostridial neurotoxin receptor selected from SV2a, SV2b or SV2c, preferably SV2a.

[0222] Where the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / F Hcc (preferably Hc) domain, the population of cells may express a clostridial neurotoxin receptor selected from SV2c, SV2a, or SV2b.

[0223] Where the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / G Hcc (preferably Hc) domain, the population of cells may express a clostridial neurotoxin receptor selected from SYT-II or SYT-I, preferably SYT-II.

[0224] Methods of the invention preferably detect clostridial neurotoxin activity of a composition or the presence or absence of a clostridial neurotoxin polypeptide through the use a functional luciferase expressed by the population of cells.

[0225] A cell that expresses a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain, as defined herein may not continuously express the same, e.g. throughout an entire method of the invention. However, the cell preferably expresses said single-chain polypeptide at least when the cell is contacted with a composition (e.g. comprising a clostridial neurotoxin) as described herein.

[0226] The population of cells preferably express a single-chain polypeptide comprising: (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase. The term “luciferase” as used herein refers to an enzyme that catalyses a bioluminescent reaction, e.g., by catalysing the oxidation of luciferin, emitting light and releasing oxyluciferin. Examples of luciferases include Nanoluc (SEQ ID NO: 1), firefly luciferase (e.g. Photinus pyralis luciferase), bacterial luciferase (e.g. Vibrio fischieri or Vibrio harveyi luciferase), sea pansy luciferase (e.g. Renilla reniformis luciferase), dinoflagellate luciferase, Gaussia luciferase, and copepod luciferase. Other luciferases are described US Patent No. 8,557,970, which is incorporated herein by reference. A “functional” luciferase as used herein is a luciferase that is capable of catalysing a reaction in the presence of a suitable substrate.

[0227] When the linker is intact (i.e. wherein the clostridial neurotoxin cleavage site has not been cleaved by a clostridial neurotoxin), the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase (i.e. the luciferase is capable of luciferase activity). When the linker has been cleaved (i.e. wherein the clostridial neurotoxin cleavage site has been cleaved by a clostridial neurotoxin), the linker no longer functionally joins the first and second luciferase domains, resulting in a loss of luciferase activity (i.e. the luciferase is not capable of luciferase activity). The term “single-chain” when used in the context of the functional luciferase may refer to a single polypeptide molecule having a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. In other words, each recited element of the single-chain polypeptide may be connected to the other element(s) by means of a peptide bond.

[0228] The term “functionally join” as used in the context of first and second luciferase domains refers to joining the first and second luciferase domains in such a way that any intervening sequence does not prevent the two fragments from forming an active and functional tertiary structure. The first and second luciferase domains when functionally joined exhibit luciferase activity, e.g. as they would in the absence of the linker.

[0229] The first luciferase domain is preferably N-terminal to the linker comprising the clostridial neurotoxin cleavage site. Preferably, the first luciferase domain is the most N-terminal element of the cleavable substrate. A first luciferase domain may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2. In one embodiment, a first luciferase domain may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 2. Preferably, a first luciferase domain may comprise SEQ ID NO: 2. A first luciferase domain may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2. In one embodiment, a first luciferase domain may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 2. Preferably, a first luciferase domain may consist of SEQ ID NO: 2.

[0230] The second luciferase domain is preferably C-terminal to the linker comprising the clostridial neurotoxin cleavage site. Preferably, the second luciferase domain is the most C-terminal element of the cleavable substrate. A second luciferase domain may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 3. In one embodiment, a second luciferase domain may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 3. Preferably, a second luciferase domain may comprise SEQ ID NO: 3. A second luciferase domain may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 3. In one embodiment, a second luciferase domain may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 3. Preferably, a second luciferase domain may consist of SEQ ID NO: 3.

[0231] The linker may consist of a clostridial neurotoxin cleavage site. However, it is preferred that the linker further comprises one or more spacers. Most preferably the linker comprises (even more preferably consists of) a first spacer N-terminal to the clostridial neurotoxin cleavage site and a second spacer C-terminal to the clostridial neurotoxin cleavage site. Where more than one spacer is present, the spacers may have the same or different polypeptide sequences (preferably the same polypeptide sequences).

[0232] It is well-within the skilled person’s capability to select an appropriate spacer sequence and size. A spacer may be of any suitable length, such as 3-20, 2-15, 5-15 or 4-8 amino acids in length. A spacer may be 15-20 amino acids in length, e.g. 16-19 amino acids in length. A spacer may comprise (or consist of) glycine and serine residues. In particular, a spacer may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 32. In one embodiment, a spacer may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 32. Preferably, a spacer may comprise SEQ ID NO: 32. In particular, a spacer may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 32. In one embodiment, a spacer may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 32. Preferably, a spacer may consist of SEQ ID NO: 32.

[0233] A spacer may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 72. In one embodiment, a spacer may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 72. Preferably, a spacer may comprise SEQ ID NO: 72. In particular, a spacer may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 72. In one embodiment, a spacer may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 72. Preferably, a spacer may consist of SEQ ID NO: 72.

[0234] A spacer may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 73. In one embodiment, a spacer may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 73. Preferably, a spacer may comprise SEQ ID NO: 73. In particular, a spacer may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 73. In one embodiment, a spacer may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 73. Preferably, a spacer may consist of SEQ ID NO: 73.

[0235] A first and second spacer may be present in a single-chain polypeptide of the invention. The first and second spacers may have the same polypeptide sequence. Preferably, the first and second spaces have a different polypeptide sequence.

[0236] The first spacer may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 72. In one embodiment, the first spacer may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 72. Preferably, the first spacer may comprise SEQ ID NO: 72. In particular, the first spacer may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 72. In one embodiment, the first spacer may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 72. Preferably, the first spacer may consist of SEQ ID NO: 72.

[0237] The second spacer may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 73. In one embodiment, the second spacer may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 73. Preferably, the second spacer may comprise SEQ ID NO: 73. In particular, the second spacer may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 73. In one embodiment, the second spacer may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 73. Preferably, the second spacer may consist of SEQ ID NO: 73.

[0238] Most preferably, the first spacer may consist of SEQ ID NO: 72 and the second spacer may consist of SEQ ID NO: 73. Any suitable clostridial neurotoxin cleavage site may be used e.g. a scissile bond of a SNARE. Suitable clostridial neurotoxin cleavage sites and functional luciferases may be those described in WO 2018 / 075783 A2, which is incorporated herein by reference.

[0239] The clostridial neurotoxin cleavage site may further comprise a number of amino acids N- terminal and / or C-terminal to the scissile bond in a SNARE, preferably N-terminal and C- terminal. Thus, a clostridial neurotoxin cleavage site may comprise at least amino acid residues P3P2P1 P1 ’P2’P3’ of a SNARE, wherein P1 and P1’ are the residues located N- terminal and C-terminal to the scissile bond (scissile peptide bond) cleaved by the clostridial neurotoxin, such as Gln197 and Arg198 of SNAP-25 cleaved by an L-chain of BoNT / A.

[0240] Thus, the clostridial neurotoxin cleavage site may further comprise at least 2 (e.g. the cleavage site comprises at least P3P2P1 P1 ’), 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues N- terminal of the amino acid residues forming a scissile bond in a SNARE. The clostridial neurotoxin cleavage site may further comprise at least 2 (e.g. the cleavage site comprises at least P1 P1 ’P2’P3’), 5, 10, 15, 20, 25, 30, 40, 50, or 100 amino acid residues C-terminal of the of the amino acid residues forming a scissile bond in a SNARE. For example, the clostridial neurotoxin cleavage site may further comprise: up to 5 amino acid residues N-terminal and up to 5 amino acid residues C-terminal of the amino acid residues forming a scissile bond in the SNARE; up to 10 amino acid residues N-terminal and up to 10 amino acid residues C-terminal of the amino acid residues forming a scissile bond in the SNARE; up to 25 amino acid residues N-terminal and up to 25 amino acid residues C-terminal of the amino acid residues forming a scissile bond in the SNARE; up to 50 amino acid residues N-terminal and up to 50 amino acid residues C-terminal of the amino acid residues forming a scissile bond in the SNARE; or up to 100 amino acid residues N-terminal and up to 100 amino acid residues C-terminal of the amino acid residues forming a scissile bond in the SNARE. For the avoidance of doubt, the term “up to” as used in this context encompasses the number indicated, e.g. “up to 5 amino acid residues” encompasses “5 amino acid residues”.

[0241] Preferably, a clostridial neurotoxin cleavage site comprises 8 amino acid residues C-terminal of the amino acid residues forming a scissile bond in the SNARE and up to 55 amino acid residues N-terminal of the amino acid residues forming a scissile bond in the SNARE (e.g. a clostridial neurotoxin cleavage site may comprise up to a 65 amino acid portion of a SNARE). A clostridial neurotoxin cleavage site comprising a scissile bond of a SNARE may comprise (or consist of) at least 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues of a SNARE as described herein, preferably at least 55 amino acid residues of a SNARE as described herein. A clostridial neurotoxin cleavage site comprising a scissile bond of a SNARE may comprise (or consist of) <210, <150, <100, <90, <80, <70, <60, <50, <40, <30, <20, <10, <9, <8, or <7 amino acid residues of a SNARE as described herein. For example, a clostridial neurotoxin cleavage site comprising a scissile bond of a SNARE may comprise (or consist of) 6-210, 190-210, 10-150, 20-100, 50-85, 55-80 or 60-75 amino acid residues of a SNARE as described herein. Preferably, a clostridial neurotoxin cleavage site comprising a scissile bond of a SNARE may comprise (or consist of) 60-70 amino acid residues of a SNARE as described herein. More preferably, a clostridial neurotoxin cleavage site comprising a scissile bond of a SNARE may comprise (or consist of) 190-210 amino acid residues of a SNARE as described herein.

[0242] Thus, in some embodiments, the clostridial neurotoxin cleavage site is from a SNARE (e.g. is a fragment of a SNARE). A SNARE may be a SNAP-25, a synaptobrevin (VAMP), or a Syntaxin. Thus, a clostridial neurotoxin cleavage site may comprise a scissile bond of a SNAP- 25, a synaptobrevin (VAMP), or a Syntaxin.

[0243] A SNARE for use in the invention is preferably a SNAP-25. Examples of SNAP-25 polypeptides include NCBI Gene ID: 6616, NCBI Reference Sequence: NP 570824.1 (human SNAP25b) and NCBI Reference Sequence: NP_112253 .1 (rat SNAP25b). It is preferred that the SNAP-25 is human SNAP-25. SNAP-25 may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4. In one embodiment, a SNAP-25 may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 4. Preferably, a SNAP-25 may comprise SEQ ID NO: 4. SNAP-25 may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4. In one embodiment, a SNAP-25 may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 4. Preferably, a SNAP-25 may consist of SEQ ID NO: 4.

[0244] A scissile bond of SNAP-25 (SEQ ID NO: 4) cleaved by an L-chain of BoNT / A may be Gln197- Arg198. A scissile bond of SNAP-25 (SEQ ID NO: 4) cleaved by an L-chain of BoNT / C1 may be Arg198-Ala199. A scissile bond of SNAP-25 (SEQ ID NO: 4) cleaved by an L-chain of BoNT / E may be Arg180-lle181. A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 4. Preferably, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise SEQ ID NO: 4. A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 4. Preferably, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of SEQ ID NO: 4. Preferably, the N-terminal methionine residue (residue 1) of SEQ ID NO: 4 is not present when the clostridial neurotoxin cleavage site is comprised in a single chain polypeptide of the invention.

[0245] A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 5. Preferably, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may comprise SEQ ID NO: 5. A clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5. In one embodiment, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 5. Preferably, a clostridial neurotoxin cleavage site comprising a SNAP-25 scissile bond may consist of SEQ ID NO: 5.

[0246] A SNARE may be a VAMP. A VAMP may be a VAMP1 , VAMP2, VAMP3, VAMP4, VAMP5, or YKT6. The VAMP may be a human VAMP. Exemplary VAMP polypeptide sequences are shown in the table below. Thus, a VAMP may comprise a polypeptide sequence having at least 70% sequence identity to any of SEQ ID NOs: 24-29. In one embodiment, a VAMP may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any of SEQ ID NOs: 24-29. Preferably, a VAMP may comprise any of SEQ ID NOs: 24-29. Thus, a VAMP may consist of a polypeptide sequence having at least 70% sequence identity to any of SEQ ID NOs: 24-29. In one embodiment, a VAMP may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any of SEQ ID NOs: 24-29. Preferably, a VAMP may consist of any of SEQ ID NOs: 24-29.

[0247] VAMP scissile bonds cleaved by L-chains of the clostridial neurotoxins indicated are shown in the table below:

[0248] Human VAMP1 , VAMP2, and VAMP3 may be cleaved by an L-chain of BoNT / B, BoNT / D, BoNT / F, BoNT / G, BoNT / X, and TeNT. VAMP4, VAMP5, and YKT6 may be cleaved by an L- chain of BoNT / X.

[0249] A SNARE may be a Syntaxin. A Syntaxin may be Syntaxin 1A or Syntaxin 1 B.

[0250] Syntaxin 1 A may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 30. In one embodiment, Syntaxin 1A may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 30. Preferably, Syntaxin 1A may comprise SEQ ID NO: 30. Syntaxin 1A may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 30. In one embodiment, Syntaxin 1A may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 30. Preferably, Syntaxin 1A may consist of SEQ ID NO: 30. Syntaxin 1 B may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 31. In one embodiment, Syntaxin 1 B may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 31. Preferably, Syntaxin 1 B may comprise SEQ ID NO: 31. Syntaxin 1 B may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 31. In one embodiment, Syntaxin 1 B may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 31. Preferably, Syntaxin 1 B may consist of SEQ ID NO: 31 .

[0251] Syntaxin 1A and Syntaxin 1 B may be cleaved by BoNT / C1. A scissile bond of Syntaxin 1A (SEQ ID NO: 30) cleaved by an L-chain of BoNT / C1 may be Lys253-Ala254. A scissile bond of Syntaxin 1 B (SEQ ID NO: 31) cleaved by an L-chain of BoNT / C1 may be Lys252-Ala253.

[0252] In embodiments where a polypeptide sequence of a clostridial neurotoxin cleavage site varies from a recited SNARE polypeptide sequence (SEQ ID NO) by way of sequence identity, said clostridial neurotoxin cleavage site still comprises the relevant scissile bond of said SNARE polypeptide sequence.

[0253] Preferably, a single-chain polypeptide of the invention comprises (from N-terminus to C- terminus): (i) a first luciferase domain; (ii) a first spacer; (iii) a clostridial neurotoxin cleavage site; (iv) a second spacer; and (v) a second luciferase domain. More preferably, a single chain polypeptide of the invention may consist of (from N-terminus to C-terminus): (i) a first luciferase domain; (ii) a first spacer; (iii) a clostridial neurotoxin cleavage site; (iv) a second spacer; and (v) a second luciferase domain. In such embodiments, the linker is formed of elements (ii), (iii), and (iv). The first and second spacers may have the same polypeptide sequence. Preferably, the first and second spaces have a different polypeptide sequence.

[0254] A functional luciferase may comprise (from N-terminus to C-terminus) (i) a first luciferase domain; (ii) a linker comprising a SNAP-25 clostridial neurotoxin cleavage site; and (iii) a second luciferase domain.

[0255] A functional luciferase may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. In one embodiment, a functional luciferase may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 6. In one embodiment, a functional luciferase may comprise SEQ ID NO: 6. A functional luciferase may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. In one embodiment, a functional luciferase may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 6. In one embodiment, a functional luciferase may consist of SEQ ID NO: 6.

[0256] A functional luciferase may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 70. In one embodiment, a functional luciferase may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 70. Most preferably, a functional luciferase may comprise SEQ ID NO: 70.

[0257] A functional luciferase may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 70. In one embodiment, a functional luciferase may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 70. Most preferably, a functional luciferase may consist of SEQ ID NO: 70.

[0258] A functional luciferase may comprise a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 71. In one embodiment, a functional luciferase may comprise a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 71. Most preferably, a functional luciferase may comprise a polypeptide sequence encoded by SEQ ID NO: 71.

[0259] A functional luciferase may consist of a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 71. In one embodiment, a functional luciferase may consist of a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 71. Most preferably, a functional luciferase may consist of a polypeptide sequence encoded by SEQ ID NO: 71.

[0260] The method comprises determining the clostridial neurotoxin activity of the composition or the presence and absence of a clostridial neurotoxin by measuring the luciferase activity. The luciferase activity may be the luciferase activity within the cells of the population (e.g. intact cells of the population). In such instances, a cell-permeable luciferase substrate may be added to the cells. A suitable luciferase substrate may be one described in EP2635582. The luciferase substrate may be in a precursor form that is processed by cellular esterases to produce a luciferase substrate. Suitable products include NanoBRET™ Nano-Gio® Substrate (Cat# N2160) and Nano-Gio® Vivazine™ Substrate (Cat# N2200), both of which are commercially available from Promega. Other suitable luciferase substrates are described in Craig et al (1991), Biochem J, 276, 637-641.

[0261] Preferably, the method comprises determining the clostridial neurotoxin activity of the composition or the presence and absence of a clostridial neurotoxin by measuring the luciferase activity of the cell lysate. This may be achieved by adding a suitable luciferase substrate before, during or after lysing the cells. However, it is preferred that the luciferase substrate is added during or after lysing the cells. In other words, a suitable luciferase substrate may be added during lysing the cells or to the cell lysate after the cells have been lysed. Most preferably said luciferase substrate is added after a lysis buffer is added to the cells. Preferably, the luciferase substrate is furimazine (2-furanylmethyl-deoxy-coelenterazine) or an analogue thereof.

[0262] A luciferase activity value (e.g. of the cell lysate) may be determined by measuring luminescence. For example, furimazine may be added (preferably to the cell lysate) at a concentration of 1-100 pM, preferably 10-50 pM (e.g. 37.5pM). Luminescence may be measured using any suitable technique, such as an automated plate reader (e.g. a BMG Labtech CLARIOstar plate reader).

[0263] The methods of the invention may comprise determining a measured luciferase activity value (e.g. determined by luminescence). In some embodiments, said measured luciferase activity value (e.g. luminescence value) is compared to a control value.

