Genetically modified cells susceptible to clostridial neurotoxins

Abstract

Cells that have been genetically modified to be highly sensitive to clostridial neurotoxins, such as botulinum neurotoxin and tetanus neurotoxin, or modified or recombinant versions thereof, methods for making such genetically modified cells, and methods for using such cells in assays for the activity of the modified or recombinant clostridial neurotoxins.

Description

[Technical field]

[0001] The present invention relates generally to cells that have been genetically modified to have increased sensitivity to Clostridial neurotoxins, such as botulinum neurotoxins and tetanus neurotoxins. The present invention also relates to methods of making such cells and to methods of using such cells in assays for the activity of polypeptides derived from such neurotoxins, such as modified and recombinant versions of such Clostridial neurotoxins. [Background technology]

[0002] The anaerobic Gram-positive bacterium Clostridium botulinum produces a variety of different neurotoxins, including botulinum neurotoxins (BoNTs) and tetanus neurotoxins (TeNTs).

[0003] BoNT is the most potent toxin known, with median lethal doses (LD ) in mice ranging from 0.5 to 5 ng / kg depending on the serotype. 50 ) value. BoNT is adsorbed in the gastrointestinal tract and, after entering the systemic circulation, binds to the presynaptic membrane of cholinergic nerve terminals and prevents the release of the neurotransmitter acetylcholine.

[0004] BoNT is well known for its ability to cause flaccid muscle paralysis. The muscle relaxant properties have led to its use in a variety of medical and cosmetic procedures, including the treatment of frown lines or hyperkinetic facial lines, headaches, hemifacial spasms, bladder hypermotility, excessive sweating, nasolabial folds, cervical dystonia, blepharospasm and spasticity.

[0005] There are currently at least eight different classes of BoNTs, namely BoNT serotypes A, B, C, D, E, F, G and H (known as BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G and BoNT / H, respectively), all of which share a similar structure and mode of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and such serotype classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage of amino acid sequence identity.

[0006] Different serotypes of BoNT affect different animal species with respect to the severity and duration of the paralysis they cause. For example, BoNT / A is the most lethal of all known biological agents and is 500 times more potent in rats with respect to paralysis than BoNT / B. Furthermore, the duration of paralysis after BoNT / A injection in mice is 10 times longer than that after BoNT / E injection.

[0007] In nature, clostridial neurotoxins are synthesized as single-chain polypeptides, which are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often called the activation site, located between cysteine ​​residues that provide the interchain disulfide bond. The active form of the toxin is this two-chain form. The two chains are called the heavy chain (H chain) with a molecular weight of approximately 100 kDa and the light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component known as the "targeting moiety" and an N-terminal translocation component known as the "translocation domain". The cleavage site is located between the L chain and the translocation domain component, in an exposed loop region. After binding of the targeting moiety to its target neuron and internalization of the bound toxin into the cell by the endosome, the translocation domain translocates the L chain across the endosomal membrane into the cytosol.

[0008] The L chain contains a protease component known as the "protease domain". It has a non-cytotoxic protease function and acts by proteolytically cleaving intracellular transport proteins known as SNARE proteins - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE is derived from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. The protease domain has zinc-dependent endopeptidase activity and shows high substrate specificity for SNARE proteins.

[0009] Various different clostridial neurotoxins cleave different SNARE proteins with their respective protease domains: BoNT / B, BoNT / D, BoNT / F, BoNT / G and TeNT cleave synaptobrevin, also known as vesicle-associated membrane protein (VAMP); BoNT / A, BoNT / C and BoNT / E cleave synaptosomal associated protein of 25 kDa (SNAP-25); BoNT / C cleaves syntaxin.

[0010] SNARE proteins are associated with either the membrane of secretory vesicles or the plasma membrane and mediate the fusion of secretory vesicles with the plasma membrane, thus facilitating the exocytosis of molecules by allowing the contents of the vesicle to be expelled to the outside of the cell. Cleavage of such SNARE proteins inhibits such exocytosis and thus the release of neurotransmitters from such neurons. As a result, striated muscles become paralyzed and sweat glands cease their secretion.

[0011] Thus, once delivered to a desired target cell, the Clostridial neurotoxin is capable of inhibiting cellular secretion from the target cell.

[0012] It is known in the art to modify clostridial neurotoxins to change their properties. Modifications can include amino acid modifications, such as addition, deletion and / or substitution of amino acid(s) and / or chemical modifications, such as addition of phosphate or carbohydrate or formation of disulfide bonds. Modifications can also include rearrangement of components of clostridial neurotoxins, such as flanking a protease component with a translocation component and a targeting component.

[0013] It is also known in the art to produce recombinant Clostridial neurotoxins that are genetically identical to Clostridial-derived neurotoxins or that differ from wild-type Clostridial neurotoxins in that they contain additional, fewer, or different amino acids and / or have components arranged in a different order than they are arranged in the wild-type Clostridial neurotoxin. These recombinant Clostridial neurotoxins can be chemically modified as described above.

[0014] However, differences between modified and recombinant Clostridial neurotoxins and their wild-type counterparts may affect the desired SNARE protein cleavage properties of the neurotoxins, and as such, it may be important to determine whether such differences improve, reduce or eliminate such activity.

[0015] A variety of conventional assays are available that allow a person skilled in the art to confirm whether these modified or recombinant Clostridial neurotoxins have the desired activity of cleaving the targeted SNARE protein. These assays include testing for the presence of products resulting from cleavage of the SNARE protein. For example, after contacting cells with modified or recombinant neurotoxins, the cells can be lysed and analyzed by SDS-PAGE to detect the presence of cleavage products. Alternatively, cleavage products can be detected by contacting cell lysates with antibodies.

[0016] Although native cells can be used in such assays, the assays usually require the use of high concentrations of such cells, since such native cells may have limited sensitivity to Clostridial neurotoxins. Moreover, such assays desirably use clonally stable cell lines.

[0017] Thus, there is a need for genetically modified cells with increased susceptibility to Clostridial neurotoxins for use in such assays. Summary of the Invention

[0018] The present invention relates in part to cells that are genetically modified to express or overexpress the receptor of Clostridial neurotoxin or its variant or fragment. Such receptors can be protein receptors or gangliosides. Preferably, the cells described herein (whether in the context of the cells themselves described herein or in the context of any method or use that includes the cells described herein) comprise an exogenous nucleic acid that provides the expression or overexpression (preferably overexpression) of the receptor and / or ganglioside, and said exogenous nucleic acid is under the control of a constitutive and / or inducible promoter (preferably a constitutive promoter).

[0019] The invention also relates, in part, to methods of producing such cells, the methods comprising introducing into the cells a nucleic acid encoding a Clostridial neurotoxin receptor or a variant or fragment thereof having the ability to bind to a Clostridial neurotoxin, and / or an enzyme of the ganglioside synthesis pathway or a variant or fragment thereof having the catalytic activity of such an enzyme.

[0020] The present invention further relates, in part, to an assay for determining the activity of a modified or recombinant neurotoxin, the method comprising contacting said cells with the modified or recombinant neurotoxin under conditions and for a period of time that would allow the protease domain of a wild-type Clostridial neurotoxin to cleave an indicator protein in said cells, and determining the presence of a product resulting from cleavage of such indicator protein.

[0021] The present invention encompasses a method for testing / assessing the activity of a batch of clostridial neurotoxins for therapeutic / cosmetic use. Such a method advantageously finds utility in toxin activity monitoring during storage and tracking activity over time. An additional advantage is the ability to determine optimal storage conditions (e.g., that do not reduce activity levels). The method is particularly advantageous for characterizing the activity (e.g., cell binding / SNARE cleavage ability) of recombinant clostridial neurotoxins.

[0022] An embodiment of the present invention provides an in vitro method for characterizing the activity of or identifying a Clostridial neurotoxin (preferably, BoNT) preparation for therapeutic (and / or cosmetic) use, said method comprising: a. providing a cell (e.g., a genetically modified cell) having an exogenous nucleic acid that provides expression or overexpression of a receptor and / or ganglioside (preferably a receptor) having binding affinity for a Clostridial neurotoxin and an exogenous nucleic acid that provides expression or overexpression of an indicator protein (preferably an indicator protein comprising a SNARE domain) that is cleavable by a Clostridial neurotoxin, preferably wherein the cell does not express the receptor and / or ganglioside (preferably the receptor) in the absence of said exogenous nucleic acid; b. contacting the cell with a Clostridial neurotoxin formulation; c. comparing the level of cleavage of the indicator protein after contact with (e.g., administration of) a Clostridial neurotoxin formulation to the level of cleavage before contact with the Clostridial neurotoxin formulation; d. (i) identifying the Clostridial neurotoxin preparation as suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is increased, or (ii) identifying the presence of activity if the level of cleavage of the indicator protein after contact is increased; or e. (i) identifying the Clostridial neurotoxin preparation as not suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is not increased, or (ii) identifying the absence of activity if the level of cleavage of the indicator protein after contact is not increased.

[0023] Another aspect of the invention provides an in vitro method for characterizing the activity of a Clostridial neurotoxin (preferably, BoNT) preparation or identifying a Clostridial neurotoxin (preferably, BoNT) preparation suitable for therapeutic (and / or cosmetic) use, said method comprising: a. engineering a cell (e.g., a genetically modified cell) to incorporate an exogenous nucleic acid that provides for expression or overexpression of a receptor and / or ganglioside (preferably a receptor) having binding affinity for a Clostridial neurotoxin, and an exogenous nucleic acid that provides for expression or overexpression of an indicator protein (preferably an indicator protein comprising a SNARE domain) that is cleavable by a Clostridial neurotoxin, preferably wherein the cell does not express the receptor and / or ganglioside (preferably the receptor) in the absence of said exogenous nucleic acid; b. contacting the cell with a Clostridial neurotoxin formulation; c. comparing the level of cleavage of the indicator protein after contact with (e.g., administration of) a Clostridial neurotoxin formulation to the level of cleavage before contact with the Clostridial neurotoxin formulation; d. (i) identifying the Clostridial neurotoxin preparation as suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is increased, or (ii) identifying the presence of activity if the level of cleavage of the indicator protein after contact is increased; or e. (i) identifying the Clostridial neurotoxin preparation as not suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is not increased, or (ii) identifying the absence of activity if the level of cleavage of the indicator protein after contact is not increased.

[0024] Another aspect of the invention provides an in vitro method for characterizing the activity of a Clostridial neurotoxin (preferably, BoNT) preparation or for identifying a Clostridial neurotoxin (preferably, BoNT) preparation suitable for therapeutic (and / or cosmetic) use, said method comprising: a. providing a cell (e.g., a genetically modified cell) having an exogenous nucleic acid that provides expression or overexpression of a receptor and / or ganglioside (preferably a receptor) having binding affinity for a Clostridial neurotoxin and an exogenous nucleic acid that provides expression or overexpression of an indicator protein (preferably an indicator protein comprising a SNARE domain) that is cleavable by a Clostridial neurotoxin, preferably wherein the cell does not express the receptor and / or ganglioside (preferably the receptor) in the absence of said exogenous nucleic acid; b. contacting the cell with a Clostridial neurotoxin formulation; c. comparing the level of cleavage of the indicator protein following contact with (e.g., administration of) a Clostridial neurotoxin formulation with the level of cleavage following contact with a control Clostridial neurotoxin formulation; d. (i) identifying the Clostridial neurotoxin formulation as suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is increased relative to or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin formulation, or (ii) identifying the presence of activity if the level of cleavage of the indicator protein after contact is increased relative to or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin formulation; or e. (i) identifying the Clostridial neurotoxin preparation as not suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is not increased relative to or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin preparation, or (ii) identifying the absence of activity if the level of cleavage of the indicator protein after contact is not increased relative to or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin preparation.