[0264] A suitable control may be a negative control. The negative control may be provided by carrying out the method in the same way but wherein the composition tested does not comprise clostridial neurotoxin polypeptides. The negative control may be a negative reference standard. The negative reference standard may correspond to a value that has been either theoretically or experimentally determined and which represents a negative result in a method of the invention. The value may have been determined prior to carrying out a method of the invention or may be determined simultaneously with, or subsequent to, carrying out a method of the invention.

[0265] The method may comprise comparing the luciferase activity (e.g. of the cell lysate) with a luciferase activity of a negative control in which the population of cells has been contacted with a composition that does not comprise a clostridial neurotoxin, or in which the population of cells has not been contacted with a composition. The method may comprise comparing the measured luciferase activity value with a luciferase activity value of a negative control in which the population of cells has been contacted with a composition that does not comprise a clostridial neurotoxin, or in which the population of cells has not been contacted with a composition.

[0266] When the control is a negative control, a lower level of luminescence when compared to the negative control indicates that a composition tested comprises clostridial neurotoxin polypeptides. In such instances, this may allow for a determination to made that a composition comprises clostridial neurotoxin polypeptides. Clostridial neurotoxin activity may be determined to be present when the luciferase activity (e.g. luminescence) is less than the luciferase activity of the negative control. Clostridial neurotoxin activity may be determined to be present when the measured luciferase activity value (e.g. luminescence) is less than the luciferase activity value of the negative control. Clostridial neurotoxin activity is determined to be absent when the luciferase activity is the same or greater than the luciferase activity of the negative control. Clostridial neurotoxin activity is determined to be absent when the measured luciferase activity value is the same or greater than the luciferase activity value of the negative control.

[0267] In one embodiment, when the control is a negative control, the same luminescence when compared to the negative control indicates that a composition tested does not comprise clostridial neurotoxin polypeptides (e.g., clostridial neurotoxin polypeptides are absent from the composition). In such instances, this may allow for a determination to made that a composition does not comprise clostridial neurotoxin polypeptides (e.g., clostridial neurotoxin polypeptides are absent from the composition).

[0268] In one embodiment, when the control is a negative control, a lower level of luminescence when compared to the negative control indicates that a composition tested comprises active clostridial neurotoxin polypeptides (e.g., clostridial neurotoxin polypeptides are present in the composition). The extent by which the level is lower may be quantified to indicate the activity level of the composition comprising clostridial neurotoxin polypeptides. Preferably, the lower the level of luminescence compared to the negative control, the higher the number of active clostridial neurotoxin polypeptides. In one embodiment, when the control is a negative control, the same luminescence when compared to the negative control may indicate that a composition tested comprises inactive clostridial neurotoxin polypeptides.

[0269] A suitable control may be a positive control. The positive control may be provided by carrying out the method in the same way but wherein the composition comprises clostridial neurotoxin polypeptides, preferably associated with a known activity level. The positive control may be a positive reference standard. The positive reference standard may correspond to a value that has been either theoretically or experimentally determined and which represents a positive result in a method of the invention. The positive result may represent an ideal activity level for a composition comprising clostridial neurotoxin polypeptides. Such a value may be used for quality-control purposes. The value may have been determined prior to carrying out a method of the invention or may be determined simultaneously with, or subsequent to, carrying out a method of the invention. Thus, where a “luminescence” (e.g. a level of luminescence) or “luciferase activity value” is used herein in reference to a control, this may mean a “luminescence” (e.g. a level of luminescence) or “luciferase activity value” represented by said control.

[0270] The method may comprise comparing the luciferase activity with a luciferase activity of a positive control (e.g. of known potency) in which the population of cells has been contacted with a composition that comprises a clostridial neurotoxin. The method may comprise comparing the measured luciferase activity value with a luciferase activity value of a positive control (e.g. of known potency) in which the population of cells has been contacted with a composition that comprises a clostridial neurotoxin.

[0271] In one embodiment, when the control is a positive control, the same (or a lower level of) luminescence when compared to the positive control indicates that a composition tested comprises clostridial neurotoxin polypeptides (e.g., clostridial neurotoxin polypeptides are present in the composition). In such instances, this may allow for a determination to be made that a composition comprises clostridial neurotoxin polypeptides (e.g., clostridial neurotoxin polypeptides are present in the composition).

[0272] In one embodiment, when the control is a positive control, the same (or a lower level of) luminescence when compared to the positive control indicates that a composition tested comprises active clostridial neurotoxin polypeptides. The same level of luminescence when compared to the positive control may indicate that the composition tested comprises the same number of active clostridial neurotoxin polypeptides as the positive control. The same level of luminescence when compared to the positive control may indicate that the composition has the same activity level as the positive control.

[0273] A lower level of luminescence when compared to the positive control may indicate that the composition tested comprises a higher number of active clostridial neurotoxin polypeptides than the positive control. A lower level of luminescence when compared to the positive control may indicate that the composition has a higher activity level than the positive control. The extent by which the level is lower may be quantified to indicate the activity level of the composition comprising clostridial neurotoxin polypeptides. Preferably, the lower the level of luminescence compared to the positive control, the higher the number of active clostridial neurotoxin polypeptides. Preferably, the lower the level of luminescence compared to the positive control, the higher the activity level of the composition comprising clostridial neurotoxin polypeptides.

[0274] In one embodiment, when the control is a positive control, a higher level of luminescence when compared to the positive control indicates that a composition tested does not comprise clostridial neurotoxin polypeptides (e.g. clostridial neurotoxin polypeptides are absent in the composition). In such instances, this may allow for a determination to made that a composition does not comprise clostridial neurotoxin polypeptides (e.g. clostridial neurotoxin polypeptides are absent in the composition). To make a more definitive determination, a comparison may also be made to a negative control, as described herein.

[0275] In one embodiment, when the control is a positive control, a higher level of luminescence when compared to the positive control indicates that a composition tested comprises inactive clostridial neurotoxin polypeptides. A higher level of luminescence when compared to the positive control may indicate that the composition tested comprises fewer active clostridial neurotoxin polypeptides than the positive control. A higher level of luminescence when compared to the positive control may indicate that the composition has a lower activity level than the positive control. The extent by which the level is higher may be quantified to indicate the activity level of the composition comprising clostridial neurotoxin polypeptides. Preferably, the higher the level of luminescence compared to the positive control, the lower the number of active clostridial neurotoxin polypeptides. Preferably, the higher the level of luminescence compared to the positive control, the lower the activity level of the composition comprising clostridial neurotoxin polypeptides. The term “different” (and associated terms such as “change”, “changed”, and “difference”, and synonyms thereof) as used herein may mean a difference that is substantially different to a comparator (e.g. a control as described herein). A difference (and associated terms such as “change”, “changed”, and “difference”, and synonyms thereof) may mean a statistically- significant difference when compared to a comparator (e.g. a control as described herein). A “substantial difference” may be a difference of at least 5%, 10%, 15%, 20%, 25% or 30% when compared to a comparator (e.g. a control as described herein). The term “no difference” (and associated terms such as “unchanged” and “the same”, and synonyms thereof) may mean that there is no substantial difference when compared to a comparator (e.g. a control as described herein). No difference (and associated terms such as “unchanged” and “the same”, and synonyms thereof) may mean that there is no statistically-significant difference when compared to a comparator (e.g. a control as described herein). The term “lower” (and associated terms such as “less than”) as used herein may mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% lower when compared to a comparator (e.g. a control as described herein). The term “lower” (and associated terms such as “less than”) may mean statistically- significantly lower when compared to a comparator (e.g. a control as described herein). The term “higher” (and associated terms such as “higher than”) as used herein may mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% higher when compared to a comparator (e.g. a control as described herein). The term “higher” (and associated terms such as “higher than”) may mean statistically-significantly higher when compared to a comparator (e.g. a control as described herein).

[0276] A method of the invention may be used to determine a potency value (EC5o) of a composition comprising clostridial neurotoxin polypeptides. In order to obtain said potency value, the method may be carried out using a composition comprising the clostridial neurotoxin at a first concentration, and determining the activity value for the first concentration. The method may further comprise repeating these steps using a composition comprising the clostridial neurotoxin at different concentrations and determining the activity value for each concentration until a potency value (EC5o) may be determined. For each concentration a further population of cells (e.g. a population of cells identical to the population of cells used for the first concentration) may be used. The potency value may be determined by comparing the measured luciferase activity value (for the first concentration) with the measured luciferase activity value(s) for the subsequent concentrations (e.g. a second, third and / or fourth concentration). In other words, the method may further comprise contacting at least a second population of cells (e.g. a population of cells identical to the population of cells contacted with a first composition) with at least a second composition comprising a different concentration of the clostridial neurotoxin, and determining the clostridial neurotoxin activity of the at least second composition by measuring luciferase activity to obtain a measured luciferase activity value(s) and determining a potency value (EC5o) of the composition by comparing the measured luciferase activity value with the measured luciferase activity value(s) for the at least second composition, preferably further comprising contacting at least a third population of cells (e.g. a population of cells identical to the population of cells contacted with a first composition) with at least a third composition comprising a different concentration of the clostridial neurotoxin, and determining the clostridial neurotoxin activity of the at least third composition by measuring luciferase activity to obtain a measured luciferase activity value(s) and determining a potency value (EC5O) of the composition by comparing the measured luciferase activity value with the measured luciferase activity value(s) for the at least third and / or second compositions. The skilled person will appreciate that at least a fourth population of cells and at least a fourth composition, etc. may be employed as necessary to determine the EC5o value.

[0277] The “EC5o” may refer to a clostridial neurotoxin (e.g. amount or concentration thereof in a composition) which induces a response halfway between the baseline and maximum after some specified exposure time. It is commonly used as a measure of potency. The EC50 of a graded dose response curve may represent the concentration of a clostridial neurotoxin where 50% of its maximal effect is observed.

[0278] The methods of the invention involve contacting the population of cells (preferably neuronal progenitor-like cells) with a composition. In this context “contacting the population of cells with the composition” (and the like) means that the cell (e.g. a component thereof, such a clostridial neurotoxin receptor) and the composition are allowed to physically and / or chemically interact. Contacting may comprise a surface of the cell (e.g. a component thereof) being contacted with a composition. Contacting may be carried out under conditions and for a period of time which is sufficient to allow binding of a clostridial neurotoxin to the cell, internalisation, and translocation of the L-chain into the cytosol. Contacting may be carried out under conditions and for a period of time which is sufficient to allow interaction of any clostridial neurotoxin polypeptide (e.g. the L-chain thereof) in the composition with the single-chain polypeptide comprising a clostridial neurotoxin cleavage site within the cells. The composition may be added to fresh medium (e.g. cell culture medium). Said medium may replace existing medium (e.g. cell culture medium). In other words, the existing medium may be removed (e.g. aspirated) and fresh medium may be added which contains the composition diluted to the appropriate concentration. Alternatively or additionally, the composition may be added to the existing cell culture medium. The step of contacting the composition with the population of cells may occur by adding the composition directly to a medium in which the cell is present, for example a cell culture medium. The composition may be diluted in medium (e.g. cell culture medium). Said dilution may occur prior to adding the composition to a cell culture vessel comprising the population of cells, e.g. a cell culture flask or a well within a multi-well plate. The medium within the vessel may be removed prior to contacting the population of cells with the composition. In this case, contacting may occur when fresh media comprising the composition is added to the culture vessel. The cell culture medium may be removed (e.g. aspirated) and replaced by fresh cell culture medium comprising the composition but does not require complete removal of the cell culture medium. In other words, the cell culture medium may be partially removed (e.g. aspirated) prior to contacting and replaced with fresh cell culture medium comprising the composition, or substantially all of the cell culture medium may be removed (e.g. aspirated) prior to contacting cell and replaced with fresh cell culture medium comprising the composition. The term “partially removed” in this context may mean that some but not all of the medium may be removed, e.g. at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55% or 60% of the medium may be removed. The term “substantially all” means that at least 90%, 95%, 97%, 98%, or 99% of the medium may be removed. In some instances, 100% of the ganglioside(s) may be removed or replaced.

[0279] The population of cells and composition may be incubated for a period of time to enable any clostridial neurotoxin (e.g. L-chain thereof) within the composition to come into contact with the single-chain polypeptide comprising a clostridial neurotoxin cleavage site within the cells. The incubation time (e.g. the time period the population of cells and the composition are contacted for) may be at least 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 75 hours, 80 hours, or 85 hours. The incubation time (e.g. the time period the population of cells and the composition are contacted for) may be <85 hours, <80 hours, <75 hours, or <72 hours. The incubation time (e.g. the time period the population of cells and the composition are contacted for) may be SOBS hours, 50-80 hours, 50-75 hours, 60-85 hours, 60-80 hours, 60-75 hours, 65-80 hours, 65- 75 hours, or 68-72 hours. Preferably, the incubation time may be 68 to 72 hours, more preferably 70 hours. Once the population of cells have been contacted with the composition the cells may be incubated at any suitable temperature. In one embodiment, the population of cells are incubated at 15-40°C, 20°C-40°C, 25-40°C, 30-40°C or 35-40°C. Preferably, the population of cells are incubated with the composition at a 37°C ±2°C.

[0280] The composition may be present or absent in a medium (e.g. a cell culture medium) during a step of lysing the population of cells. For example, the medium (e.g. a cell culture medium) comprising the composition may be removed and replaced with a suitable lysis buffer. When the composition is absent from the medium (e.g. a cell culture medium), the medium (e.g. a cell culture medium) containing the composition may be replaced with fresh medium (e.g. a cell culture medium) which does not contain the composition or may be replaced with a suitable lysis buffer. In one embodiment, the composition is present in the medium (e.g. a cell culture medium) during a step of lysing the cells, (e.g. the composition may not be removed from the medium prior to lysing the cells). Preferably, the composition is not present in the medium (e.g. a cell culture medium) (e.g. the medium, such as cell culture medium, may be replaced with a suitable lysis buffer) during the step of lysing the population of cells.

[0281] A suitable lysis buffer may comprise 150 mM NaCI, 1.0% octylphenoxypolyethoxyethanol, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, e.g. RIPA lysis buffer.

[0282] Thus, following contact of the population of cells with the composition (e.g. after the incubation time), said composition may be removed from the population of cells, e.g. prior to lysing the cells. For example, the medium (e.g. a cell culture medium) comprising the composition may be removed and replaced with fresh medium (e.g. cell culture media). In this context the term “removed” may mean that the medium (e.g. cell culture medium) comprising the composition is taken out (e.g. aspirated) from the cell culture vessel in which the cells are grown. However, it is not necessary for all of the medium (e.g. cell culture medium) comprising the composition to be removed or replaced. Some medium (e.g. cell culture medium) comprising the composition may still be present in the cell culture vessel. The medium (e.g. cell culture medium) comprising the composition may be partially removed e.g. at least 20%, 30%, 35%, 40%, 45%, or 50% of the medium (e.g. cell culture medium) comprising the composition may be removed. For example, substantially all of the medium (e.g. cell culture medium) comprising the composition may be removed or replaced. “Substantially all” means that at least 85%, 90%, 95%, 97%, 98% or 99% of the medium (e.g. cell culture medium) comprising the composition has been removed. In some instances, 100% of the medium (e.g. cell culture medium) comprising the composition may be removed or replaced. By removing the medium (e.g. cell culture medium) comprising the composition from the cell culture vessel, the composition may no longer be in contact with the population of cells. In one embodiment, most or all of the composition may no longer be in contact with the population of cells. The composition may be partially removed from the population of cells, e.g. at least 20%, 30%, 35%, 40%, 45%, or 50% of the composition may no longer be in contact with the population of cells. In one embodiment, substantially all of the composition may no longer be in contact with the population of cells, e.g. at least 85%, 90%, 95%, 97%, 98% or 99% may no longer be in contact with the population of cells. In some instances, 100% of the composition may no longer be in contact with the population of cells.

[0283] A composition employed in the present invention may be one which is known to comprise a clostridial neurotoxin polypeptide, or a composition which is suspected to comprise a clostridial neurotoxin polypeptide. The composition may comprise a clostridial neurotoxin polypeptide at a concentration of 0.01 ng / mL to 400ng / ml_, 0.01 ng / mL to 300ng / ml_, 0.01 ng / mL to 200ng / ml_, 0.05ng / ml_ to 400ng / ml_, 0.05ng / ml_ to 300ng / ml_, 0.05ng / ml_ to 200ng / ml_, 0.05ng / ml_ to 100ng / ml_, 0.05ng / ml_ to 50ng / ml_, O.l ng.mL to 400ng / ml_, O.l ng.mL to 300ng / ml_, O.l ng.mL to 200ng / ml_, O.l ng.mL to 50ng / mL, 1 ng / mL to 400ng / mL, 1 ng / mL to 300ng / mL, 1 ng / mL to 200ng / mL, 1 ng / mL to 100ng / mL, 1 ng / mL to 50ng / mL, 10 ng / mL to 400ng / mL, 10 ng / mL to 300ng / mL, 10 ng / mL to 200ng / mL, 10 ng / mL to 50ng / mL, 50ng / mL to 400 ng / mL, 100 ng / mL to 400ng / mL, 200ng / mL to 400ng / mL, or 100 ng / mL to 300ng / mL. The composition may comprise a clostridial neurotoxin polypeptide at a concentration of 0.01 ng / mL to 400ng / mL, 0.05ng / mL to 400ng / mL, O.l ng.mL to 400ng / mL, 1 ng / mL to 400ng / mL, 0.01 ng / mL to 200ng / mL, 0.05ng / mLto 200ng / mL, O.l ng.mLto 200ng / mL, 1 ng / mL to 200ng / mL or 200ng / mL to 400 ng / mL. The composition may comprise a clostridial neurotoxin polypeptide at a concentration of 0.05ng / mL to 400ng / mL, 0.05ng / mL to 50ng / mL, O.l ng.mL to 50ng / mL, 50ng / mL to 400ng / mL or 200ng / mL to 400 ng / mL. The composition may comprise 0.05 ng / mL to 50 ng / mL of a clostridial neurotoxin polypeptide. Preferably, the composition comprises 2 ng / mL to 200 ng / mL of clostridial neurotoxin polypeptide.

[0284] The composition may comprise a clostridial neurotoxin polypeptide at a concentration of 0.05pM to 3000pM, 0.05 pM to 2500 pM, 0.05 pM to 2000 pM, 0.05 pM to 1500 pM, 0.05 pM to 1000 pM, 0.05 pM to 500 pM, 0.1 pM to 3000 pM, 0.1 pM to 2500 pM, 0.1 pM to 2000 pM, 0.1 pM to 1500 pM, 0.1 pM to 1000 pM, 0.1 pM to 500 pM, 0.333 pM to 333 pM, 1 pM to 3000pM, 1 pM to 2500 pM, 1 pM to 2000 pM, 1 pM to 1500 pM, 10pM to 3000pM, 10 pM to 2500 pM, 10 pM to 2000 pM, or 10 pM to 1500 pM,. The composition may comprise 0.333pM to 333pM of a clostridial neurotoxin polypeptide. Preferably, the composition comprises 13.3pM to 1332pM of a clostridial neurotoxin polypeptide. During contacting of the population of cells with the composition, the clostridial neurotoxin polypeptide to cell ratio may be in the range of 80 to 5.3 million, 80 to 4 million, 80 to 3 million, 80 to 2.5 million, 80 to 2 million, 80 to 1 .6 million, 80 to 1 million, or 80 to 500,000. Preferably, the clostridial neurotoxin polypeptide to cell ratio may be in the range of 80 to 1 .6 million or 80 to 500,000. Preferably, the clostridial neurotoxin polypeptide to cell ratio may be in the range of 500 to 500,000.

[0285] When a population of cells is seeded at a concentration of 500,000 cells / mL the ratio range may be 80 to 3.18 million, 80 to 2.39 million, 80 to 1.6 million, 80 to 800,000, 80 to 400,000, 80 to 80,000, 400 to 3.18 million, 400 to 2.39 million, 400 to 1 .6 million, 400 to 800,000, 400 to 400,000, 400 to 80,000, 800 to 3.18 million, 800 to 2.39 million, 800 to 1.6 million, 800 to 800,000, 800 to 400,000, 800 to 80,000, 8,000 to 3.18 million, 8,000 to 2.39 million, 8,000 to 1 .6 million, 8,000 to 800,000, 8,000 to 400,000, 8,000 to 80,000, 80,000 to 3.18 million, 80,000 to 2.39 million, 80,000 to 1.6 million, 80,000 to 800,000, 80,000 to 400,000, 400,000 to 3.18 million, or 800,000 to 3.18 million. In one embodiment, when a population of cells has been seeded at a concentration of 500,000 cells / mLthe ratio range may be 80 to 3.18 million, 80 to 2.39 million, 80 to 1.6 million, 80 to 400,000, 400 to 3.18 million, 400 to 1.6 million, 400 to 400,000, 800 to 3.18 million, 800 to 1.6 million, 8,000 to 3.18 million, 8,000 to 1.6million, 80,000 to 800,000, or 8,000 to 400,000. In another embodiment, when a population of cells has been seeded at a concentration of 500,000 cells / mL the ratio range may be 80 to 3.18 million, 80 to 1.6 million, 80 to 400,000, 400 to 3.18 million, 400 to 1.6 million, or 400 to 400,000. Preferably, when a population of cells has been seeded at a concentration of 500,000 cells / mL the ratio range may be 400 to 400,000.

[0286] When a population of cells is seeded at a concentration of 450,000 cells / mL, the ratio range may be 88.88 to 3.53 million, 88.88 to 2.66 million, 88.88 to 1.77 million, 88.88 to 888,888, 88.88 to 444,444, 88.88 to 88,888, 400 to 3.53 million, 444 to 2.66 million, 444 to 1 .77 million, 444 to 888,888, 444 to 444,444, 444 to 88,888, 888 to 3.53 million, 888 to 2.66 million, 888 to 1 .77 million, 888 to 888,888, 888 to 444,444, 888 to 88,888, 8,888 to 3.53 million, 8, 888 to 2.66 million, 8,888 to 1.77 million, 8,888 to 888,888, 8,888 to 444,444, 8,888 to 88,888, 88,888 to 3.53 million, 88,888 to 2.66 million, 88,888 to 1.77 million, 88,888 to 888,888, 88,888 to 444,444, 444,444 to 3.53 million, or 888,888 to 3.53 million. In one embodiment, when a population of cells is seeded at a concentration of 450,000 cells / mL, the ratio range may be 88.88 to 3.53 million, 88.88 to 2.66 million, 88.88 to 1 .77 million, 88.88 to 444,444, 400 to 3.53 million, 444 to 1.77 million, 444 to 444,444, 888 to 3.53 million, 888 to 1.77 million, 8,888 to 3.53 million, 8,888 to 1.77million, 8,888 to 888,888, or 8,888 to 444,444. In one embodiment, when a population of cells is seeded at a concentration of 450,000 cells / mL, the ratio range may be 88.88 to 3.53 million, 88.88 to 1 .77 million, 88.88 to 444,444, 444 to 3.53 million, 444 to 1.77 million, 444 to 444,444. Preferably, when a population of cells is seeded at a concentration of 450,000 cells / mL the ratio range may be 444.4 to 444,444.

[0287] When a population of cells is seeded at a concentration of 400,000 cells / mL, the ratio range may be 100 to 3.97 million, 100 to 2.99 million, 100 to 2 million, 100 to 1 million, 100 to 500,000, 100 to 100,000, 500 to 3.97 million, 500 to 2.99 million, 500 to 2 million, 500 to 1 million, 500 to 500,000, 500 to 100,000, 1000 to 3.97 million, 1000 to 2.99 million, 1000 to 2 million, 1000 to 1 million, 1000 to 500,000, 1000 to 100,000, 10,000 to 3.97 million, 10,000 to 2.99 million, 10,000 to 2 million, 10,000 to 1 million, 10,000 to 500,000, 10,000 to 100,000, 100,000 to 3.97 million, 100,000 to 2.99 million, 100,000 to 2 million, 100,000 to 1 million, 100,000 to 500,000, 500,000 to 3.97 million, or 1 million to 3.97 million. In one embodiment, when a population of cells is seeded at a concentration of 400,000 cells / mL, the ratio range may be 100 to 3.97 million, 100 to 2.99 million, 100 to 2 million, 100 to 500,000, 500 to 3.97 million, 500 to 2 million, 500 to 500,000, 1000 to 3.97 million, 1000 to 2 million, 10,000 to 3.97 million, 10,000 to 2 million, 100,000 to 1 million, or 10,000 to 500,000. In one embodiment, when a population of cells is seeded at a concentration of 400,000 cells / mL, the ratio range may be 100 to 3.97 million, 100 to 2 million, 100 to 500,000, 500 to 3.97 million, 500 to 2 million, or 500 to 500,000. Preferably, when a population of cells is seeded at a concentration of 400,000 cells / mL the ratio range may be 500 to 500,000.

[0288] When a population of cells is seeded at a concentration of 350,000 cells / mL, the ratio range may be 114.3 to 4.54 million, 114.3 to 3.53 million, 114.3 to 2.28 million, 114.3 to 1.14 million, 114.3 to 572,000, 114.3 to 114,300, 572 to 4.54 million, 572 to 3.53 million, 572 to 2.28 million, 572 to 1 .14 million, 572 to 572,000, 572 to 114,300, 1143 to 4.54 million, 1143 to 3.53 million, 1143 to 2.28million, 1143 to 1.14million, 1143 to 572,000, 1143 to 114,300, 11 ,430 to 4.54 million, 11 ,430 to 3.53 million, 11 , 430 to 2.28 million, 11 ,430 to 1.14 million, 11 ,430 to 572,000, 11 ,430 to 114,300, 114,300 to 4.54 million, 114,300 to 3.53 million, 114,300 to 2.28million, 114,300 to 1.14million, 114,300 to 572,000, 572,000to 4.54 million, or 1.14million to 4.54 million. In one embodiment, when a population of cells is seeded at a concentration of 350,000 cells / mL, the ratio range may be 114.3 to 4.54 million, 114.3 to 3.53 million, 114.3 to 2.28million, 114.3 to 572,000, 572 to 4.54 million, 572 to 2.28 million, 572 to 572,000, 1143 to 4.54 million, 1143 to 2.28 million, 11 ,430 to 4.54 million, 11 ,430 to 2.28 million, 114,300 to 1.14 million, or 11 ,430 to 572,000. In one embodiment, when a population of cells is seeded at a concentration of 350,000 cells / mL, the ratio range may be 114.3 to 4.54 million, 114.3 to 2.28 million, 114.3 to 572,000, 572 to 4.54 million, 572 to 2.28 million, 572 to 572,000. Preferably, when a population of cells is seeded at a concentration of 350,000 cells / mL the ratio range may be 572 to 572,000.

[0289] When a population of cells is seeded at a concentration of 300,000 cells / mL, the ratio range may be 133.33 to 5.3 million, 133.33 to 3.99 million, 133.33 to 2.66 million, 133.33 to 1 .33m il lion , 133.33 to 666,666, 133.33 to 133,333, 666.66 to 5.3 million, 666.66 to 3.99 million, 666.66 to 2.66 million, 666.66 to 1.33 million, 666.66 to 666,666, 666.66 to 133,333, 1333 to 5.3 million, 1333 to 3.99 million, 1333 to 2.66 million, 1333 to 1.33 million, 1333 to 666,666, 1333 to 133,333, 13,333 to 5.3 million, 13,333 to 3.99 million, 13,333 to 2.66 million, 13,333 to

[0290] 1.33 million, 13,333 to 666,666, 13,333 to 133,333, 133,333 to 5.3 million, 133,333 to 3.99 million, 133,333 to 2.66 million, 133,333 to 1.33 million, 133,333 to 666,666, 666,666 to 5.3 million, or 1.33 million to 5.3 million. In one embodiment, when a population of cells is seeded at a concentration of 300,000 cells / mL, the ratio range may be 133.33 to 5.3 million, 133.33 to 3.99 million, 133.33 to 2.66 million, 133.33 to 666,666, 666.66 to 5.3 million, 666.66 to 2.66 million, 666.66 to 666,666, 1333 to 5.3 million, 1333 to 2.66 million, 13,333 to 5.3 million, 13,333 to 2.66 million, 13, 333 to 1.33 million, or 13,333 to 666,666. In one embodiment, when a population of cells is seeded at a concentration of 300,000 cells / mL, the ratio range may be

[0291] 133.33 to 5.3 million, 133.33 to 2.66 million, 133.33 to 666,666, 666.66 to 5.3 million, 666.66 to 2.66 million, or 666.66 to 666,666. Preferably, when a population of cells is seeded at a concentration of 300,000 cells / mL the ratio range may be 666.6 to 666,666.

[0292] The term “clostridial neurotoxin polypeptide to cell ratio” as used herein refers to the concentration of clostridial neurotoxin polypeptide (toxin molecules per mL) within the composition divided by the concentration of cells seeded (number of cells / mL). For example, for a population of cells seeded at a concentration of 400,000 cells / mL contacted with a composition comprising a clostridial neurotoxin polypeptide at a concentration of 2x107toxin molecules per mL (or 0.05ng / mL) the ratio would be 80 (e.g. the ratio would be 80:1 or 80 toxin molecules per 1 cell).

[0293] In some instances herein, “clostridial neurotoxin polypeptides” are referred to. However, this is to indicate that more than one clostridial neurotoxin polypeptide may be present (e.g. in a composition) when carrying out the method. It is not intended to necessarily indicate that two or more different types of clostridial neurotoxin polypeptides are present, although this is encompassed. Thus, the clostridial neurotoxin polypeptides may be of one type (e.g. all the clostridial neurotoxin polypeptides are BoNT / A polypeptides) or multiple types (e.g. a portion of the clostridial neurotoxin polypeptides are BoNT / B polypeptides and a portion are BoNT / A polypeptides). Preferably, the clostridial neurotoxin polypeptides are of one type. The above applies analogously to some instances herein where “botulinum neurotoxin polypeptides” are referred to.

[0294] A clostridial neurotoxin according to the invention may comprise a botulinum neurotoxin or a tetanus neurotoxin (TeNT) Hcc domain. A clostridial neurotoxin of the invention may comprise a BoNT / A Hcc domain, a BoNT / B Hcc domain, a BoNT / C1 Hcc domain, a BoNT / D Hcc domain, a BoNT / E Hcc domain, a BoNT / F Hcc domain, a BoNT / G Hcc domain, a BoNT / X Hcc domain, or TeNT Hcc domain. Preferably, a clostridial neurotoxin of the invention comprises a BoNT / B Hcc domain or a BoNT / A Hcc domain, more preferably a BoNT / B Hcc domain. Preferably, a clostridial neurotoxin of the invention comprises a BoNT / E Hcc domain.

[0295] A clostridial neurotoxin according to the invention may comprise a botulinum neurotoxin or a tetanus neurotoxin (TeNT) Hcdomain. A clostridial neurotoxin of the invention may comprise a BoNT / A Hcdomain, a BoNT / B Hcdomain, a BoNT / C1 Hcdomain, a BoNT / D Hcdomain, a BoNT / E Hcdomain, a BoNT / F Hcdomain, a BoNT / G Hcdomain, a BoNT / X Hcdomain, or TeNT Hcdomain. Preferably, a clostridial neurotoxin of the invention comprises a BoNT / B Hcdomain or a BoNT / A Hcdomain, more preferably a BoNT / B Hcdomain. Preferably, a clostridial neurotoxin of the invention comprises a BoNT / E Hcc domain.

[0296] The term “clostridial neurotoxin” embraces toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, Ci, D, E, F, G, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F). A reference BoNT / A sequence is shown as SEQ ID NO: 12. A reference BoNT / B sequence is shown as SEQ ID NO: 13. A reference BoNT / C1 (also referred to as BoNT / C herein) sequence is shown as SEQ ID NO: 33. A reference BoNT / D sequence is shown as SEQ ID NO: 34. A reference BoNT / E sequence is shown as SEQ ID NO: 35. A reference BoNT / F sequence is shown as SEQ ID NO: 36. A reference BoNT / G sequence is shown as SEQ ID NO: 37. A reference TeNT sequence is shown as SEQ ID NO: 38. A reference BoNT / X sequence is shown as SEQ ID NO: 39. The term “clostridial neurotoxin” may also embrace newly discovered botulinum neurotoxin protein family members expressed by non-clostridial microorganisms, such as the Enterococcus encoded toxin which has closest sequence identity to BoNT / X, the Weissella oryzae encoded toxin called B0NT / W0 (NCBI Ref Seq: WP_027699549.1), which cleaves VAMP2 at W89-W90, the Enterococcus faecium encoded toxin (GenBank: OTO22244.1), which cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero encoded toxin (NCBI Ref.Seq: WP_034687872.1).

[0297] Thus, a clostridial neurotoxin may be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Thus, a composition of the invention may comprise BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT. Preferably, a clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X. In one embodiment, the composition may comprise a botulinum neurotoxin selected from BoNT / A, BoNT / B, or BoNT / E.

[0298] Clostridial neurotoxins are formed from two polypeptide chains, the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises a C-terminal targeting component (receptor binding domain or Hcdomain) and an N-terminal translocation component (HNdomain). Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex, consisting of BoNT itself complexed to a number of accessory proteins. There are at present eight different classes of botulinum neurotoxin, namely: botulinum neurotoxin serotypes A, B, C1 , D, E, F, G, and X all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralising anti-sera, with such classification by serotype correlating with percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes on the basis of amino acid percentage sequence identity.

[0299] BoNTs are absorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of their neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP); BoNT / C1 , BoNT / A and BoNT / E cleave the synaptosomal-associated protein of 25 kDa (SNAP-25); and BoNT / C1 cleaves syntaxin. BoNT / X has been found to cleave SNAP-25, VAMP1 , VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1. Tetanus toxin is produced in a single serotype by C. tetani. C. butyricum produces BoNT / E, while C. baratii produces BoNT / F.

[0300] Examples of L-chain reference sequences include:

[0301] Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440

[0302] Botulinum type C1 neurotoxin: amino acid residues 1-441

[0303] Botulinum type D neurotoxin: amino acid residues 1-445 Botulinum type E neurotoxin: amino acid residues 1-422 Botulinum type F neurotoxin: amino acid residues 1-439 Botulinum type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457

[0304] For recently identified BoNT / X, the L-chain has been reported as corresponding to amino acids 1-439 thereof, with the L-chain boundary potentially varying by approximately 25 amino acids (e.g. 1-414 or 1-464).

[0305] A BoNT / A L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-448 of SEQ ID NO: 12. A BoNT / B L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-440 of SEQ ID NO: 13. A BoNT / C1 L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-441 of SEQ ID NO: 33. A BoNT / D L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-445 of SEQ ID NO: 34. A BoNT / E L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-422 of SEQ ID NO: 35. A BoNT / E L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-422 of SEQ ID NO: 80. A BoNT / F L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1- 439 of SEQ ID NO: 36. A BoNT / G L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-441 of SEQ ID NO: 37. A BoNT / X L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-439 of SEQ ID NO: 39. ATeNT L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-457 of SEQ ID NO: 38.

[0306] The above-identified reference sequences should be considered a guide, as slight variations may occur according to sub-serotypes. By way of example, US 2007 / 0166332 (hereby incorporated by reference in its entirety) cites slightly different clostridial sequences: Botulinum type A neurotoxin: amino acid residues M1-K448

[0307] Botulinum type B neurotoxin: amino acid residues M1-K441

[0308] Botulinum type C1 neurotoxin: amino acid residues M1-K449

[0309] Botulinum type D neurotoxin: amino acid residues M1-R445 Botulinum type E neurotoxin: amino acid residues M1-R422 Botulinum type F neurotoxin: amino acid residues M1-K439 Botulinum type G neurotoxin: amino acid residues M1-K446 Tetanus neurotoxin: amino acid residues M1-A457

[0310] The translocation domain is a fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or the domain corresponding to that fragment in the intact H-chain. In one embodiment the Hcfunction of the H-chain may be removed by deletion of the Hcamino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the Hcfunction may be inactivated by chemical or biological treatment. Thus, in some embodiments the H-chain may be incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.

[0311] Examples of suitable (reference) Translocation Domains include:

[0312] Botulinum type A neurotoxin - amino acid residues (449-871)

[0313] Botulinum type B neurotoxin - amino acid residues (441-858) Botulinum type C neurotoxin - amino acid residues (442-866) Botulinum type D neurotoxin - amino acid residues (446-862) Botulinum type E neurotoxin - amino acid residues (423-845) Botulinum type F neurotoxin - amino acid residues (440-864) Botulinum type G neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879)

[0314] A BoNT / A HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 449-871 of SEQ ID NO: 12. ABoNT / B HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 441-858 of SEQ ID NO: 13. A BoNT / C1 HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 442-866 of SEQ ID NO: 33. ABoNT / D HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 446-862 of SEQ ID NO: 34. A BoNT / E HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 423-845 of SEQ ID NO: 35. A BoNT / E HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 423-845 of SEQ ID NO: 80. A BoNT / F HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 440-864 of SEQ ID NO: 36. A BoNT / G HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 442-863 of SEQ ID NO: 37. A BoNT / X HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 440-892 of SEQ ID NO: 39. A TeNT HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 458-879 of SEQ ID NO: 38.