[0025] The control Clostridial neurotoxin preparation is a preparation of a Clostridial neurotoxin (e.g., a batch of Clostridial neurotoxin) that is known to have cleavage activity and / or is known to be suitable for therapeutic (and / or cosmetic) use (in other words, the control is a positive control). Preferably, the (test) Clostridial neurotoxin preparation and the control neurotoxin preparation are preparations of the same type / serotype of Clostridial neurotoxin, e.g., if the (test) Clostridial neurotoxin preparation is a preparation of BoNT / E, then the control Clostridial neurotoxin is also preferably a preparation of BoNT / E.

[0026] Another aspect of the invention provides a method for modifying cells suitable for use in assays for characterizing the activity of a Clostridial neurotoxin (preferably, BoNT) formulation or for identifying a Clostridial neurotoxin (preferably, BoNT) formulation suitable for therapeutic (and / or cosmetic) use, the method comprising:

[0027] This includes engineering the cells to incorporate: (i) an exogenous nucleic acid that provides expression or overexpression of a receptor and / or ganglioside having binding affinity for a Clostridial neurotoxin, preferably, that does not express the receptor and / or ganglioside (preferably, the receptor) in the absence of said exogenous nucleic acid; and (ii) an exogenous nucleic acid that provides for the expression or overexpression of an indicator protein that is cleavable by a Clostridial neurotoxin, preferably an indicator protein that contains a SNARE domain.

[0028] The following are optional embodiments of any of the aspects of the invention described herein.

[0029] In one embodiment, the cell does not normally express the receptor and / or ganglioside (preferably the receptor, preferably the receptor is SV2A). In other words, in a preferred embodiment, the cell does not express the receptor and / or ganglioside (preferably the receptor, preferably the receptor is SV2A) in the absence of said exogenous nucleic acid.

[0030] In one embodiment, the cell is of a different cell type than the natural target of the clostridial neurotoxin. In other words, in one embodiment, the cell is not the natural target of the clostridial neurotoxin. For example, the cell may not be the natural target of BoNT / A. Additionally or alternatively, the cell may not be the natural target of BoNT / E.

[0031] In one embodiment, the cell is not a neural cell (eg, a neuron).

[0032] Advantageously, by providing expression or overexpression of receptors and / or gangliosides with binding affinity for Clostridial neurotoxins, the methods and cells of the present invention allow for a reduction in false negative results (e.g., when low cleavage activity is inaccurately detected as a result of low affinity of Clostridial neurotoxins for binding and translocation to the cells used in the assay, rather than low activity of the protease domain). Furthermore, the present invention provides a wide range of cell types that can be used in such assays, reducing the reliance on neural cells (e.g., naturally expressing sufficient levels of receptors / gangliosides) that may be difficult to culture in vitro. These advantages are provided in the context of cell-based assays (allowing for characterization of binding, translocation and protease activity) as opposed to cell-free systems (characterizing only protease activity).

[0033] The term "if the level of cleavage of the indicator protein after contact is not increased" means that the level of cleavage is not substantially increased. The term "substantially" as used herein in the context of the term "if the level of cleavage of the indicator protein after contact is not increased" preferably means that there is no statistically significant increase. The increase (not substantial) may be less than 30%, 25%, 20%, 15%, 10%, 5% or 1%, preferably less than 20% increase. The increase (not substantial) may be less than 5%, 2%, 1% or 0.5%, preferably less than 0.1% increase. More preferably, the term "if the level of cleavage of the indicator protein after contact is not increased" as used herein means that there is no decrease at all in the level of cleavage of the indicator protein after contact (i.e., the increase in the level of cleavage is 0%).

[0034] The level of receptor and / or ganglioside (preferably receptor) expressed in the cells described herein is preferably equal to or higher than the level of receptor and / or ganglioside (preferably receptor) expressed in the natural target (e.g., neuronal cell) of Clostridial neurotoxin.For example, the level of receptor and / or ganglioside (preferably receptor) expressed in the cells described herein can be ≧10%, ≧20%, ≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90% or ≧100% of the level of receptor and / or ganglioside (preferably receptor) expressed in the natural target (e.g., neuronal cell) of Clostridial neurotoxin.

[0035] The level of receptor and / or ganglioside (preferably receptor) expressed in the cells described herein may be preferably higher than the level of receptor and / or ganglioside (preferably receptor) expressed in the cells lacking said exogenous nucleic acid.For example, the level of receptor and / or ganglioside (preferably receptor) expressed in the cells described herein may be ≧10%, ≧20%, ≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90% or ≧100% of the level of receptor and / or ganglioside (preferably receptor) expressed in the cells lacking said exogenous nucleic acid (e.g., otherwise equivalent cells).

[0036] In one embodiment, the term "overexpression", when used in conjunction with any aspect or embodiment described herein, preferably means expression of ≧10%, ≧20%, ≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90% or ≧100% relative to the level of receptor and / or ganglioside (preferably receptor) expressed in a natural target (e.g., neuronal cell) of the Clostridial neurotoxin. In one embodiment, the term "overexpression", when used in conjunction with any aspect or embodiment described herein, preferably means expression of ≧10%, ≧20%, ≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90% or ≧100% relative to the level of receptor and / or ganglioside (preferably receptor) expressed in a cell (e.g., an otherwise equivalent cell) lacking said exogenous nucleic acid.

[0037] In one embodiment, the Clostridial neurotoxin is BoNT / A (or BoNT / AH). CC The receptor may be a BoNT containing a SV2A domain, and preferably the receptor may be SV2A, SV2B and / or SV2C (preferably SV2A).

[0038] Additionally or alternatively, the clostridial neurotoxin may be BoNT / B (or BoNT / BH CC The receptor may be a BoNT containing a receptor domain, and preferably the receptor may be Syt-I and / or Syt-II.

[0039] Additionally or alternatively, the clostridial neurotoxin may be BoNT / E (or BoNT / EH CC The receptor may be a BoNT containing a SV2A domain, and preferably the receptor may be SV2A and / or SV2B (preferably SV2A).

[0040] Additionally or alternatively, the clostridial neurotoxin may be BoNT / G (or BoNT / GH CCThe receptor may be a BoNT containing a receptor domain, and preferably the receptor may be Syt-I and / or Syt-II.

[0041] For further information regarding suitable receptors / gangliosides, see Binz and Rummel (Journal of Neurochemistry, volume 109, Issue 6, June 2009, Pages 1584-1595), which is incorporated herein by reference. [Brief description of the drawings]

[0042] [Figure 1A] Figure 1A depicts a Western blot using an anti-SNAP-25 antibody following treatment of N2a cells with BoNT / A at 0.1 nM, 1 nM, or 10 nM for 8 hours, or at 1 nM, 0.1 nM, or 0.01 nM for 24 hours. The presence of a lower band indicates the presence of cleavage products. [Figure 1B] Figure IB depicts a Western blot using an anti-SNAP-25 antibody following treatment of M17 cells with BoNT / A at 0.1 nM, 1 nM, or 10 nM for 8 hours, or at 1 nM, 0.1 nM, or 0.01 nM for 24 hours. The presence of a lower band indicates the presence of cleavage products. [Figure 1C] Figure 1C depicts a Western blot using an anti-SNAP-25 antibody following treatment of IMR-32 cells with BoNT / A at 0.1 nM, 1 nM, or 10 nM for 8 hours, or at 1 nM, 0.1 nM, or 0.01 nM for 24 hours. The presence of a lower band indicates the presence of cleavage products. [Figure 1D] Figure ID depicts a Western blot using an anti-SNAP-25 antibody following treatment of NG108 cells with BoNT / A at 0.1 nM, 1 nM, or 10 nM for 8 hours, or at 1 nM, 0.1 nM, or 0.01 nM for 24 hours. The presence of a lower band indicates the presence of cleavage products. [Figure 2A]FIG. 2A depicts a fluorescent micrograph of NG108 cells one day after transfection with a plasmid containing the mScarlet-SNAP25-GeNluc construct. [Figure 2B] FIG. 2B depicts a fluorescent micrograph of M17 cells one day after transfection with a plasmid containing the mScarlet-SNAP25-GeNluc construct. [Diagram 3] FIG. 3 depicts a fluorescent micrograph of puromycin-resistant N108 cells stably transfected with plasma containing the mScarlet-SNAP25-GeNluc construct. [Figure 4] FIG. 4 shows a bar graph depicting the average number of green fluorescent cells per HPF following treatment with 0, 0.1 nM or 1 nM BoNT / A. [Figure 5A] FIG. 5A depicts a scatter plot of flow cytometry data of NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct, showing granularity / complexity on the x-axis and cell size on the y-axis. [Figure 5B] FIG. 5B depicts a histogram of emission fluorescence intensity measured at 525 nM of NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 5C] FIG. 5C depicts a histogram of emission fluorescence intensity measured at 585 nM of NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 5D] FIG. 5D depicts a histogram of emission fluorescence intensity measured at 617 nM of NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 5E] FIG. 5E depicts a histogram of emission fluorescence intensity measured at 665 nM of NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 5F] FIG. 5F depicts a histogram of emission fluorescence intensity measured at 785 nM of NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 5G] FIG. 5G depicts a scatter plot of flow cytometry data of NG108 cells stably transfected with mScarlet-SNAP-25-GeNluc measured at 665 nM on the x-axis and side scatter (SS) on the y-axis. [Figure 6A] FIG. 6A depicts a scatter plot of flow cytometry data of M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct, showing granularity / complexity on the x-axis and cell size on the y-axis. [Figure 6B] FIG. 6B depicts a histogram of emission fluorescence intensity measured at 525 nM of M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 6C] FIG. 6C depicts a histogram of emission fluorescence intensity measured at 585 nM of M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 6D] FIG. 6D depicts a histogram of emission fluorescence intensity measured at 617 nM of M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 6E] FIG. 6E depicts a histogram of emission fluorescence intensity measured at 665 nM of M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 6F] FIG. 6F depicts a histogram of emission fluorescence intensity measured at 785 nM of M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. [Figure 6G]FIG. 6G depicts a scatter plot of flow cytometry data of M17 cells stably transfected with mScarlet-SNAP-25-GeNluc measured at 665 nM on the x-axis and side scatter (SS) on the y-axis. [Figure 7A] FIG. 7A depicts a histogram of emission fluorescence intensity measured at 525 nM of control NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct. [Figure 7B] FIG. 7B depicts a histogram of emission fluorescence intensity measured at 525 nM of NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated with 0.1 nM BoNT / A. [Figure 7C] FIG. 7C depicts a histogram of emission fluorescence intensity measured at 525 nM of NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated with 1.0 nM BoNT / A. [Figure 8A] FIG. 8A depicts a histogram of emission fluorescence intensity measured at 785 nM of control NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct. [Figure 8B] FIG. 8B depicts a histogram of emission fluorescence intensity measured at 785 nM of NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated with 0.1 nM BoNT / A. [Figure 8C] FIG. 8C depicts a histogram of emission fluorescence intensity measured at 785 nM of NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated with 1.0 nM BoNT / A. [Figure 9]FIG. 9 depicts a Western blot performed on NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated with no toxin (control), 1 nM or 8 nM BoNT / A or 0 (control), 1 nM, 10 nM or 100 nM BoNT / E. [Figure 10A] FIG. 10A depicts flow cytometry data of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and not treated with toxin. [Figure 10B] FIG. 10B depicts flow cytometry data of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT / A for 72 hours. [Figure 10C] FIG. 10C depicts flow cytometry data of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 1 nM BoNT / A for 72 hours. [Figure 10D] FIG. 10D depicts flow cytometry data of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 0.1 nM BoNT / A for 72 hours. [Figure 10E] FIG. 10E depicts flow cytometry data of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT / E for 72 hours. [Figure 10F] FIG. 10F depicts a fluorescent micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and not treated with toxin. [Figure 10G] FIG. 10G depicts a fluorescent micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT / A for 72 hours. [Figure 10H]FIG. 10H depicts a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 1 nM BoNT / A for 72 hours. [Figure 10I] FIG. 10I depicts a fluorescent micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 0.1 nM BoNT / A for 72 hours. [Figure 10J] FIG. 10J depicts a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT / E for 72 hours. [Figure 11A] FIG. 11A depicts flow cytometry data from wild-type NG108 cells. [Figure 11B] FIG. 11B depicts flow cytometry data of genetically modified NG108 cells selected for high expression of indicator proteins and sensitivity to 1,000 pM BoNT / A and not further treated with BoNT / A. [Figure 11C] FIG. 11C depicts flow cytometry data from genetically modified NG108 cells selected for high expression of indicator proteins and sensitivity to 1,000 pM BoNT / A and treated with 100 pM BoNT / A for 48 hours. [Figure 11D] FIG. 11D depicts flow cytometry data from genetically modified NG108 cells selected for high expression of indicator proteins and sensitivity to 1,000 pM BoNT / A and treated with 100 pM BoNT / A for 96 hours. [Figure 11E] FIG. 11E depicts flow cytometry data from genetically modified NG108 cells that were selected for high expression of the indicator protein but not for sensitivity to BoNT / A and were not further treated with BoNT / A. [Figure 11F]FIG. 11F depicts flow cytometry data from genetically modified NG108 cells selected for high expression of an indicator protein but not for sensitivity to BoNT / A and treated with 100 pM BoNT / A for 96 hours. [Figure 12A] FIG. 12A depicts flow cytometry data of genetically modified NG108 cells selected for high expression of indicator proteins and sensitivity to 100 pM BoNT / A and were not further treated with BoNT / A. [Figure 12B] FIG. 12B depicts flow cytometry data from genetically modified NG108 cells selected for high expression of indicator proteins and sensitivity to 100 pM BoNT / A and treated with 100 pM BoNT / A for 96 hours. [Figure 13A] FIG. 13A shows a plot of the percentage amount of cleaved indicator protein after treatment of NG108 cells genetically modified to express the indicator protein and SV2A or SV2C with various concentrations of BoNT / A for various times. [Figure 13B] FIG. 13B shows a plot of the percentage amount of cleaved indicator protein after treatment of NG108 cells genetically modified to express the indicator protein and SV2A or SV2C with various concentrations of BoNT / E for various times. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] It should be understood that the present invention is not limited to the embodiments described herein. Indeed, numerous variations, modifications, and substitutions will be apparent to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used in practicing the present invention.