[0315] The above-identified reference sequence should be considered a guide as slight variations may occur according to sub-serotypes. By way of example, US 2007 / 0166332 (hereby incorporated by reference thereto) cites slightly different clostridial sequences:

[0316] Botulinum type A neurotoxin - amino acid residues (A449-K871) Botulinum type B neurotoxin - amino acid residues (A442-S858) Botulinum type C neurotoxin - amino acid residues (T450-N866) Botulinum type D neurotoxin - amino acid residues (D446-N862) Botulinum type E neurotoxin - amino acid residues (K423-K845) Botulinum type F neurotoxin - amino acid residues (A440-K864) Botulinum type G neurotoxin - amino acid residues (S447-S863) Tetanus neurotoxin - amino acid residues (S458-V879)

[0317] In the context of the present invention, a variety of clostridial neurotoxin HNregions comprising a translocation domain can be useful in aspects of the present invention. The HNregions from the heavy chains of clostridial neurotoxins are approximately 410-430 amino acids in length and comprise a translocation domain. Research has shown that the entire length of a HNregion from a clostridial neurotoxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include clostridial neurotoxin HNregions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include clostridial neurotoxin HNregions comprising a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.

[0318] For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

[0319] The term HNembraces naturally-occurring neurotoxin HNportions, and modified HNportions having amino acid sequences that do not occur in nature and / or synthetic amino acid residues. In one embodiment said modified HNportions still demonstrate the above-mentioned translocation function.

[0320] Examples of clostridial neurotoxin receptor binding domain (Hc) reference sequences include:

[0321] BoNT / A- N872-L1296

[0322] BoNT / B - E859-E1291

[0323] BoNT / C1 - N867-E1291

[0324] BoNT / D - S863-E1276

[0325] BoNT / E - R846-K1252

[0326] BoNT / F - K865-E1274 BoNT / G - N864-E1297 TeNT - I880-D1315

[0327] For recently-identified BoNT / X, the Hcdomain has been reported as corresponding to amino acids 893-1306 thereof, with the domain boundary potentially varying by approximately 25 amino acids (e.g. 868-1306 or 918-1306).

[0328] A BoNT / A Hcdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 872-1296 of SEQ ID NO: 12. A BoNT / B Hcdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 859-1291 of SEQ ID NO: 13. A BoNT / C1 He domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 867-1291 of SEQ ID NO: 33. A BoNT / D He domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 863-1276 of SEQ ID NO: 34. A BoNT / E Hcdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 846-1252 of SEQ ID NO: 35. A BoNT / E Hcdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 846-1252 of SEQ ID NO: 80. A BoNT / F Hcdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 865-1274 of SEQ ID NO: 36. A BoNT / G He domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 864-1297 of SEQ ID NO: 37. A BoNT / X Hcdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 893-1306 of SEQ ID NO: 39. ATeNT Hcdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 880-1315 of SEQ ID NO: 38.

[0329] A clostridial neurotoxin H-chain (e.g. the Hcdomain portion) may further comprise a translocation facilitating domain (or a fragment thereof may be translocation facilitating domain fragment). Said domain facilitates delivery of the L-chain into the cytosol of the target cell and are described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is herein incorporated by reference thereto.

[0330] By way of example, a translocation facilitating domain may comprise a clostridial neurotoxin HCN domain or a fragment or variant thereof. In more detail, a clostridial neurotoxin HCN translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, a clostridial neurotoxin HCN translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include:

[0331] Botulinum type A neurotoxin - amino acid residues (872-1110)

[0332] Botulinum type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111) Botulinum type D neurotoxin - amino acid residues (863-1098) Botulinum type E neurotoxin - amino acid residues (846-1085) Botulinum type F neurotoxin - amino acid residues (865-1105) Botulinum type G neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127) The above sequence positions may vary a little according to serotype / sub-type, and further examples of suitable (reference) clostridial neurotoxin HCN domains include:

[0333] Botulinum type A neurotoxin - amino acid residues (874-1110)

[0334] Botulinum type B neurotoxin - amino acid residues (861-1097)

[0335] Botulinum type C neurotoxin - amino acid residues (869-1111)

[0336] Botulinum type D neurotoxin - amino acid residues (865-1098)

[0337] Botulinum type E neurotoxin - amino acid residues (848-1085)

[0338] Botulinum type F neurotoxin - amino acid residues (867-1105) Botulinum type G neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127)

[0339] Any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain may be combined with a non- clostridial translocation domain peptide or with clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin HCN translocation facilitating domain may be combined with a non-clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin HCN facilitating domain may be combined with a clostridial translocation domain peptide, examples of which include:

[0340] Botulinum type A neurotoxin - amino acid residues (449-1110)

[0341] Botulinum type B neurotoxin - amino acid residues (442-1097)

[0342] Botulinum type C neurotoxin - amino acid residues (450-1111)

[0343] Botulinum type D neurotoxin - amino acid residues (446-1098)

[0344] Botulinum type E neurotoxin - amino acid residues (423-1085)

[0345] Botulinum type F neurotoxin - amino acid residues (440-1105) Botulinum type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127)

[0346] The Hcpeptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25kDa each, namely the N-terminal region (commonly referred to as the HCN peptide or domain) and the C- terminal region (commonly referred to as the HCc peptide or domain). This fact is confirmed by the following publications, each of which is herein incorporated in its entirety by reference thereto: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751- 1759; and Rummel A (2004) Mol. Microbiol. 51 (3), 631-643. Moreover, it has been well documented that the C-terminal region (Hcc), which constitutes the C-terminal 160-200 amino acid residues, is responsible for binding of a clostridial neurotoxin to its natural cell receptors, namely to nerve terminals at the neuromuscular junction - this fact is also confirmed by the above publications. Thus, reference throughout this specification to a clostridial heavy-chain lacking a functional heavy chain Hcpeptide (or domain) such that the heavy-chain is incapable of binding to cell surface receptors to which a native clostridial neurotoxin binds means that the clostridial heavy-chain simply lacks a functional Hcc peptide. In other words, the Hcc peptide region may be either partially or wholly deleted, or otherwise modified (e.g. through conventional chemical or proteolytic treatment) to reduce its native binding ability for nerve terminals at the neuromuscular junction.

[0347] Hcc reference sequences are presented below:

[0348] Botulinum type A neurotoxin - amino acid residues (Y1111 -L1296)

[0349] Botulinum type B neurotoxin - amino acid residues (Y1098-E1291) Botulinum type C neurotoxin - amino acid residues (Y1112-E1291) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086-K1252) Botulinum type F neurotoxin - amino acid residues (Y1106-E1274) Botulinum type G neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y1128-D1315).

[0350] The above-identified reference sequences should be considered a guide as slight variations may occur according to sub-serotypes.

[0351] A BoNT / A Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1111-1296 of SEQ ID NO: 12. A BoNT / B Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1098-1291 of SEQ ID NO: 13. A BoNT / C1 Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1112-1291 of SEQ ID NO: 33. A BoNT / D Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1099-1276 of SEQ ID NO: 34. A BoNT / E Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1086-1252 of SEQ ID NO: 35. A BoNT / E Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1086-1252 of SEQ ID NO: 80. A BoNT / F Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1106-1274 of SEQ ID NO: 36. A BoNT / G Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1106-1297 of SEQ ID NO: 37. A TeNT Hcc domain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1128-1315 of SEQ ID NO: 38.

[0352] The term “clostridial neurotoxin” is also intended to embrace modified clostridial neurotoxins and derivatives thereof, including but not limited to those described below. A modified clostridial neurotoxin or derivative may contain one or more amino acids that has been modified as 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. By way of example, a modified clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify functional aspects of the toxin, for example biological activity or persistence. Thus, in one embodiment, the clostridial neurotoxin of the invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.

[0353] A modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified Hcdomain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial neurotoxin. Such modifications in the Hcdomain can include modifying residues in the ganglioside binding site of the Hcdomain or in the protein (SV2 or synaptotagmin) binding site that alter binding to the ganglioside receptor and / or the protein receptor of the target nerve cell. Examples of such modified clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are hereby incorporated by reference in their entirety.

[0354] Thus, a BoNT / A Hcc domain is preferably a modified BoNT / A Hcc domain, more preferably a modified BoNT / A Hc domain. Therefore, preferably, a clostridial neurotoxin in accordance with the present invention is a modified BoNT / A. Preferably, said modified clostridial neurotoxin comprises one or more modifications that increases the isoelectric point of the clostridial neurotoxin when compared to an equivalent unmodified clostridial neurotoxin lacking said one or more modifications. Suitable modified clostridial neurotoxins are described below and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1 , which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 14-17 (preferably SEQ ID NO: 14 - mrBoNT / A) described herein.

[0355] A modified BoNT / A may be one that comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991 , GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081 , GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277. Such a modified BoNT / A may demonstrate a reduction in, or absence of, side effects compared to the use of known BoNT / A. Said modified BoNT / A may exhibit increased tissue retention properties, thereby providing increased potency and / or duration of action and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional adverse effects), thus providing further advantages.

[0356] The modification may be a modification when compared to a BoNT / A shown as SEQ ID NO: 12, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 12. As the presence of a methionine residue at position 1 of SEQ ID NO: 12 (as well as the SEQ ID NOs corresponding to modified BoNT / A polypeptides described herein) is optional, the skilled person will take the presence / absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 12 includes a methionine, the position numbering will be as defined above (e.g. ASN 886 will be ASN 886 of SEQ ID NO: 12). Alternatively, where the methionine is absent from SEQ ID NO: 12 the amino acid residue numbering should be modified by -1 (e.g. ASN 886 will be ASN 885 of SEQ ID NO: 12). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present / absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.

[0357] An alignment described herein for determining amino acid residue numbering may be carried out using any of the methods described herein for determining sequence homology and / or % sequence identity. The amino acid residue(s) indicated for modification above are surface exposed amino acid residue(s).

[0358] A modified BoNT / A may comprise a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991 , ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277.

[0359] The term “one or more amino acid residue(s)” when used in the context of a modified BoNT / A preferably means at least 2, 3, 4, 5, 6 or 7 of the indicated amino acid residue(s). Thus, a modified BoNT / A may comprise at least 2, 3, 4, 5, 6 or 7 (preferably 7) modifications at the indicated amino acid residue(s). A modified BoNT / A may comprise 1-30, 3-20, or 5-10 amino acid modifications. More preferably, the term “one or more amino acid residue(s)” when used in the context of a modified BoNT / A means all of the indicated amino acid residue(s).

[0360] Preferably, beyond the one or more amino acid modification(s) at the indicated amino acid residue(s), the modified BoNT / A does not contain any further amino acid modifications when compared to SEQ ID NO: 12.

[0361] The modification may comprise (e.g. may be selected from): iv. substitution of an acidic surface exposed amino acid residue with a basic amino acid residue; ii. substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue;

[0362] Hi. substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; or v. deletion of an acidic surface exposed amino acid residue.

[0363] A modification as indicated above may result in a modified BoNT / A that has an increased positive surface charge and increased isoelectric point when compared to the corresponding unmodified BoNT / A (e.g. SEQ ID NO: 12). Without wishing to be bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and cell surface thus increasing retention at a site of administration and / or duration of action. The isoelectric point (pl) is a specific property of a given protein. As is well known in the art, proteins are made from a specific sequence of amino acids (also referred to when in a protein as amino acid residues). Each amino acid of the standard set of twenty has a different side chain (or R group), meaning that each amino acid residue in a protein displays different chemical properties such as charge and hydrophobicity. These properties may be influenced by the surrounding chemical environment, such as the temperature and pH. The overall chemical characteristics of a protein will depend on the sum of these various factors.

[0364] Certain amino acid residues (detailed below) possess ionisable side chains that may display an electric charge depending on the surrounding pH. Whether such a side chain is charged or not at a given pH depends on the pKa of the relevant ionisable moiety, wherein pKa is the negative logarithm of the acid dissociation constant (Ka) fora specified proton from a conjugate base.

[0365] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values of approximately 4.1 (precise pKa values may depend on temperature, ionic strength and the microenvironment of the ionisable group). Thus, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as “physiological pH”). At low pH values, these side chains will become protonated and lose their charge.

[0366] Conversely, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values of approximately 10-12. These side chains therefore exhibit a positive charge at a pH of 7.4. These side chains will become de-protonated and lose their charge at high pH values.

[0367] The overall (net) charge of a protein molecule therefore depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and on the surrounding pH. Changing the surrounding pH changes the overall charge on the protein. Accordingly, for every protein there is a given pH at which the number of positive and negative charges is equal and the protein displays no overall net charge. This point is known as the isoelectric point (pl). The isoelectric point is a standard concept in protein biochemistry with which the skilled person would be familiar.

[0368] The isoelectric point (pl) is therefore defined as the pH value at which a protein displays a net charge of zero. An increase in pl means that a higher pH value is required for the protein to display a net charge of zero. Thus, an increase in pl represents an increase in the net positive charge of a protein at a given pH. Conversely, a decrease in pl means that a lower pH value is required for the protein to display a net charge of zero. Thus, a decrease in pl represents a decrease in the net positive charge of a protein at a given pH.

[0369] Methods of determining the pl of a protein are known in the art and would be familiar to a skilled person. By way of example, the pl of a protein can be calculated from the average pKa values of each amino acid present in the protein (“calculated pl”). Such calculations can be performed using computer programs known in the art, such as the Compute pl / MW Tool from ExPASy (https: / / web.expasy.org / compute_pi / ), which is the preferred method for calculating pl in accordance with the present invention. Comparisons of pl values between different molecules should be made using the same calculation technique / program.

[0370] Where appropriate, the calculated pl of a protein can be confirmed experimentally using the technique of isoelectric focusing (“observed pl”). This technique uses electrophoresis to separate proteins according to their pl. Isoelectric focusing is typically performed using a gel that has an immobilised pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches the pH at which it has zero net charge, this point being the pl of the protein. Results provided by isoelectric focusing are typically relatively low- resolution in nature, and thus the present inventors believe that results provided by calculated pl (as described above) are more appropriate to use.

[0371] Throughout the present specification, “pl” means “calculated pl” unless otherwise stated.

[0372] The pl of a protein may be increased or decreased by altering the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pl may be provided by reducing the number of acidic residues, or by increasing the number of basic residues.

[0373] A modified BoNT / A of the invention may have a pl value that is at least 0.2, 0.4, 0.5 or 1 pl units higher than that of BoNT / A (e.g. SEQ ID NO: 12). Preferably, a modified BoNT / A may have a pl of at least 6.6, e.g. at least 6.8.

[0374] The properties of the 20 standard amino acids are indicated in the table below:

[0375] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive). At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues. The following amino acids are considered uncharged, polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0376] The following amino acids are considered uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine. In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into the BoNT / A polypeptide sequence, thus increasing the total number of amino acid residues in said sequence. In an amino acid deletion, an amino acid residue is removed from the clostridial toxin amino acid sequence, thus reducing the total number of amino acid residues in said sequence.

[0377] Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence is replaced with a different amino acid residue. The replacement amino acid residue may be one of the 20 standard amino acids, as described above. Alternatively, the replacement amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the standard set of 20 described above). By way of example, the replacement amino acid may be a basic non-standard amino acid, e.g. L-Ornithine, L-2-amino-3-guanidinopropionic acid, or D-isomers of Lysine, Arginine and Ornithine). Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using E. coli auxotrophic expression hosts.

[0378] In one embodiment, the substitution is selected from: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, wherein the substitution is a substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e. aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine).

[0379] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is substitution with lysine or arginine. Most preferably, the modification is substitution with lysine.

[0380] Following modification in accordance with the invention, the modified BoNT / A is preferably capable of binding to the target cell receptors that unmodified BoNT / A (e.g. SEQ ID NO: 12) binds.

[0381] Preferably, a modified BoNT / A for use in the invention comprises between 4 and 40 amino acid modifications located in the clostridial toxin HCN domain. Said modified BoNT / A preferably also has pl of at least 6.6. Said modified BoNT / A preferably comprises modifications of at least 4 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991 , ASN 1025, ASN 1026, and ASN 1052, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue. Preferably, said modified BoNT / A further comprises at least one modification located in the Hcc domain (e.g. at GLN 1229). For example, said modified BoNT / A may comprise modifications of at least 5 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991 , ASN 1025, ASN 1026, ASN 1052, and GLN 1229, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue.

[0382] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. By way of example, amino acid modifications may be introduced by modification of a DNA sequence encoding a polypeptide (e.g. encoding unmodified BoNT / A or a fragment thereof). This can be achieved using standard molecular cloning techniques, for example by site-directed mutagenesis where short strands of DNA (oligonucleotides) coding for the desired amino acid(s) are used to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting parts of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, a modified gene sequence can be chemically synthesised.

[0383] A modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 14-17. In one embodiment, a modified BoNT / A may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 14-17. Preferably, a modified BoNT / A may comprise any one of SEQ ID NOs: 14-17. A modified BoNT / A may consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 14-17. In one embodiment, a modified BoNT / A may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 14-17. Preferably, a modified BoNT / A may consist of any one of SEQ ID NOs: 14-17. Of the recited SEQ ID NOs, SEQ ID NO: 14 is most preferred. The skilled person will appreciate that where the polypeptide sequence of a modified BoNT / A varies compared to a given SEQ ID NO by way of % sequence identity, that the at least one of the modifications (e.g. that increase pl) are still present (e.g. unmodified) in the variant modified BoNT / A.

[0384] Thus, a composition of the invention preferably comprises a modified BoNT / A, such as a modified BoNT / A as described above. In some embodiments, the L-chain is a BoNT / A L-chain, the HNdomain is a BoNT / A HNdomain, and the Hcc domain (e.g. Hcdomain) is a modified BoNT / A Hcc domain (e.g. a modified BoNT / A Hcc domain).