[0044] In describing the invention, when a range of values ​​is provided for an embodiment, it is understood that each intervening value is encompassed within the embodiment.

[0045] As used herein, a "variant" of a protein or polypeptide refers to a protein or polypeptide having an amino acid sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the amino acid sequence of a reference protein or polypeptide.

[0046] "Sequence identity," as used herein, refers to the identity between a reference amino acid or nucleotide sequence and a query amino acid or nucleotide sequence, where the sequences are aligned for the highest order match, and can be calculated using published techniques or methods codified in computer programs such as BLASTP, BLASTN, FASTA (Altschul 1990, J. Mol. Biol. 215:403).

[0047] As used herein, a "fragment" of a protein or polypeptide refers to a truncated form of the protein or polypeptide or a truncated form of a variant of the protein or polypeptide.

[0048] The present invention relates, in part, to cells that have been genetically modified to have increased susceptibility to Clostridial neurotoxins.

[0049] Clostridial neurotoxins are neurotoxins produced naturally by the bacterium Clostridium botulinum.

[0050] In certain embodiments of the invention, the Clostridial neurotoxin is a botulinum neurotoxin (BoNT) or a tetanus neurotoxin (TeNT). As used herein, the terms "Clostridial neurotoxin," "BoNT," and "TeNT" refer to wild-type Clostridial neurotoxins, including those produced by strains other than Clostridium botulinum, and modified and recombinant Clostridial neurotoxins, respectively.

[0051] The modified clostridial neurotoxin may contain one or more modifications, including amino acid modifications and / or chemical modifications, compared to the wild-type clostridial neurotoxin. The amino acid modifications include the deletion, substitution, or addition of one or more amino acid residues. The chemical modifications include modifications made to one or more amino acid residues, such as the addition of phosphate or carbohydrate or the formation of disulfide bonds.

[0052] In certain embodiments, modifications can be made to alter the properties of a Clostridial neurotoxin. Modifications to a Clostridial neurotoxin can increase or decrease its biological activity.

[0053] The biological activity of clostridial neurotoxins encompasses at least three distinct activities. The first activity is the "proteolytic activity" present in the protease component of the neurotoxin, which is responsible for hydrolysis of peptide bonds of one or more SNARE proteins involved in the regulation of cell membrane fusion. The second activity is the "translocation activity" present in the translocation component of the neurotoxin, which is responsible for transporting the neurotoxin across the endosomal membrane and into the cytoplasm. The third activity is the "receptor binding activity" present in the targeting component of the neurotoxin, which is responsible for binding of the neurotoxin to a receptor on the target cell.

[0054] In certain embodiments, the modification of a neurotoxin may include a truncated component(s) of a Clostridial neurotoxin while still maintaining the activity of such component(s). For example, a neurotoxin may be modified to include only the portion(s) of the protease component that is necessary for proteolytic activity, only the portion(s) of the translocation component that is necessary for translocation activity, and / or only the portion(s) of the targeting component that is necessary for receptor binding activity.

[0055] Clostridial neurotoxins are initially produced as inactive single-chain polypeptides and are deployed in their active two-chain form following cleavage of the neurotoxin at its activation site, resulting in a two-chain protein having a heavy chain (H chain) that contains the translocation and targeting component and a light chain (L chain) that contains the protease component.

[0056] In certain embodiments, the biological activity of a Clostridial neurotoxin is modified by modifying the activation site of the neurotoxin. The ability of the neurotoxin to be activated can be increased, decreased, or remain the same. In certain embodiments, the biological activity of a Clostridial neurotoxin is increased or caused by modifying the activation site so that it is more easily cleaved, thus activating the neurotoxin. In embodiments where activation is desired only in certain environments or cells, the activation site can be modified so that it is only cleaved by proteases present in such environments or cells. In certain other environments, the biological activity of the neurotoxin is reduced or inactivated by modifying the activation site so that it is less easily cleaved.

[0057] In certain embodiments, the biological activity of a clostridial neurotoxin is modified by modifying the protease component of the neurotoxin. The proteolytic activity of the neurotoxin can be increased, decreased, or remain the same. In certain embodiments, the protease component can be replaced with a protease component from a different clostridial neurotoxin or a variant or fragment thereof. For example, BoNT / A can be modified by replacing its protease component with that of BoNT / E.

[0058] In certain embodiments, the biological activity of a clostridial neurotoxin is modified by modifying the translocation component of the neurotoxin. The translocation activity of the neurotoxin can be increased, decreased, or remain the same. In certain embodiments, the translocation component can be replaced with a translocation component from a different clostridial neurotoxin or a variant or fragment thereof. For example, BoNT / A can be modified by replacing its translocation component with that of BoNT / E.

[0059] In certain embodiments, the biological activity of a clostridial neurotoxin is modified by modifying the targeting moiety of the neurotoxin. The targeting ability of the neurotoxin can be increased, decreased, or remain the same. In certain embodiments, the targeting moiety can be replaced with a targeting moiety from a different clostridial neurotoxin or a variant or fragment thereof. For example, BoNT / A can be modified by replacing its targeting moiety with that of BoNT / E. In certain other embodiments, the targeting moiety can be replaced with a non-clostridial polypeptide, such as an antibody.

[0060] Modifications can also include rearrangements of the components of a Clostridial neurotoxin, for example, flanking a protease component with a translocation component and a targeting component.

[0061] Recombinant Clostridial neurotoxins are produced genetically. They may be genetically identical to wild-type Clostridial neurotoxins, or may differ from wild-type Clostridial neurotoxins in that they contain additional, fewer or different amino acids. For example, recombinant Clostridial neurotoxins can be produced that reflect any of the modified Clostridial neurotoxins described above. Recombinant Clostridial neurotoxins may also have components arranged in a different order than they are arranged in the wild-type Clostridial neurotoxin. Recombinant Clostridial neurotoxins can also be chemically modified as described above.

[0062] In certain embodiments, a modified or recombinant Clostridial neurotoxin is a polypeptide having an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity to a wild-type Clostridial neurotoxin, e.g., a BoNT or TeNT of serotype A, B, C, D, E, F, G or H.

[0063] A series of programs based on various algorithms for comparing different sequences are available to those skilled in the art.In this context, the algorithms of Needleman and Wunsch or Smith and Waterman provide particularly reliable results.To carry out sequence alignment and calculate the sequence identity values ​​listed herein, the commercially available program DNASTAR Lasergene MegAlign version 7.1.0 based on the algorithm Clustal W is used over the entire sequence region with the following settings: pairwise alignment parameters, gap penalty, 10.00, gap length penalty, 0.10, protein weight matrix Gonnet 250, which should always be used as the standard settings for sequence alignment unless otherwise stated.

[0064] The BoNT / A serotype is divided into at least six subserotypes (also known as subtypes), BoNT / A1 through BoNT / A6, which share at least 84% and up to 98% amino acid sequence identity. BoNT / A proteins within a given subtype share a higher percentage of amino acid sequence identity.

[0065] Clostridial neurotoxins target neurons by binding to receptors, which include protein receptors and cell membrane gangliosides.

[0066] Gangliosides are oligoglycosylceramides derived from lactosylceramide and contain sialic acid residues, such as N-acetylneuraminic acid (Neu5Ac), N-glycolyl-neuraminic acid (Neu5Gc) or 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN). Gangliosides are present and concentrated on the cell surface, with the two carbohydrate chains of the ceramide moiety embedded in the cell membrane and the oligosaccharides located on the extracellular surface, where they present recognition points for extracellular molecules or the surface of adjacent cells. Gangliosides also bind specifically to viruses and to bacterial toxins, such as Clostridial neurotoxins.

[0067] Gangliosides are defined by a nomenclature system 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 gangliosides in thin-layer chromatography. For example, the order of migration of monosialogangliosides is GM3>GM2>GM1. Additional terms are added to indicate variations within the basic structure, e.g., GM1a, GD1b, etc. Glycosphingolipids with 0, 1, 2 and 3 sialic acid residues linked to an internal galactose unit are referred to as asialo- (or 0-), a-, b- and c-series gangliosides, respectively, while gangliosides with sialic acid residues linked to an internal N-galactosamine residue are classified as a-series gangliosides. The pathway for the biosynthesis of 0-, a-, b- and c-series gangliosides involves sequential activity of sialotransferases and glycosyltransferases, as exemplified, for example, by Ledeen et al., Trends in Biochemical Sciences, 40: 407-418 (2015). Further sialylation at different positions in the carbohydrate chain of each series can occur, resulting in an increasingly complex and heterogeneous range of products, such as a-series gangliosides with sialic acid residue(s) linked to an internal N-acetylgalactosamine residue. Gangliosides are transferred to the outer leaflet of the cell membrane by a transport system involving vesicle formation.