[0385] A modified BoNT / A may be encoded by a nucleotide sequence comprising at least 70% sequence identity to SEQ ID NO: 40. In one embodiment, a modified BoNT / A may be encoded by a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 40. Preferably, a modified BoNT / A may be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 40. The skilled person will appreciate that where the nucleotide sequence encoding a modified BoNT / A varies compared to a given SEQ ID NO by way of % sequence identity, that the modified BoNT / A encoded still comprises at least one of the modifications (e.g. that increase pl), thus the relevant regions of the nucleotide sequence encoding said at least one of the modifications are still present (e.g. unmodified) in the variant nucleotide sequence.

[0386] A modified BoNT / A may comprise a substitution at one or more (preferably two or more, three or more, four or more, five or more or six or more, more preferably at all) of positions 930, 955, 991 , 1026, 1052, 1229, and 886. Preferably, a modified BoNT / A comprises lysine or arginine (more preferably lysine) at one or more of positions 930, 955, 991 , 1026, 1052, 1229, and 886. In one embodiment, the modified BoNT / A comprises lysine or arginine (more preferably lysine) at least two, three, four, five, six or all of positions 930, 955, 991 , 1026, 1052, 1229, and 886. Most preferably, the modified BoNT / A comprises lysine or arginine (more preferably lysine) at all of positions 930, 955, 991 , 1026, 1052, 1229, and 886.

[0387] A clostridial neurotoxin may comprise (or consist of) a hybrid or chimeric clostridial neurotoxin. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof, and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment the hybrid clostridial neurotoxin may comprise the entire light chain of a light chain from one clostridial neurotoxin subtype and the heavy chain from another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin may contain a portion (e.g. the binding domain) of the heavy chain of one clostridial neurotoxin subtype, with another portion of the heavy chain being from another clostridial neurotoxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain portions 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 subjects who are immunologically resistant to a given clostridial neurotoxin subtype, to subjects who may have a lower than average concentration of receptors to a given clostridial neurotoxin heavy chain binding domain, or to subjects who may have a protease-resistant variant of the membrane or vesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in US 8,071 ,110, which publication is hereby incorporated by reference in its entirety.

[0388] A clostridial neurotoxin of the invention may be one comprising a BoNT / B Hcc domain (e.g. a chimeric clostridial neurotoxin comprising a BoNT / B Hcc domain), preferably a chimeric clostridial neurotoxin comprising a BoNT / B Hcdomain. Thus, in a particularly preferred embodiment, a clostridial neurotoxin of the invention may be a chimeric clostridial neurotoxin comprising (preferably consisting of) a BoNT / A light-chain and translocation domain (LHNdomain), and a BoNT / B receptor binding domain (Hcdomain). Most preferably, said BoNT / B Hcdomain comprises the following substitutions E1191 M and S1199Y. A suitable chimeric clostridial neurotoxin may be one taught in WO 2017 / 191315 A1 , which is incorporated herein by reference. Such preferred sequences include SEQ ID NOs: 7-11 , with SEQ ID NO: 7 being most preferred.

[0389] The BoNT / A LHNdomain may be covalently linked to the BoNT / B Hcdomain. Said chimeric BoNT / A is also referred to herein as “BoNT / AB” or a “BoNT / AB chimera”.

[0390] The C-terminal amino acid residue of the LHNdomain may correspond to the first amino acid residue of the 3™ helix separating the LHNand Hcdomains of BoNT / A, and the N-terminal amino acid residue of the Hcdomain may correspond to the second amino acid residue of the 3w helix separating the LHNand Hcdomains in BoNT / B.

[0391] Reference herein to the “first amino acid residue of the 3™ helix separating the LHNand Hcdomains of BoNT / A” means the N-terminal residue of the 3™ helix separating the LHNand Hcdomains.

[0392] Reference herein to the “second amino acid residue of the 3™ helix separating the LHNand Hcdomains of BoNT / B” means the amino acid residue following the N-terminal residue of the 3™ helix separating the LHNand Hcdomains.

[0393] A “3 helix” is a type of secondary structure found in proteins and polypeptides, along with a- helices, p-sheets and reverse turns. The amino acids in a 3™ helix are arranged in a right- handed helical structure where each full turn is completed by three residues and ten atoms that separate the intramolecular hydrogen bond between them. Each amino acid corresponds to a 120° turn in the helix (i.e., the helix has three residues per turn), and a translation of 2.0 A (= 0.2 nm) along the helical axis, and has 10 atoms in the ring formed by making the hydrogen bond. Most importantly, the N-H group of an amino acid forms a hydrogen bond with the C = O group of the amino acid three residues earlier; this repeated i + 3 — ► i hydrogen bonding defines a 3w helix. A 3™ helix is a standard concept in structural biology with which the skilled person is familiar.

[0394] This 3w helix corresponds to four residues which form the actual helix and two cap (or transitional) residues, one at each end of these four residues. The term “3 helix separating the LHNand Hcdomains” as used herein consists of those 6 residues.

[0395] Through carrying out structural analyses and sequence alignments, a 3™ helix separating the LHNand Hcdomains was identified. This 3™ helix is surrounded by an a-helix at its N-terminus (i.e. at the C-terminal part of the LHNdomain) and by a p-strand at its C-terminus (i.e. at the N-terminal part of the Hcdomain). The first (N-terminal) residue (cap or transitional residue) of the 3w helix also corresponds to the C-terminal residue of this a-helix.

[0396] The 3w helix separating the LHNand Hcdomains can be for example determined from publicly available crystal structures of botulinum neurotoxins, for example 3BTA (http: / / www.rcsb. org / pdb / explore / explore.do?structureld=3BTA) and 1 EPW

[0397] (http: / / www.rcsb. org / pdb / explore / explore.do?structureld=1 EPW) for botulinum neurotoxins A1 and B1 respectively.

[0398] In silica modelling and alignment tools which are publicly available can also be used to determine the location of the 3™ helix separating the LHNand Hcdomains in other neurotoxins, for example the homology modelling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and a number of other tools / services listed at the Internet Resources for Molecular and Cell Biologists (http: / / molbiol- tools.ca / ). In particular that the region around the “HN / HCN” junction is structurally highly conserved which renders it an ideal region to superimpose different serotypes. For example, the following methodology may be used to determine the sequence of this 3™ helix in other neurotoxins:

[0399] 1. The structural homology modelling tool LOOP (http: / / loopp.org) was used to obtain a predicted structure of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb);

[0400] 2. The structural (pdb) files thus obtained were edited to include only the N-terminal end of the HCN domain and about 80 residues before it (which are part of the HNdomain), thereby retaining the “HN / HCN” region which is structurally highly conserved;

[0401] 3. The protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) was used to superpose each serotype onto the 3BTA.pdb structure;

[0402] 4. The superposed pdb files were inspected to locate the 3™ helix at the start of the Hcdomain of BoNT / A1 , and corresponding residues in the other serotype were then identified; 5. The other BoNT serotype sequences were aligned with Clustal Omega in order to check that corresponding residues were correct.

[0403] Examples of LHN, Hcand 3 helix domains determined by this method are presented below:

[0404] Using structural analysis and sequence alignments, it was found that the p-strand following the 3w helix separating the LHNand Hcdomains is a conserved structure in all botulinum and tetanus neurotoxins and starts at the 8thresidue when starting from the first residue of the 3™ helix separating the LHNand Hcdomains (e.g., at residue 879 for BoNT / A1).

[0405] A BoNT / AB chimera may comprise an LHNdomain from BoNT / A covalently linked to a Hcdomain from BoNT / B,

[0406] • wherein the C-terminal amino acid residue of the LHNdomain corresponds to the eighth amino acid residue N-terminally to the p-strand located at the beginning (N-term) of the

[0407] Hcdomain of BoNT / A, and

[0408] • wherein the N-terminal amino acid residue of the Hcdomain corresponds to the seventh amino acid residue N-terminally to the p-strand located at the beginning (N- term) of the Hcdomain of BoNT / B. A BoNT / AB chimera may comprise an LHNdomain from BoNT / A covalently linked to a Hcdomain from BoNT / B,

[0409] • wherein the C-terminal amino acid residue of the LHNdomain corresponds to the C- terminal amino acid residue of the a-helix located at the end (C-term) of LHNdomain of BoNT / A, and

[0410] • wherein the N-terminal amino acid residue of the Hcdomain corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the a-helix located at the end (C-term) of LHNdomain of BoNT / B.

[0411] The rationale of the design process of the BoNT / AB chimera was to try to ensure that the secondary structure was not compromised and thereby minimise any changes to the tertiary structure. Without wishing to be bound by theory, it is hypothesized that by not disrupting the four central amino acid residues of the 3™ helix in the BoNT / AB chimera ensures an optimal conformation for the chimeric neurotoxin.

[0412] The LHNdomain from BoNT / A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 12, or a polypeptide sequence having at least 70% sequence identity thereto. The LHNdomain from BoNT / A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 12, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. Preferably, the LHNdomain from BoNT / A corresponds to amino acid residues 1 to 872 of SEQ ID NO: 12.

[0413] The Hcdomain from BoNT / B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 13, or a polypeptide sequence having at least 70% sequence identity thereto. The Hcdomain from BoNT / B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 13, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. Preferably, the Hcdomain from BoNT / B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 13.

[0414] Preferably, the LHNdomain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 12) and the Hcdomain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO: 13).

[0415] Preferably, a BoNT / B Hcdomain further comprises at least one amino acid residue substitution, addition or deletion in the Hcc domain (e.g. subdomain) which has the effect of increasing the binding affinity of BoNT / B neurotoxin for human SYT-II as compared to the natural BoNT / B sequence. Suitable amino acid residue substitution, addition or deletion in the BoNT / B Hcc domain have been disclosed in WO 2013 / 180799 and in WO 2016 / 154534 (both herein incorporated by reference).

[0416] Suitable amino acid residue substitution, addition or deletion in the BoNT / B Hcc domain include substitution mutations selected from: V1118M; Y1183M; E1191 M; E1191 I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191 L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0417] Suitable amino acid residue substitution, addition or deletion in the BoNT / B Hcc domain further include combinations of two substitution mutations selected from the: E1191 M and S1199L, E1191 M and S1199Y, E1191 M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191 M and S1199W, E1191 M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0418] Suitable amino acid residue substitution, addition or deletion in the BoNT / B Hcc domain also include a combination of three substitution mutations which are E1191 M, S1199W and W1178Q.

[0419] Preferably, the suitable amino acid residue substitution, addition or deletion in the BoNT / B Hcc domain includes a combination of two substitution mutations which are E1191 M and S1199Y.

[0420] The modification may be a modification when compared to unmodified BoNT / B shown as SEQ ID NO: 13, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 13. As the presence of a methionine residue at position 1 of SEQ ID NO: 13 is optional, the skilled person will take the presence / absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 13 includes a methionine, the position numbering will be as defined above (e.g. E1191 will be E1191 of SEQ ID NO: 13). Alternatively, where the methionine is absent from SEQ ID NO: 13 the amino acid residue numbering should be modified by -1 (e.g. E1191 will be E1190 of SEQ ID NO: 13). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present / absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.

[0421] A chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 7-11. In one embodiment, a chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 7-11. Preferably, a chimeric clostridial neurotoxin may comprise any one of SEQ ID NOs: 7-11. Achimeric clostridial neurotoxin may consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 7-11. In one embodiment, a chimeric clostridial neurotoxin may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 7-11. Preferably, a chimeric clostridial neurotoxin may consist of any one of SEQ ID NOs: 7-11. Of the recited SEQ ID NOs, SEQ ID NO: 7 is most preferred. The skilled person will appreciate that where the polypeptide sequence of a chimeric clostridial neurotoxin comprising at least one BoNT / B Hcc domain mutation varies compared to a given SEQ ID NO byway of % sequence identity, that the at least one BoNT / B Hcc domain mutations (preferably E1191 M and S1199Y) are present (e.g. are unmodified) in the variant chimeric clostridial neurotoxin.

[0422] Thus, a composition of the invention most preferably comprises a chimeric clostridial neurotoxin, such as a chimeric clostridial neurotoxin as described above. In some embodiments, the L-chain is a BoNT / A L-chain, the HNdomain is a BoNT / A HNdomain, and the Hcc domain (e.g. Hcdomain) is a BoNT / B Hcc domain (e.g. a BoNT / B Hcc domain).

[0423] In another embodiment, a clostridial neurotoxin of the invention may be a chimeric clostridial neurotoxin comprising a BoNT / X light-chain and translocation domain (LHNdomain), and a receptor binding domain (Hcdomain) or a portion thereof from a different (i.e. non-BoNT / X) clostridial neurotoxin. A suitable chimeric and / or hybrid clostridial neurotoxin may be one taught in WO 2020 / 065336 A1 , which is incorporated herein by reference.

[0424] A clostridial neurotoxin of the invention may be one comprising a BoNT / E Hcc domain. A clostridial neurotoxin of the invention may be BoNT / E. A clostridial neurotoxin may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 35 or SEQ ID NO: 80. In one embodiment, a clostridial neurotoxin may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 35 or SEQ ID NO: 80. Preferably, a clostridial neurotoxin may comprise SEQ ID NO: 35 or SEQ ID NO: 80. A clostridial neurotoxin may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 35 or SEQ ID NO: 80. In one embodiment, a clostridial neurotoxin may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 35 or SEQ ID NO: 80. Preferably, a clostridial neurotoxin may consist of SEQ ID NO: 35 or SEQ ID NO: 80. A composition of the invention may comprise a clostridial neurotoxin having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 35 or SEQ ID NO: 80. A composition of the invention may comprise a clostridial neurotoxin that comprises SEQ ID NO: 35 or SEQ ID NO: 80. A composition of the invention may comprise a clostridial neurotoxin that consists of SEQ ID NO: 35 or 80. Of SEQ ID NOs: 35 and 80, SEQ ID NO: 80 is most preferred.

[0425] In embodiments where a clostridial neurotoxin described herein has a tag for purification (e.g. a His-tag) and / or a linker, said tag and / or linker are optional.

[0426] The clostridial neurotoxins of the present invention may be free from the complexing proteins that are present in a naturally occurring clostridial neurotoxin complex.

[0427] The clostridial neurotoxins of the present invention can be produced using recombinant nucleic acid technologies. Thus, in one embodiment, a clostridial neurotoxin (as described above) is a recombinant clostridial neurotoxin.

[0428] In one embodiment a nucleic acid (for example, a DNA) comprising a nucleic acid sequence encoding a clostridial neurotoxin is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence may be selected from any of the nucleic acid sequences described herein.

[0429] In a preferred embodiment, the vector has a promoter selected from:

[0430] Promoter Induction Agent Typical Induction Condition

[0431] Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM)

[0432] AraBAD L-arabinose 0.2% (0.002-0.4%)

[0433] T7-lac operator IPTG 0.2 mM (0.05-2.0mM)

[0434] In another preferred embodiment, the vector has a promoter selected from:

[0435] Promoter Induction Agent Typical Induction Condition

[0436] Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM)

[0437] AraBAD L-arabinose 0.2% (0.002-0.4%)

[0438] T7-lac operator IPTG 0.2 mM (0.05-2.0mM)

[0439] T5-lac operator IPTG 0.2 mM (0.05-2.0mM) The nucleic acid molecules may be made using any suitable process known in the art. Thus, the nucleic acid molecules may be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention may be made using molecular biology techniques.

[0440] The DNA construct of the present invention is preferably designed in silico, and then synthesised by conventional DNA synthesis techniques.

[0441] The above-mentioned nucleic acid sequence information is optionally modified for codonbiasing according to the ultimate host cell (e.g. E. coli) expression system that is to be employed.

[0442] The terms “nucleotide sequence” and “nucleic acid” are used synonymously herein. Preferably the nucleotide sequence is a DNA sequence.

[0443] A clostridial neurotoxin of the invention is preferably present as a di-chain clostridial neurotoxin in which the L-chain is linked to the H-chain (or component thereof, e.g. the HNdomain) via a di-sulphide bond. Thus, a clostridial neurotoxin of the invention may be any clostridial neurotoxin or variant (expressed by way of % sequence identity to a given SEQ ID NO) herein that has been cleaved by a protease in its activation loop (at one or more sites).

[0444] A clostridial neurotoxin preferably comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 14 with a protease in its activation loop at one or more sites. In one embodiment, a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 14 with a protease in its activation loop at one or more sites. Preferably, a clostridial neurotoxin comprises an L-chain and H- chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising SEQ ID NO: 14 with a protease in its activation loop at one or more sites.

[0445] A clostridial neurotoxin most preferably comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 7 with a protease in its activation loop at one or more sites. In one embodiment, a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 7 with a protease in its activation loop at one or more sites. Preferably, a clostridial neurotoxin comprises an L-chain and H- chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising SEQ ID NO: 7 with a protease in its activation loop at one or more sites.

[0446] A clostridial neurotoxin preferably comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 35 or 80 with a protease in its activation loop at one or more sites. A clostridial neurotoxin comprises an L-chain and H- chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 35 or 80 with a protease in its activation loop at one or more sites. Preferably, a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising SEQ ID NO: 35 or 80 with a protease in its activation loop at one or more sites.

[0447] In a particularly preferred embodiment, a di-chain clostridial neurotoxin comprises (or consists of) a light-chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 66 or 67 (preferably SEQ ID NO: 66) and a heavychain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 68, wherein the light-chain and heavy-chain are joined together by a di-sulphide bond. More preferably, a di-chain clostridial neurotoxin comprises (or consists of) a light-chain comprising SEQ ID NO: 66 or 67 (preferably SEQ ID NO: 66) and a heavy-chain comprising SEQ ID NO: 68, wherein the light-chain and heavy-chain are joined together by a di-sulphide bond. Even more preferably, a di-chain clostridial neurotoxin comprises (or consists of) a light-chain having SEQ ID NO: 66 and a heavy-chain having SEQ ID NO: 68, wherein the light-chain and heavy-chain are joined together by a di-sulphide bond. The di-sulphide bond is preferably formed by and / or is between the cysteine residue at position 429 of SEQ ID NO: 66 or 67 and the cysteine residue at position 6 of SEQ ID NO: 68. Said di- chain clostridial neurotoxin may correspond to a di-chain form of SEQ ID NO: 7. The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving activation loops to produce di-chain clostridial neurotoxins are taught in WO 2014 / 080206, WO2014 / 079495, and EP2677029A2, which are incorporated herein by reference.