[0068] To date, nearly 200 gangliosides have been identified in vertebrate tissues. Common gangliosides include GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, and GQ1.

[0069] Clostridial neurotoxins have two independent binding regions in the HCC domain of gangliosides and neuronal protein receptors. BoNT / A, BoNT / B, BoNT / E, BoNT / F and BoNT / G have a conserved ganglioside-binding site in the HCC domain composed of an "E(Q)...H(K)...SXWY...G" motif, whereas BoNT / C and BoNT / D display two independent ganglioside-binding sites. Lam et al., Progress in Biophysics and Molecular Biology, 117:225-231 (2015). Most BoNTs bind only to gangliosides that have 2,3-linked N-acetylneuraminic acid residues (designated Sia5) attached to Gal4 of the oligosaccharide core, but the corresponding ganglioside-binding pocket on TeNT can also bind to GM1a, a ganglioside lacking the Sia5 sugar residue. BoNT / D has been shown to bind to GM1a and GD1a. See Kroken et al., Journal of Biological Chemistry, 286:26828-26837 (2011). Combining data from ganglioside-deficient mice and biochemical assays, BoNT / A, BoNT / E, BoNT / F and BoNT / G show a preference for the terminal NAcGal-Gal-NAcNeu moiety present in GD1a and GT1b, whereas BoNT / B, BoNT / C, BoNT / D and TeNT require the disialyl motif found in GD1b, GT1b and GQ1b. Thus, large amounts of complex polysialogangliosides, e.g., GD1a, GD1b and GT1b, appear to be essential for the specific accumulation of all BoNT serotypes and TeNT on the surface of neuronal cells as the first step in intoxication.See Rummel, Andreas, “Double receptor anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity,” Botulinum Neurotoxins, Springer Berlin Heidelberg (2012) 61-90.

[0070] In view of the above, in certain embodiments of the present invention, cells are genetically modified to express or overexpress ganglioside.In certain embodiments, cells are genetically modified to express or overexpress GM1a, GD1a, GD1b, GT1b and / or GQ1b.In certain embodiments, cells are modified to express or overexpress GD1a, GD1b and / or GT1b.In certain embodiments, cells are modified to express or overexpress GD1b and / or GT1b.

[0071] Gangliosides are synthesized starting from ceramide. From ceramide, one pathway involves the addition of a glucose unit by glucosylceramide synthase to form glucosylceramide (GlcCer). β1,4-galactosyltransferase I (GalT-I) then catalyzes the addition of a galactose unit to GlcCer to form lactosylceramide (LacCer). From LacCer, GalNAc-transferase (GalNAcT) may add N-acetylgalactosamine to form GA1, and GM3 synthase may add sialic acid to form GM3. From GM3, GD3 can be formed by the addition of additional sialic acid by GD3 synthase. GT3 can be formed from GD3 by the addition of yet another sialic acid by GT3 synthase. In a separate pathway, a galactose unit is added to LacCer by galactosylceramide synthase to form galactosylceramide (GalCer). Additional carbohydrate groups are then added by GM4 synthase to form GM4. GM3, GD3 and GT3 can then be modified to form more complex gangliosides of the "a", "b" or "c" series, respectively. Such reactions are catalyzed by GalNAcT, β1,3-galactosyltransferase II (GalT-II), α2,3-sialyltransferase IV (ST-IV) or α2,8-sialyltransferase V (ST-V). For example, from GD3, the "b" series gangliosides GD1b, GT1b and GQ1b are formed.

[0072] Thus, the cells of the present invention can be modified to express or overexpress desired gangliosides by being modified to express or overexpress the enzymes of the biosynthetic pathway that leads to gangliosides.For example, the cells can be modified (i.e., by transfection) to contain exogenous nucleic acids that code for such enzymes.Thus, in certain embodiments, the cells are modified to express or overexpress glucosylceramide synthase, GalT-I, GalNAcT, GM3 synthase, GD3 synthase, GT3 synthase, galactosylceramide synthase, GM4 synthase, GalT-II, ST-IV and / or ST-V.

[0073] Those skilled in the art will appreciate that variants or fragments of such enzymes that retain their desired catalytic activity may play an important role in the synthesis of the ganglioside of interest. Thus, in certain embodiments, cells are genetically modified to express or overexpress variants or fragments of enzymes of the ganglioside synthesis pathway that retain the enzymatic capacity of the enzyme. For example, in certain embodiments, the cells are genetically modified to express or overexpress a variant or fragment of glucosylceramide synthase capable of adding glucose to ceramide, a variant or fragment of GalT-I capable of adding a galactose unit to GlcCer, a variant or fragment of GalNAcT capable of adding N-acetylgalactosamine to LacCer, a variant or fragment of GM3 synthase capable of adding sialic acid to LacCer, a variant or fragment of GD3 synthase capable of adding sialic acid to GM3, a variant or fragment of GT3 synthase capable of adding sialic acid to GD3, a variant or fragment of galactosylceramide synthase capable of adding a galactose unit to LacCer, and / or a variant of GM4 synthase capable of adding a carbohydrate group to GalCer.

[0074] In certain embodiments, the variants are proteins having an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with that of an enzyme of a biosynthetic pathway leading to a ganglioside and that retain the desired catalytic activity of such an enzyme. In certain such embodiments, the variants are proteins having an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with glucosylceramide synthase, GalT-I, LacCer, GalNAcT, GD3 synthase, GT3 synthase, galactosylceramide, GM4 synthase, GalT-II, ST-IV and / or ST-V and that retain the desired catalytic activity of that enzyme.