[0448] Suitable activation loop sequences are shown in the table below:

[0449] Lys-C may cleave an activation loop C-terminal to one or more of the lysine residues present therein. Where Lys-C cleaves the activation loop more than once, the skilled person will appreciate that a small peptide of the activation loop of a di-chain clostridial neurotoxin may be absent when compared to a SEQ ID NO shown herein. For example, SEQ ID NO: 64 or 65 may be absent.

[0450] Where the clostridial neurotoxin used in a method of the invention is BoNT / E, a protease used to cleave the activation loop is preferably trypsin, for example bovine trypsin (see WO2018 / 002348 and W02022 / 069903 both of which are incorporated herein by reference).

[0451] The invention provides, in one aspect, a method of producing a single-chain clostridial neurotoxin having a light chain and a heavy chain, the method comprising expressing a nucleic acid described herein in an expression host, lysing the host cell to provide a host cell homogenate containing the single-chain clostridial neurotoxin, and isolating the single-chain clostridial neurotoxin. In one aspect, the present invention provides a method of proteolytically processing a clostridial neurotoxin described herein, the method comprising contacting the clostridial neurotoxin with a protease that hydrolyses a peptide bond in the activation loop of the clostridial neurotoxin, thereby converting the (single-chain) clostridial neurotoxin into a corresponding di-chain clostridial neurotoxin (e.g. wherein the light chain and heavy chain are joined together by a disulphide bond).

[0452] The present invention therefore provides a di-chain clostridial neurotoxin obtainable by a method of the invention.

[0453] Where an initial methionine amino acid residue or a corresponding initial codon is indicated in any of the SEQ ID NOs disclosed herein, said residue / codon is optional. Preferably, said initial methionine amino acid residue or corresponding initial codon is absent.

[0454] In one aspect, the invention provides a method for producing a therapeutic and / or cosmetic clostridial neurotoxin composition, the method comprising:

[0455] (a) obtaining the results of a method according to the invention; and

[0456] (b) formulating and / or packaging the composition for therapeutic or cosmetic use when the clostridial neurotoxin activity (e.g. an activity level) is the same as or higher than a positive control; or

[0457] (c) subjecting the composition to further purification when the clostridial neurotoxin activity is lower than a positive control (e.g. a positive reference standard), and formulating and / or packaging the further purified composition for therapeutic or cosmetic use.

[0458] In one aspect, the invention provides a therapeutic and / or cosmetic clostridial neurotoxin composition obtainable by a method of the invention, optionally wherein the therapeutic or cosmetic clostridial neurotoxin composition is packaged.

[0459] The term “obtainable” as used herein also encompasses the term “obtained”.

[0460] In one aspect, the invention provides a therapeutic clostridial neurotoxin composition of the invention for use in medicine.

[0461] In one aspect, the invention provides use of a therapeutic clostridial neurotoxin composition of the invention in the manufacture of a medicament.

[0462] In one aspect, the invention provides a method for treating a subject in need thereof, comprising the step of administering an effective amount of a therapeutic clostridial neurotoxin composition of the invention. By "effective amount", it is meant that the chimeric neurotoxin or therapeutic (or pharmaceutical) composition is administered in a quantity sufficient to provide the effect for which it is indicated. As used herein, the term "subject" preferably refers to a human being or an animal, more preferably to a human being.

[0463] In one aspect, the invention provides a therapeutic clostridial neurotoxin composition of the invention for use in treating a disease or disorder. In one aspect, the invention provides use of a therapeutic clostridial neurotoxin composition of the invention in the manufacture of a medicament for treating a disease or disorder. In one aspect, the invention provides a method for treating a disease or disorder in a subject in need thereof, comprising the step of administering an effective amount of a therapeutic clostridial neurotoxin composition of the invention.

[0464] The disease or disorder may be selected from a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), neuromuscular disorder or condition of ocular motility (e.g. concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, Charley horse, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache or migraine), phantom pain (e.g. phantom limb pain), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and a smooth muscle disorder. Preferably, the disease or disorder may be selected from: limb spasticity (upper or lower); cervical dystonia; headache disorders (preferably migraine); blepharospasm; hemifacial spasm; and lower urinary tract disorders (e.g. bladder pain syndrome (preferably interstitial cystitis); overactive bladder; and detrusor overactivity (e.g. neurogenic detrusor overactivity).

[0465] In one aspect, the invention provides a non-therapeutic use of a cosmetic clostridial neurotoxin composition of the invention, for treating an aesthetic or cosmetic condition. In other words, the invention provides a method for treating an aesthetic or cosmetic condition in a subject in need thereof, comprising the step of administering an effective amount of a cosmetic clostridial neurotoxin composition of the invention to said subject. Preferably, said subject is a healthy subject, i.e. a subject which is not suffering from any disease.

[0466] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0467] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside for at least 16 hours e.g. 18-22 hours, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0468] (i) a first luciferase domain;

[0469] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0470] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0471] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours;

[0472] (c) lysing the cells, thereby providing a cell lysate; and

[0473] (d) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value.

[0474] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin within a composition, the method comprising:

[0475] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside for at least 16 hours e.g. 18-22 hours, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0476] (i) a first luciferase domain;

[0477] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0478] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0479] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours;

[0480] (c) lysing the cells, thereby providing a cell lysate; and

[0481] (d) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value. In one embodiment, the population of cells (preferably neuronal progenitor-like cells) may be undifferentiated or partially differentiated and may not be co-cultured with another cell type.

[0482] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0483] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside for at least 16 hours e.g. 18-22 hours, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0484] (i) a first luciferase domain;

[0485] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0486] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0487] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours;

[0488] (c) lysing the cells, thereby providing a cell lysate; and

[0489] (d) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value; and wherein:

[0490] (i) the population of cells (preferably neuronal progenitor-like cells) are undifferentiated or partially differentiated and not co-cultured with another cell type.

[0491] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin within a composition, the method comprising:

[0492] (a) contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside for at least 16 hours e.g. 18-22 hours, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0493] (i) a first luciferase domain;

[0494] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0495] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0496] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours; (c) lysing the cells, thereby providing a cell lysate; and

[0497] (d) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value and wherein:

[0498] (i) the population of cells (preferably neuronal progenitor-like cells) are undifferentiated or partially differentiated and not co-cultured with another cell type.

[0499] In one embodiment, the population of cells may not be incubated (preferably in a neural differentiation media containing neural differentiation supplements e.g. B27) for more than 115 minutes (preferably no more than 95 minutes) prior to contact with a ganglioside. In one embodiment, the population of cells may not be incubated in a neural differentiation media comprising a neural differentiation supplement selected from the group consisting of retinoic acid, purmorphamine, B18, N2, N21 or GS21. The population of cells may be undifferentiated. The population of cells may be partially differentiated.

[0500] The ganglioside contacted with the population of cells may be GT1 b at a concentration of 50- 70 pg / ml e.g. 60 pg / ml; the ganglioside (GT1 b) may be removed from the cells prior to contacting the population of cells with the composition; and / or a luciferase substrate (e.g. Furimazine) may be added during or after lysing the cells.

[0501] Prior to contacting with a ganglioside at least 30,000 cells (preferably neuronal progenitor-like cells) may be seeded. The population of cells (preferably neuronal progenitor-like cells) may be NG108-cells. The cells (preferably neuronal progenitor-like cells) may express a ganglioside (preferably GT1 b) and a clostridial neurotoxin receptor (preferably SV2a, SV2c and / or SYT-II), wherein the clostridial neurotoxin receptors may be expressed at normal (e.g. wild type) levels for cells (i.e. the cells [preferably neuronal progenitor-like cells] are not genetically engineered to express the receptors). Where the cells express the clostridial neurotoxin receptors SV2c and / or SYT-II, the clostridial neurotoxin within the composition may be a BoNT / A, BoNT / B, or a chimeric clostridial neurotoxin comprising a botulinum neurotoxin A (BoNT / A) light-chain and translocation domain (HNdomain) and a BoNT / B receptor binding domain (Hcdomain). Where the cells express the clostridial neurotoxin receptors SV2a or SV2b, the clostridial neurotoxin within the composition may be a BoNT / E. During contact of the population of cells (preferably neuronal progenitor-like cells) with a ganglioside, further ganglioside(s) may be contacted with the population of cells (e.g. the cells may be contacted with at least one additional ganglioside selected from GT1a, GD1a and GM1). The step of determining the clostridial neurotoxin activity of the composition may comprise comparing the measured luciferase activity value with a luciferase activity value of a negative control. The negative control may be a control in which the population of cells (preferably neuronal progenitor-like cells) has been contacted with a composition that does not comprise a clostridial neurotoxin, or a control in which the population of cells (preferably neuronal progenitor-like cells) has not been contacted with a composition. Clostridial neurotoxin activity may be determined to be present when the measured luciferase activity value is less than the luciferase activity value of the negative control; or clostridial neurotoxin activity may be determined to be absent when the measured luciferase activity value is the same or greater than the luciferase activity value of the negative control. Determining the clostridial neurotoxin activity of the composition may further comprise comparing the measured luciferase activity value with a luciferase activity value of a positive control (e.g. of known potency) in which the population of cells has been contacted with a composition that comprises a clostridial neurotoxin. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be higher when the measured luciferase activity value is less than the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be the same when the measured luciferase activity value is the same as the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be lower when the measured luciferase activity value is greater than the luciferase activity value of the positive control.

[0502] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0503] (a) contacting a population of neuronal progenitor-like cells with a ganglioside for at least 18 hours e.g. 18-22 hours, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0504] (i) a first luciferase domain;

[0505] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0506] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0507] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours;

[0508] (c) lysing the cells, thereby providing a cell lysate; and (d) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value; and wherein:

[0509] (i) the population of neuronal progenitor-like cells are NG 108-15 cells and the cells are undifferentiated or partially differentiated and not co-cultured with another cell type;

[0510] (ii) the ganglioside contacted with the population of neuronal progenitor-like cells is GT1 b at a concentration of 50-70 pg / ml e.g. 60 pg / ml;

[0511] (iii) the ganglioside (GT1 b) is removed from the cells prior to contacting the population of cells with the composition; and

[0512] (iv) a luciferase substrate (e.g. Furimazine) is added during or after lysing the cells.

[0513] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin within a composition, the method comprising:

[0514] (a) contacting a population of neuronal progenitor-like cells with a ganglioside for at least 18 hours e.g. 18-22 hours, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0515] (i) a first luciferase domain;

[0516] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0517] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0518] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours;

[0519] (c) lysing the cells, thereby providing a cell lysate; and

[0520] (d) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value; and wherein:

[0521] (v) the population of neuronal progenitor-like cells are NG 108-15 cells and the cells are undifferentiated or partially differentiated and not co-cultured with another cell type;

[0522] (vi) the ganglioside contacted with the population of neuronal progenitor-like cells is GT1 b at a concentration of 50-70 pg / ml e.g. 60 pg / ml;

[0523] (vii) the ganglioside (GT1 b) is removed from the cells prior to contacting the population of cells with the composition; and

[0524] (viii) a luciferase substrate (e.g. Furimazine) is added during or after lysing the cells. In one embodiment, prior to contacting with a ganglioside at least 30,000 NG 108-15 cells may be seeded, and the cells may not be incubated for more than 115 minutes (preferably no more than 95 minutes) prior to contact with a ganglioside. In one embodiment, prior to contacting with a ganglioside at least 30,000 NG 108-15 cells may be seeded, and optionally the cells are not incubated for more than 95 minutes prior to contact with a ganglioside. The incubation step prior to contact with a ganglioside may involve incubating the cells in a neuronal differentiation media containing neural differentiation supplements (e.g. B27) for no more than 115 minutes (preferably no more than 95 minutes). In one embodiment, the population of neuronal progenitor-like cells may not be incubated in a neural differentiation media containing neural differentiation supplements selected from the group consisting of retinoic acid, purmorphamine, B18, N2, N21 and GS21 . The population of cells may be undifferentiated. The population of cells may be partially differentiated. The NG108-15 cells may express a ganglioside (preferably GT1 b) and a clostridial neurotoxin receptor (preferably SV2c and / or SYT-II), wherein the clostridial neurotoxin receptors may be expressed at normal (e.g. wild type) levels for NG108-15 cells (i.e. the NG108-15 cells are not genetically engineered to express the receptors). Where the cells express the clostridial neurotoxin receptors SV2c and / or SYT-II, the clostridial neurotoxin within the composition may be a BoNT / A, BoNT / B, or a chimeric clostridial neurotoxin comprising a botulinum neurotoxin A (BoNT / A) light-chain and translocation domain (HNdomain) and a BoNT / B receptor binding domain (Hcdomain). During contact of the population of NG108-15 cells with a ganglioside, further ganglioside(s) may be contacted with the population of cells (e.g. the cells may be contacted with at least one additional ganglioside selected from GT1a, GD1a and GM1).

[0525] The step of determining the clostridial neurotoxin activity of the composition may comprise comparing the measured luciferase activity value with a luciferase activity value of a negative control. The negative control may be a control in which the population of NG108-15 cells has been contacted with a composition that does not comprise a clostridial neurotoxin, or a control in which the population of NG 108-15 cells has not been contacted with a composition. Clostridial neurotoxin activity may be determined to be present when the measured luciferase activity value is less than the luciferase activity value of the negative control; or clostridial neurotoxin activity may be determined to be absent when the measured luciferase activity value is the same or greater than the luciferase activity value of the negative control. Determining the clostridial neurotoxin activity of the composition may further comprise comparing the measured luciferase activity value with a luciferase activity value of a positive control (e.g. of known potency) in which the population of cells has been contacted with a composition that comprises a clostridial neurotoxin. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be higher when the measured luciferase activity value is less than the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be the same when the measured luciferase activity value is the same as the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be lower when the measured luciferase activity value is greater than the luciferase activity value of the positive control.

[0526] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0527] (a) providing a population of cells (preferably neuronal progenitor-like cells), wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a singlechain polypeptide comprising:

[0528] (i) a first luciferase domain;

[0529] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0530] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0531] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours, wherein at least 30,000 cells are seeded prior to contacting;

[0532] (c) lysing the cells, thereby providing a cell lysate; and

[0533] (d) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value.

[0534] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin within a composition, the method comprising:

[0535] (a) providing a population of cells (preferably neuronal progenitor-like cells), wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a singlechain polypeptide comprising:

[0536] (i) a first luciferase domain;

[0537] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0538] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours, wherein at least 30,000 cells are seeded prior to contacting;

[0539] (c) lysing the cells, thereby providing a cell lysate; and

[0540] (d) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value.

[0541] In one embodiment, the population of cells may be undifferentiated or partially differentiated and may not be co-cultured with another cell type.

[0542] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0543] (a) providing a population of cells (preferably neuronal progenitor-like cells), wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a singlechain polypeptide comprising:

[0544] (i) a first luciferase domain;

[0545] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0546] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0547] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours, wherein at least 30,000 cells are seeded prior to contacting;

[0548] (c) lysing the cells, thereby providing a cell lysate; and

[0549] (d) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value; and wherein:

[0550] (i) the population of cells are undifferentiated or partially differentiated and not cocultured with another cell type.

[0551] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin within a composition, the method comprising:

[0552] (a) providing a population of cells (preferably neuronal progenitor-like cells), wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a singlechain polypeptide comprising:

[0553] (i) a first luciferase domain;

[0554] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0555] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0556] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours, wherein at least 30,000 cells are seeded prior to contacting;

[0557] (c) lysing the cells, thereby providing a cell lysate; and

[0558] (d) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value; and wherein:

[0559] (i) the population of cells are undifferentiated or partially differentiated and not cocultured with another cell type

[0560] In one embodiment, the population of cells may not be incubated (preferably in a neural differentiation media containing neural differentiation supplements e.g. B27) for more than 24 hours prior to contact with the composition. In one embodiment, the population of cells may not be incubated in a neural differentiation media containing neural differentiation supplements selected from the group consisting of retinoic acid, purmorphamine, B18, N2, N21 and GS21. A luciferase substrate (e.g. Furimazine) may be added during or after lysing the cells. The population of cells may be undifferentiated. The population of cells may be partially differentiated.

[0561] Before contacting the cells with the composition, the method may further comprise contacting a population of cells (preferably neuronal progenitor-like cells) with a ganglioside for at least 16 hours e.g. 18-22 hours, and prior to contact with the ganglioside the cells may be incubated (preferably in a neuronal differentiation media containing neural differentiation supplements e.g. B27) for no more than 115 minutes (preferably no more than 95 minutes). The ganglioside contacted with the population of cells (preferably neuronal progenitor-like cells) may be GT1 b (preferably at a concentration of 50-70 pg / ml e.g. 60 pg / ml). After contacting the cells with a ganglioside but prior to contacting the cells with the composition, the ganglioside (GT1 b) may be removed. Further ganglioside(s) may be contacted with the population of cells (e.g. the cells may be contacted with at least one additional ganglioside selected from GT1a, GD1a and GM1). In a further embodiment, the cells express a ganglioside (preferably GT1 b) and a clostridial neurotoxin receptor (preferably SV2a, SV2c and / or SYT-II), wherein the clostridial neurotoxin receptors are expressed at wild type levels for the cells (preferably neuronal progenitor-like cells) (i.e. the cells are not genetically engineered to express the receptors). Where the cells express the clostridial neurotoxin receptors SV2c and / or SYT-II, the clostridial neurotoxin within the composition may be a BoNT / A, BoNT / B, or a chimeric clostridial neurotoxin comprising a botulinum neurotoxin A (BoNT / A) light-chain and translocation domain (HNdomain) and a BoNT / B receptor binding domain (Hcdomain). The neuronal progenitor-like cells may express a ganglioside (preferably GT1 b) and a clostridial neurotoxin receptor (preferably SV2a or SV2b), wherein the clostridial neurotoxin receptors are expressed at wild type levels for the cells (preferably neuronal progenitor-like cells) (i.e. the cells are not genetically engineered to express the receptors). Where the cells express the clostridial neurotoxin receptors SV2a or SV2b, the clostridial neurotoxin within the composition may be a BoNT / E.

[0562] The step of determining the clostridial neurotoxin activity of the composition may comprise comparing the measured luciferase activity value with a luciferase activity value of a negative control. The negative control may be a control in which the population of neuronal progenitorlike cells has been contacted with a composition that does not comprise a clostridial neurotoxin, or a control in which the population of cells (preferably neuronal progenitor-like cells) has not been contacted with a composition. Clostridial neurotoxin activity may be determined to be present when the measured luciferase activity value is less than the luciferase activity value of the negative control; or clostridial neurotoxin activity may be determined to be absent when the measured luciferase activity value is the same or greater than the luciferase activity value of the negative control. Determining the clostridial neurotoxin activity of the composition may further comprise comparing the measured luciferase activity value with a luciferase activity value of a positive control (e.g. of known potency) in which the population of cells has been contacted with a composition that comprises a clostridial neurotoxin. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be higher when the measured luciferase activity value is less than the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be the same when the measured luciferase activity value is the same as the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be lower when the measured luciferase activity value is greater than the luciferase activity value of the positive control.

[0563] In one aspect, the invention provides a method for determining the clostridial neurotoxin activity of a composition, the method comprising:

[0564] (a) providing a population of neuronal progenitor-like cells, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0565] (i) a first luciferase domain; (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0566] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0567] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours, wherein at least 30,000 cells are seeded prior to contacting;

[0568] (c) lysing the cells, thereby providing a cell lysate; and

[0569] (d) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value; and wherein:

[0570] (i) the population of neuronal progenitor-like cells are NG 108-15 cells and the cells are undifferentiated or partially differentiated and not co-cultured with another cell type;

[0571] (ii) a luciferase substrate (e.g. Furimazine) is added during or after lysing the cells.

[0572] In one aspect, the invention provides a method for determining the presence or absence of a clostridial neurotoxin within a composition, the method comprising:

[0573] (a) providing a population of neuronal progenitor-like cells, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:

[0574] (i) a first luciferase domain;

[0575] (ii) a linker comprising a clostridial neurotoxin cleavage site; and

[0576] (iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;

[0577] (b) contacting the population of cells with the composition for greater than 60 hours e.g. 65-75 hours, wherein at least 30,000 cells are seeded prior to contacting;

[0578] (c) lysing the cells, thereby providing a cell lysate; and

[0579] (d) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate to obtain a measured luciferase activity value; and wherein:

[0580] (i) the population of neuronal progenitor-like cells are NG 108-15 cells and the cells are undifferentiated or partially differentiated and not co-cultured with another cell type;

[0581] (ii) a luciferase substrate (e.g. Furimazine) is added during or after lysing the cells. In one embodiment, before contacting the cells with the composition, the method may further comprise contacting a population of neuronal progenitor-like cells with a ganglioside for at least 18 hours e.g. 18-22 hours, wherein (i) the ganglioside contacted with the population of neuronal progenitor-like cells is GT1 b at a concentration of 50-70 pg / ml e.g. 60 pg / ml. Prior to contacting with a ganglioside, the neuronal progenitor-like cells may be incubated (preferably in a neuronal differentiation media containing neural differentiation supplements e.g. B27) for no more than 115 minutes (preferably no more than 95 minutes). In one embodiment, the population of neuronal progenitor-like cells may not be incubated in a neural differentiation media containing neural differentiation supplements selected from the group consisting of retinoic acid, purmorphamine, B18, N2, N21 and GS21. The population of cells may be undifferentiated. The population of cells may be partially differentiated. After contacting the cells with a ganglioside but prior to contacting the cells with the composition, the ganglioside (GT1 b) may be removed. In a further embodiment, further ganglioside(s) may be contacted with the population of cells (e.g. the cells may be contacted with at least one additional ganglioside selected from GT1a, GD1a and GM1). In a further embodiment, the NG108-15 cells express a ganglioside (preferably GT1 b) and a clostridial neurotoxin receptor (preferably SV2c and / or SYT-II), wherein the clostridial neurotoxin receptors are expressed at wild type levels for NG 108-15 cells (i.e. the NG 108-15 cells are not genetically engineered to express the receptors). Where the cells express the clostridial neurotoxin receptors SV2c and / or SYT- II, the clostridial neurotoxin within the composition may be a BoNT / A, BoNT / B, or a chimeric clostridial neurotoxin comprising a botulinum neurotoxin A (BoNT / A) light-chain and translocation domain (HNdomain) and a BoNT / B receptor binding domain (Hcdomain). The NG 108- 15 cells may express a ganglioside (preferably GT1 b) and a clostridial neurotoxin receptor (preferably SV2a), wherein the clostridial neurotoxin receptors are expressed at wild type levels for NG108-15 cells (i.e. the NG108-15 cells are not genetically engineered to express the receptors). Where the cells express the clostridial neurotoxin receptor SV2a or Sv2b (preferably SV2a), the clostridial neurotoxin within the composition may be a BoNT / E.

[0582] The step of determining the clostridial neurotoxin activity of the composition may comprise comparing the measured luciferase activity value with a luciferase activity value of a negative control. The negative control may be a control in which the population of NG108-15 cells has been contacted with a composition that does not comprise a clostridial neurotoxin, or a control in which the population of NG 108-15 cells has not been contacted with a composition. Clostridial neurotoxin activity may be determined to be present when the measured luciferase activity value is less than the luciferase activity value of the negative control; or clostridial neurotoxin activity may be determined to be absent when the measured luciferase activity value is the same or greater than the luciferase activity value of the negative control. Determining the clostridial neurotoxin activity of the composition may further comprise comparing the measured luciferase activity value with a luciferase activity value of a positive control (e.g. of known potency) in which the population of cells has been contacted with a composition that comprises a clostridial neurotoxin. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be higher when the measured luciferase activity value is less than the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be the same when the measured luciferase activity value is the same as the luciferase activity value of the positive control. The level of clostridial neurotoxin activity and / or the potency of the composition may be determined to be lower when the measured luciferase activity value is greater than the luciferase activity value of the positive control.

[0583] In one aspect, the invention provides a kit comprising:

[0584] (a) a population of cells (preferably neuronal progenitor-like cells) described herein; and

[0585] (b) a ganglioside described herein;

[0586] (c) optionally means for detecting luminescence of functional luciferase; and / or

[0587] (d) optionally instructions for the use of the same.

[0588] Where an initial methionine amino acid residue or a corresponding initial codon is indicated in any of the SEQ ID NOs described herein, said residue / codon is optional. Preferably, said initial methionine amino acid residue or corresponding initial codon is absent. For example, said initial methionine amino acid residue may be removed during production of a polypeptide described herein in a host cell.

[0589] The methods described herein are preferably in vitro methods.

[0590] Embodiments related to the various methods of the invention are intended to be applied equally to alternative methods and / or products, and vice versa.

[0591] SEQUENCE HOMOLOGY

[0592] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., 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, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0593] Thus, percent 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 the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1 , and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes); preferably this method is used to align a sequence with a SEQ ID NO described herein to define amino acid position numbering, as described herein.

[0594] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides I amino acids divided by the total number of nucleotides I amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

[0595] A 4

[0596] R-1 5

[0597] N-2 0 6

[0598] D-2-2 1 6

[0599] C 0-3 -3 -3 9

[0600] Q-1 1 0 0-3 5

[0601] E-1 0 0 2-4 2 5

[0602] G 0-2 0-1 -3 -2 -2 6

[0603] H -2 0 1 -1 -3 0 0-2 8

[0604] I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

[0605] L-1 -2 -3 -4-1 -2 -3 -4 -3 2 4

[0606] K-1 2 0-1 -3 1 1 -2-1 -3-2 5

[0607] M -1 -1 -2-3-1 0-2 -3 -2 1 2-1 5

[0608] F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0-3 0 6

[0609] P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

[0610] S 1 -1 1 0-1 0 0 0-1 -2-2 0-1 -2-1 4

[0611] T 0 -1 0-1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2-1 1 5

[0612] W-3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -211

[0613] Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

[0614] V 0-3-3 -3 -1 -2 -2 -3-3 3 1 -2 1 -1 -2 -2 0-3-1 4

[0615] The percent identity is then calculated as:

[0616] Total number of identical matches

[0617] > x 100

[0618] [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

[0619] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0620] CONSERVATIVE AMINO ACID SUBSTITUTIONS

[0621] Basic: arginine lysine histidine

[0622] Acidic: glutamic acid aspartic acid

[0623] Polar: glutamine asparagine

[0624] Hydrophobic: leucine isoleucine valine

[0625] Aromatic: phenylalanine tryptophan tyrosine

[0626] Small: glycine alanine serine threonine methionine

[0627] In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of 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 may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.

[0628] Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAare known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, 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 a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271 :19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0629] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.

[0630] 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). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction 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 identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention. Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable 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).

[0631] 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 belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.

[0632] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0633] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

[0634] Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein", as used herein, 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 instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0635] Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.

[0636] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0637] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a clostridial neurotoxin” includes a plurality of such candidate agents and reference to “the clostridial neurotoxin” includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0638] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0639] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0640] Figure 1 shows a schematic diagram of an exemplary single chain polypeptide providing a functional luciferase having a first luciferase domain; a linker comprising a SNAP-25 cleavage site flanked by spacers; and a second luciferase domain.

[0641] Figure 2 shows the effect of expressing a single chain polypeptide of Figure 1 on sensitivity to BoNT activity of a composition containing (A) mrBoNT / A or (B) mrBoNT / AB.

[0642] Figure 3 shows the effect of addition of GT1 b to NG108-15 cells prior to addition of a BoNT composition on sensitivity to BoNT activity of a composition comprising (A) mrBoNT / AB or (B) mrBoNT / A. Tables (C) and (D) show that addition of GT1 b improves the EC5o values relative to control (no supplementation or DMSO) for compositions comprising (C) mrBoNT / AB or (D) mrBoNT / A. BoNT activity was determined by measuring luminescence of cells expressing a single chain polypeptide of Figure 1 .

[0643] Figure 4 shows the effect of overexpressing the clostridial neurotoxin receptors SV2c and SYT-II on cell morphology(A) cell morphology for NG108-15 cells expressing SV2c; and (B) cell morphology for NG108-15 cells expressing SYT-II.

[0644] Figure 5 shows the effect of cell seeding density on the assay sensitivity to BoNT / A at various concentrations: top concentration = 200 ng / ml mrBoNT / AB (A), top concentration = 300 ng / ml mrBoNT / AB (B), and top concentration = 400 ng / ml mrBoNT / AB (C). Table (D) shows EC50 values for cell densities of 30,00 cells, 40,000 cells and 50,000 cells. A higher cell density was found to increase sensitivity to BoNT / AB regardless of BoNT / AB concentration.

[0645] SEQUENCE LISTING

[0646] Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue / codon is optional. Preferably, said initial Met amino acid residue or corresponding initial codon is absent. SEQ ID NO: 1 - Polypeptide Sequence of Luciferase

[0647] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPY

[0648] EGLSADQMAQIEEVFKWYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKI

[0649] TVTGTLWNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL

[0650] SEQ ID NO: 2 - Polypeptide Sequence of Luciferase Domain 1

[0651] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPY

[0652] EGLSADQMAQIEEVFKWYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKI

[0653] TVTGTLWNGNKIIDERLITPDGSMLFRVTINS

[0654] SEQ ID NO: 3 - Polypeptide Sequence of Luciferase Domain 2

[0655] VTGYRLFEEIL

[0656] SEQ ID NO: 4 Polypeptide Sequence of Full-Length SNAP-25

[0657] MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIE

[0658] EGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGWASQPARW

[0659] DEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIME KADSNKTRIDEANQRATKMLGSG

[0660] SEQ ID NO: 5 - Polypeptide Sequence of 65 Amino Acid SNAP-25

[0661] RENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRAT KMLGSG

[0662] SEQ ID NO: 6 - Polypeptide Sequence of Reporter Construct (N-term luciferase domain, spacer, 65 Amino Acid SNAP-25 cleavage site, spacer, C-term luciferase domain)

[0663] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPY

[0664] EGLSADQMAQIEEVFKWYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKI

[0665] TVTGTLWNGNKIIDERLITPDGSMLFRVTINSSGGGGSRENEMDENLEQVSGIIGNLRHMAL

[0666] DMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSGSGGGGSVTGYRLFEEIL

[0667] SEQ ID NO: 7 - Polypeptide Sequence of mrBoNT / AB

[0668] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP

[0669] EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTE

[0670] LKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPD

[0671] FTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMS

[0672] GLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVF KEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPK

[0673] VNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDK

[0674] GYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNF

[0675] DNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSV

[0676] NEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYI

[0677] GPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNE

[0678] KWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDD

[0679] LSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIG

[0680] QVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAK

[0681] VEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHN

[0682] EYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNA

[0683] KIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYS

[0684] EYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGE

[0685] KFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTI

[0686] QIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPY NLKLGCNWQFIPKDEGWTE

[0687] SEQ ID NO: 8 - Polypeptide Sequence of BoNT / AB Variant 2

[0688] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0689] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0690] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0691] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0692] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0693] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0694] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0695] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0696] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0697] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0698] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0699] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0700] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0701] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0702] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE

[0703] VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYI

[0704] HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT

[0705] ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK

[0706] YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES

[0707] GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0708] SEQ ID NO: 9 - Polypeptide Sequence of BoNT / AB Variant 3

[0709] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0710] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0711] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0712] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0713] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0714] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0715] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0716] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0717] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0718] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0719] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0720] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0721] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0722] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0723] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN

[0724] LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI

[0725] PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED

[0726] ISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI

[0727] WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK

[0728] DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL

[0729] NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE

[0730] IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH HHHHHHH

[0731] SEQ ID NO: 10 - Polypeptide Sequence of BoNT / AB Variant 4

[0732] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0733] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0734] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0735] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0736] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0737] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0738] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0739] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0740] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0741] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0742] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0743] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0744] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV

[0745] YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH

[0746] NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI

[0747] TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS

[0748] QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY

[0749] NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK

[0750] KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG

[0751] IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0752] SEQ ID NO: 11 - Polypeptide Sequence of BoNT / AB Variant 5

[0753] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0754] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0755] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0756] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0757] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0758] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0759] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0760] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0761] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0762] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0763] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0764] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0765] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0766] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0767] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV

[0768] YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH

[0769] NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS

[0770] QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY

[0771] NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK

[0772] KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG

[0773] IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0774] SEQ ID NO: 12 - Polypeptide Sequence of Native BoNT / A (BoNT / A)

[0775] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP

[0776] EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTE

[0777] LKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPD

[0778] FTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAG...

Claims

1. CLAIMS1. A method for determining the clostridial neurotoxin activity of a composition, the method comprising:(a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:(i) a first luciferase domain;(ii) a linker comprising a clostridial neurotoxin cleavage site; and(iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and(b) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity.

2. The method according to claim 1 , comprising:(a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:(i) a first luciferase domain;(ii) a linker comprising a clostridial neurotoxin cleavage site; and(iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;(b) lysing the cells, thereby providing a cell lysate; and(c) determining the clostridial neurotoxin activity of the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

3. A method for determining the presence or absence of a clostridial neurotoxin in a composition, the method comprising:(a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:(i) a first luciferase domain;(ii) a linker comprising a clostridial neurotoxin cleavage site; and(iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase; and(b) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity.

4. The method according to claim 3, comprising:(a) contacting a population of cells (preferably neuronal progenitor-like cells) with the composition, wherein the population of cells express: a clostridial neurotoxin receptor and / or a ganglioside; and a single-chain polypeptide comprising:(i) a first luciferase domain;(ii) a linker comprising a clostridial neurotoxin cleavage site; and(iii) a second luciferase domain; wherein the linker functionally joins the first and second luciferase domains, thereby providing a functional luciferase;(b) lysing the cells, thereby providing a cell lysate; and(c) determining the presence or absence of the clostridial neurotoxin in the composition by measuring luciferase activity of the cell lysate; wherein a luciferase substrate is added before, during or after lysing the cells (preferably during or after lysing the cells).

5. The method according to any one of the preceding claims, wherein the population of cells has been contacted with a ganglioside prior to contacting with the composition.

6. The method according to any one of the preceding claims, comprising contacting the population of cells with a ganglioside prior to contacting with the composition.

7. The method according to any one of the preceding claims, wherein the population of cells seeded prior to contacting comprises at least 30,000 cells, at least 35,000 cells, at least 40,000 cells, at least 45,000 cells, or at least 50,000 cells, preferably greater than 30,000 cells, more preferably wherein the population of cells seeded prior to containing comprises 50,000 cells.

8. The method according to any one of claims 5-7, wherein the population of cells when contacted with the ganglioside comprises at least 30,000 cells, 40,000 cells or 45,000 cells,preferably wherein the population of cells when contacted with the ganglioside comprises greater than 30,000 cells, more preferably wherein the population of cells when contacted with the ganglioside comprises 50,000 cells.

9. The method according to any one of the preceding claims, wherein the population of cells when contacted with the composition comprises at least 30,000 cells, 40,000 cells or 45,000 cells, preferably wherein the population of cells when contacted with the composition comprises greater than 30,000 cells, more preferably wherein the population of cells when contacted with the composition comprises 50,000 cells.

10. The method according to any one of claims 5-9, wherein the population of cells when contacted with the ganglioside comprises 30,000-100,000 cells, 30,000-60,000 cells, or 35,000-55,000 cells, preferably 45,000-55,000 cells, more preferably 50,000 cells.

11. The method according to any one of the preceding claims, wherein the population of cells when contacted with the composition comprises 30,000-100,000 cells, 30,000-60,000 cells, or 35,000-55,000 cells, preferably 45,000-55,000 cells, more preferably 50,000 cells.

12. The method according to claim 5 or claim 6, wherein the population of cells when contacted with the ganglioside is at a concentration of at least 300,000 cells / ml, at least 350,000 cells / ml, at least 400,000 cells / ml, at least 450,000 cells / ml, or at least 500,000 cells / ml, preferably greater than 300,000 cells / ml, more preferably at least 450,000 cells / ml.

13. The method according to any one of claims 5, 6 or 12, wherein the population of cells when contacted with the ganglioside is at a concentration of 300,000-1 ,000,000 cells / ml, 300,000-600,000 cells / ml, or 350,000-550,000 cells / ml, preferably, at a concentration of 450,000-550,000 cells / ml, more preferably 500,000 cells / ml.

14. The method according to any one of the preceding claims, wherein the population of cells when contacted with the composition is at a concentration of at least 300,000 cells / ml, at least 350,000 cells / ml, at least 400,000 cells / ml, at least 450,000 cells / ml, or at least 500,000 cells / ml, preferably greaterthan 300,000 cells / ml, more preferably at least 450,000 cells / ml.

15. The method according to any one of the preceding claims, wherein the population of cells when contacted with the composition is at a concentration of 300,000-1 ,000,000 cells / ml,300,000-600,000 cells / ml, or 350,000-550,000 cells / ml, preferably, at a concentration of 450,000-550,000 cells / ml, more preferably 500,000 cells / ml.

16. The method according to any one of claims 1 , 2, or 5-15, wherein determining the clostridial neurotoxin activity of the composition by measuring luciferase activity (e.g. of the cell lysate) further comprises obtaining a measured luciferase activity value and comparing the measured luciferase activity value with a luciferase activity value of a negative control in which the population of cells has been contacted with a composition that does not comprise a clostridial neurotoxin, or in which the population of cells has not been contacted with a composition.

17. The method according to claim 16, wherein: (i) clostridial neurotoxin activity is determined to be present when the measured luciferase activity value is less than the luciferase activity value of the negative control; or (ii) clostridial neurotoxin activity is determined to be absent when the measured luciferase activity value is the same or greater than the luciferase activity value of the negative control.

18. The method according to any one of claims 1 , 2, or 5-17, wherein determining the clostridial neurotoxin activity of the composition by measuring luciferase activity (e.g. of the cell lysate) further comprises obtaining a measured luciferase activity value and comparing the measured luciferase activity value with a luciferase activity value of a positive control (e.g. of known potency) in which the population of cells has been contacted with a composition that comprises a clostridial neurotoxin.

19. The method according to claim 18, wherein: (i) the level of clostridial neurotoxin activity and / or the potency of the composition is determined to be higher when the measured luciferase activity value is less than the luciferase activity value of the positive control; (ii) the level of clostridial neurotoxin activity and / or the potency of the composition is determined to be the same when the measured luciferase activity value is the same as the luciferase activity value of the positive control; or (iii) the level of clostridial neurotoxin activity and / or the potency of the composition is determined to be lower when the measured luciferase activity value is greater than the luciferase activity value of the positive control.

20. The method according to any one of claims 1 , 2, or 5-19, wherein determining the activity of the clostridial neurotoxin composition further comprises comparing the determined clostridial neurotoxin activity with a pre-determined activity value, optionally wherein theclostridial neurotoxin composition is selected for therapeutic and / or cosmetic use when the activity of the clostridial neurotoxin composition is the same or greater than the activity level corresponding to the pre-determined activity value.21 . The method according to any one of claims 1 , 2, or 5-20: further comprising contacting at least a second population of cells (e.g. a population of cells identical to the population of cells of any one of the preceding claims) with at least a second composition comprising a different concentration of the clostridial neurotoxin, and determining the clostridial neurotoxin activity of the at least second composition by measuring luciferase activity to obtain a measured luciferase activity value(s) and determining a potency value (EC5o) of the composition by comparing the measured luciferase activity value with the measured luciferase activity value(s) for the at least second composition, preferably further comprising contacting at least a third population of cells (e.g. a population of cells identical to the population of cells of any one of the preceding claims) with at least a third composition comprising a different concentration of the clostridial neurotoxin, and determining the clostridial neurotoxin activity of the at least third composition by measuring luciferase activity to obtain a measured luciferase activity value(s) and determining a potency value (EC5o) of the composition by comparing the measured luciferase activity value with the measured luciferase activity value(s) for the at least third and / or second compositions.

22. The method according to any one of claims 3-15, wherein:(a) the presence of a clostridial neurotoxin is indicated by a change (e.g. a statistically significant change) in the luciferase activity (e.g. of the cell lysate) overtime; and / or(b) absence of a clostridial neurotoxin is indicated by no change (e.g. no statistically significant change) in the luciferase activity (e.g. of the cell lysate) over time.

23. The method according to claim 22, wherein the presence or absence of clostridial neurotoxin in the composition is determined by comparing the measured luciferase activity to a control.

24. The method according to any one of the preceding claims, wherein the population of cells is contacted with the clostridial neurotoxin composition for greater than 60 hours, e.g. 65- 75 hours.

25. The method according to any one of claims 5-24, wherein the population of cells is contacted with one or more ganglioside, e.g. one or more, two or more, three or more, four or more, or five or more gangliosides.

26. The method according to any one of claims 5-25, wherein the ganglioside contacted with the population of cells comprises GM1 (e.g. GM1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, GD3, preferably GT1 b.

27. The method according to any one of claims 5-26, wherein the ganglioside (preferably GT1 b) is / was present (e.g. in a medium in which the cells are being cultured) at concentration of 10-100 pg / ml, 10-80 pg / ml, 25-100 pg / ml, 25-80 pg / ml, 30-90 pg / ml, 40- 100 pg / ml, 40-80 pg / ml or 50-70 pg / ml, preferably 50-70 pg / ml, preferably 60 pg / ml.

28. The method according to any one of claims 5-27, wherein the population of cells is contacted with the ganglioside for at least 10 hours, at least 15 hours, at least 18 hours.

29. The method according to any one of claims 5-28, wherein the population of cells is contacted with the ganglioside for up to 48 hours, up to 35 hours, 24 hours, up to 22 hours, up to 20 hours, up to 18 hours or up to 16 hours, preferably up to 24 hours, more preferably up to 22 hours.

30. The method according to any one of claims 5-29, wherein the population of cells is contacted with the ganglioside for a period of 16 to 24 hours, 17 to 23 hours, or 18 to 22 hours, preferably 18-22 hours.31 . The method according to any one of claims 5-30, wherein the ganglioside is contacted with the population of cells at 15-45°C, 15-40°C, 20°C-40°C, 25-40°C, 30-40°C or 35-40°C, preferably 37°C ±2°C.

32. The method according to any one of claims 5-31 , wherein the ganglioside is present in the medium (e.g. cell culture medium) during contacting with the composition.

33. The method according to any one of claims 5-31 , wherein the ganglioside is removed prior to contacting the population of cells with the composition.

34. The method according to any one of the preceding claims, wherein the population of cells is incubated for at least 20 minutes, 40 minutes, 50 minutes, 60 minutes, 65 minutes, 70 minutes, 80 minutes, 85 minutes, 90 minutes, 2 hours, 4 hours, 6 hours, 8 hours or 10 hours prior to any contacting step.

35. The method according to any one of the preceding claims, wherein the population of cells is incubated (e.g. in a neural differentiation media containing neural differentiation supplements) for no more than 24 hours (preferably no more than 18-24 hours) prior to any contacting step.

36. The method according to any one of the preceding claims, wherein the population of cells is incubated (e.g. in a neural differentiation media containing neural differentiation supplements) for no more than 24 hours (preferably no more than 18-24 hours) prior to contacting with the composition.

37. The method according to any one of claims 5-36, wherein the population of cells is incubated (e.g. in a neural differentiation media containing neural differentiation supplements) for no more than 115 minutes (preferably no more than 95 minutes, more preferably 60-90 minutes) prior to contacting with a ganglioside.

38. The method according to any one of claims 34-37, wherein the population of cells are incubated in a neural differentiation media.

39. The method according to claim 38, wherein the neural differentiation media comprises the neural differentiation supplement B27.

40. The method according to claim 38 or claim 39, wherein the neural differentiation media does not contain neural differentiation supplement selected from the group consisting of retinoic acid, purmorphamine, B18, N2, N21 or GS21.

41. The method according to any one of the preceding claims, wherein the population of cells are neuronal progenitor-like cells.

42. The method according to any one of the preceding claims, wherein the population of cells are immortalized cells (e.g. immortalized neuronal cells), preferably neuroblastoma cells or neuronal hybridoma cells.

43. The method according to any one of the preceding claims, wherein the population of cells are neuronal mouse / rat hybridoma cells.

44. The method according to any one of the preceding claims, wherein the population of cells are selected from selected from BE(2)-M17, C6-BU-1 , Kelly (NB-19), LA1-55n, LA-N-2, IMR-32, N1 E-115, NB41A3, N4TG3, N18, N18TG2, Neuro-2a (N2a), NG108-15, PC12, SH-SY5Y, SiMa, and / or SK-N-BE(2)-C.

45. The method according to any one of the preceding claims, wherein the population of cells are NG108-15 cells.

46. The method according to any one of the preceding claims, wherein the population of cells are undifferentiated or partially differentiated (preferably undifferentiated).

47. The method according to any one of the preceding claims, wherein the population of cells have not undergone one or more active differentiation steps prior to contacting with a ganglioside and / or composition.

48. The method according to any one of the preceding claims, wherein the population of cells have not undergone one or more active differentiation steps (e.g. culturing in neural differentiation media comprising neural differentiation supplements) for at least 2 days, at least 5 days, at least 7 days, at least 14 days, and up to 30 days, e.g. between 5-30 days prior to contacting with a ganglioside and / or composition.

49. The method according to any one of the preceding claims, wherein the population of cells are not co-cultured with cells of a different type, e.g. Schwann cells prior to contacting with a ganglioside and / or composition.

50. The method according to any one of the preceding claims, wherein:(a) the population of cells are not terminally-differentiated during any step of the methods of the invention; or(b) the population of cells are not terminally-differentiated when the population of cells are contacted with a ganglioside; or(c) the population of cells are not terminally-differentiated when the population of cells are contacted with a ganglioside and when the population of cells are contacted with the composition.

51. The method according to any one of the preceding claims, wherein the cells are not genetically modified to express or over express (e.g. express above / higher than wild type levels) the clostridial neurotoxin receptor and / or ganglioside.

52. The method according to any one of the preceding claims, wherein the clostridial neurotoxin and / or ganglioside are under the expression of a normal (wild type) promoter and are expressed at normal (e.g. wild type or physiological levels) for the cells.

53. The method according to any one of the preceding claims, wherein the cells are not genetically modified to express or over express (e.g. express above / higher than wild type levels) a SV2a, SV2c or SYT-II clostridial neurotoxin receptor and / or GD3 ganglioside.

54. The method according to any one of the preceding claims, wherein the cells express said clostridial neurotoxin receptor and / or ganglioside at least when the cell is contacted with a ganglioside.

55. The method according to any one of the preceding claims, wherein the cells continuously express said clostridial neurotoxin receptor and / or ganglioside.

56. The method according to any one of the preceding claims, wherein the population of cells express at least one (e.g. at least two) clostridial neurotoxin receptor selected from SV2a, SV2b, SV2c, SYT-I, or SYT-II or a combination thereof.

57. The method according to claim 56, wherein the clostridial neurotoxin receptor comprises (or consists of) an extracellular portion of is SV2a, SV2b, SV2c, SYT-I, or SYT-II.

58. The method according to claim 56, wherein the clostridial neurotoxin receptor comprises (or consists of) a full length SV2a, SV2b, SV2c, SYT-I, or SYT-II.

59. The method according to claim 58, wherein the clostridial neurotoxin receptor is SV2a, optionally wherein SV2a comprises (or consists of) a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 20.

60. The method according to claim 58, wherein the clostridial neurotoxin receptor is SV2b, optionally wherein SV2b comprises (or consists of) a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 21.

61. The method according to claim 58, wherein the clostridial neurotoxin receptor is SV2c, optionally wherein SV2c comprises (or consists of) a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22, 78, or 79.

62. The method according to claim 58, wherein the clostridial neurotoxin receptor is SYT-I, optionally wherein SYT-I comprises (or consists of) a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 23, 69 or 75.

63. The method according to claim 58, wherein the clostridial neurotoxin receptor is SYT-II, optionally wherein SYT-II comprises (or consists of) a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 18, 19 or 74.

64. The method according to any one of the preceding claims, wherein the population of cells express a ganglioside, and the ganglioside is selected from GM1 (e.g. GM 1a or GM 1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT 1 b, GQ1 b, GD2, GD3 or a combination thereof.

65. The method according to any one of the preceding claims, wherein the population of cells express at least one (e.g. at least two) ganglioside selected from GM1 (e.g. GM 1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT 1 a, GT1 b, GQ1 b, GD2, GD3 or a combination thereof.

66. The method according to claim 64 or claim 65, wherein the ganglioside contacted with the population of cells is the same as the ganglioside expressed by the cells, preferably the population of cells express (e.g. at natural / wild type levels) at least GT1 b and the population of cells is contacted with at least GT 1 b.

67. The method according to claim 64 or claim 65, wherein the ganglioside contacted with the population of cells is different to the ganglioside expressed by the cells, preferably the population of cells express at least GM3, GM2, GM1 and GD1a (e.g. at natural / wild type levels) and the population of cells may also be contacted with at least GT1 b.

68. The method according to any one of the preceding claims, wherein the population of cells express:(a) a clostridial neurotoxin receptor selected from synaptic vesicle glycoprotein 2 (SV2) isoform A (SV2a), SV2 isoform B (SV2b), SV2 isoform C (SV2c), synaptotagmin I (SYT-I), synaptotagmin II (SYT-II) or a combination thereof; and(b) a ganglioside selected from GM1 (e.g. GM1a or GM1 b), GM2, GM3 (e.g. NeuAc GM3 or NeuGc GM3), GM4, GD1a, GD1 b, GalNAc-GD1a, GT1a, GT1 b, GQ1 b, GD2, GD3 or a combination thereof.

69. The method according to claim 68, wherein the population of cells express SV2c and / or SYT-II at normal (e.g. wild type or physiological) levels for the cell type and express (e.g., either at normal or overexpressed levels) at least GT1 b.

70. The method according to claim 68, the population of cells express SV2a, SV2c and / or SYT- II at normal (e.g. wild type or physiological) levels for the cell type, and GM3, GM2, GM1 and GD1a at normal (e.g. wild type or physiological) levels.71 . The method according to any one of claims 64, 65, 67, 68 or 70, wherein the population of cells do no express GT 1 b.

72. The method according to any one of the preceding claims, wherein the population of cells express a clostridial neurotoxin receptor selected from SV2c, SV2a, or SV2b, preferably SV2c, and the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / A Hcc (preferably Hc) domain.

73. The method according to any one of claims 1-71 , wherein the population of cells express a clostridial neurotoxin receptor selected from SYT-II and / or SYT-I (preferably SYT-II) and the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / B Hcc (preferably Hc) domain.

74. The method according to any one of claims 1-71 , wherein the population of cells express a clostridial neurotoxin receptor selected from SV2a or SV2b, and the clostridial neurotoxin composition comprises a clostridial neurotoxin comprising a BoNT / E Hcc (preferably Hc) domain.

75. The method according to any one of claims 1-71 , wherein the clostridial neurotoxin is a chimeric clostridial neurotoxin, and / or wherein the clostridial neurotoxin is a chimeric clostridial neurotoxin comprising a botulinum neurotoxin A (BoNT / A) light-chain and translocation domain (HNdomain) and a BoNT / B receptor binding domain (Hcdomain), preferably wherein the BoNT / B Hcdomain comprises one or more substitution mutation(s) selected from: E1191 M; S1199Y; V1118M; Y1183M; E1191 I; E1191Q; E1191T; S1199F; S1199L; S1201V; and combinations thereof, preferably wherein the BoNT / B Hcdomain comprises substitution mutations at E1191 M and S1199Y.

76. The method according to any one of claims 1-71 , wherein the clostridial neurotoxin is a BoNT / A, BoNT / B, BoNT / 01 , BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X or tetanus neurotoxin (TeNT).

77. The method according to any one of claims 1-71 , wherein the clostridial neurotoxin is a BoNT / A comprising a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991 , GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081 , GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from:(i) substitution of an acidic surface exposed amino acid residue with a basic amino acid residue;(ii) substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue;(iii) substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue;(iv) insertion of a basic amino acid residue; and(v) deletion of an acidic surface exposed amino acid residue.

78. The method according to any one of claims 1-71 , wherein the clostridial neurotoxin is a BoNT / E.

79. The method according to claim 78, wherein the clostridial neurotoxin comprises (or consists of) a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 35 or 80, preferably wherein the clostridial neurotoxin comprises (or consists of) a polypeptide sequence of (or consists of) SEQ ID NO: 35 or 80.

80. A method for producing a therapeutic and / or cosmetic clostridial neurotoxin composition, the method comprising:(a) obtaining the results of a method according to any one of claims 1-2, 5-21 , or 24- 79; and(b) formulating and / or packaging the composition for therapeutic or cosmetic use when the clostridial neurotoxin activity (e.g. an activity level) is the same as or higher than a positive control; or(c) subjecting the composition to further purification when the clostridial neurotoxin activity is lower than a positive control (e.g. a positive reference standard), and formulating and / or packaging the further purified composition for therapeutic or cosmetic use.81 . A therapeutic and / or cosmetic clostridial neurotoxin composition obtainable (e.g. obtained) by the method according to claim 80.

82. A therapeutic clostridial neurotoxin composition according to claim 81 for use in medicine.

83. Use of a therapeutic clostridial neurotoxin composition according to claim 81 in the manufacture of a medicament.

84. A method for treating a subject in need thereof, comprising the step of administering an effective amount of a therapeutic clostridial neurotoxin composition according to claim 81.

85. A non-therapeutic use of a cosmetic clostridial neurotoxin composition according to claim 81 , for treating an aesthetic or cosmetic condition.

86. A method for treating an aesthetic or cosmetic condition in a subject in need thereof, comprising the step of administering an effective amount of a cosmetic clostridial neurotoxin composition according to claim 81.

87. A therapeutic clostridial neurotoxin composition according to claim 81 for use in treating a disease or disorder selected from a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), neuromuscular disorder or condition ofocular motility (e.g. concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, Charley horse, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache or migraine), phantom pain (e.g. phantom limb pain), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and a smooth muscle disorder.

88. Use of a therapeutic clostridial neurotoxin composition according to claim 81 in the manufacture of a medicament for treating a disease or disorder, wherein the disease or disorder is selected from a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), neuromuscular disorder or condition of ocular motility (e.g. concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, Charley horse, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache or migraine), phantom pain (e.g. phantom limb pain), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and a smooth muscle disorder.

89. A method for treating a disease or disorder in a subject in need thereof, comprising the step of administering an effective amount of a therapeutic clostridial neurotoxincomposition according to claim 81 , wherein the disease or disorder is selected from a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), neuromuscular disorder or condition of ocular motility (e.g. concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, Charley horse, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache or migraine), phantom pain (e.g. phantom limb pain), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and a smooth muscle disorder.

90. A kit comprising:(a) a population of cells (preferably neuronal progenitor-like cells); and(b) a ganglioside;(c) optionally means for detecting luminescence of functional luciferase, such as a luciferase substrate; and / or(d) optionally instructions for the use of the same.

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