[0075] Fragments can have, for example, 50 or fewer amino acids, 40 or fewer amino acids, 30 or fewer amino acids, 20 or fewer amino acids, or 10 or fewer amino acids.

[0076] Assays that can be used to determine which variants or fragments have the desired catalytic activity are known in the art. For example, a person skilled in the art would be aware of assays that can be used to determine whether a variant or fragment of GD3 synthase has the ability to add sialic acid to GM3.

[0077] Those skilled in the art will understand that the above enzymes can also be encoded by nucleic acids that differ from the above exogenous nucleic acids by conservative substitutions known in the art. Those skilled in the art will also understand that enzyme variants can be encoded by nucleic acids that have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the nucleic acid that codes for the wild-type enzyme, for example. Thus, the present invention also contemplates cells that have been genetically modified to contain a nucleic acid that codes for one of the above enzymes and / or an exogenous nucleic acid that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the nucleic acid that codes for such an enzyme only by conservative substitutions, and the encoded protein is a wild-type enzyme or a variant that retains the catalytic activity of the wild-type enzyme.

[0078] In certain embodiments, cells are modified to express or overexpress an enzyme that is useful for catalyzing what is determined to be the rate-limiting step in the biosynthesis of a desired ganglioside, or a variant or fragment thereof that has the desired catalytic activity of such an enzyme. For example, GD3 synthase is an enzyme that catalyzes the rate-limiting step in the biosynthesis of "b" series gangliosides, specifically the addition of sialic acid to GM3. Thus, in embodiments in which expression or overexpression of GD1b, GT1b and / or GQ1b is desired, cells are modified to express or overexpress GD3 synthase or a variant or fragment thereof that has the ability to add sialic acid to GM3.

[0079] For example, cells can be transfected with a nucleic acid encoding GD3 synthase or a nucleic acid having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to such a nucleic acid, as described above, such as one encoding a variant of GD3 synthase that retains its catalytic activity or that differs from wild-type GD3 synthase only in conservative substitutions.

[0080] Binding of certain clostridial neurotoxins to cells may also depend on binding to protein receptors. BoNT / A, BoNT / D, BoNT / E, BoNT / F and TeNT bind to synaptic vesicle protein 2 (SV2), BoNT / A can bind to all three of its isoforms (SV2A, SV2B and SV2C), and BoNT / E can bind only to the SV2A and SV2B isoforms. BoNT / B and BoNT / G bind to both isoforms of synaptotagmin (I and II). Synaptotagmin and SV2 are localized on synaptic vesicles and become exposed to the extracellular space when the vesicles fuse with the presynaptic membrane. It is during this period that the clostridial neurotoxins bind to their protein receptors.

[0081] Thus, the cells of the invention can be modified to express or overexpress a desired protein receptor, such as SV2 (e.g., SV2A, SV2B, and SV2C) or synaptotagmin (e.g., synaptotagmin I and synaptotagmin II). For example, the cells can be modified (e.g., by transfection) to contain exogenous nucleic acid encoding such a protein receptor.

[0082] The present invention also contemplates proteins that are different from such protein receptors but still retain the ability to bind to Clostridial neurotoxins. Such proteins can be variants or fragments of such protein receptors that retain the ability of the receptor to bind to Clostridial neurotoxins. Thus, in certain embodiments, cells are modified to express or overexpress variants or fragments of SV2 that bind to BoNT / A, BoNT / D, BoNT / E, BoNT / F and / or TeNT. Also, in certain embodiments, cells are modified to express or overexpress variants or fragments of synaptotagmin that bind to BoNT / B and / or BoNT / G.

[0083] In certain embodiments, the variant is a protein having an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with that of a protein receptor that binds to a Clostridial neurotoxin. In certain such embodiments, the variant is a protein having an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SV2 (e.g., SV2A (SEQ ID NO: 8), SV2B (SEQ ID NO: 9) and SV2C (SEQ ID NO: 10)) or synaptotagmin (e.g., synaptotagmin I (SEQ ID NO: 14) and synaptotagmin II (SEQ ID NO: 15)).

[0084] Fragments can have, for example, 50 or fewer amino acids, 40 or fewer amino acids, 30 or fewer amino acids, 20 or fewer amino acids, or 10 or fewer amino acids.

[0085] In certain embodiments, the variant or fragment comprises a domain of a wild-type protein receptor that binds to a neurotoxin. For example, the variant or fragment may comprise the luminal domain(s) of wild-type SV2 (e.g., SV2A, SV2B and SV2C) or wild-type synaptotagmin (e.g., synaptotagmin I and synaptotagmin II). In certain such embodiments, the variant or fragment may comprise the fourth luminal domain of wild-type SV2, for example, the fourth luminal domain of SV2A (SEQ ID NO: 11), the fourth luminal domain of SV2B (SEQ ID NO: 12) or the fourth luminal domain of SV2C (SEQ ID NO: 13).

[0086] Assays that can be used to determine which variants or fragments have the desired Clostridial neurotoxin binding activity are known in the art. For example, one of skill in the art would know that assays can be used to determine whether a variant or fragment of SV2C has the ability to bind to BoNT / A.

[0087] Those skilled in the art will understand that the enzymes described above can also be encoded by nucleic acids that differ from the exogenous nucleic acids described above by conservative substitutions known in the art. Those skilled in the art will also understand that variants of protein receptors can be encoded by nucleic acids that have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the nucleic acid that encodes the wild-type protein receptor, for example. Thus, the present invention also contemplates cells that have been genetically modified to contain an exogenous nucleic acid that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the nucleic acid that encodes one of the above protein receptors and / or a nucleic acid that differs from the wild-type nucleic acid that encodes such a protein receptor only by conservative substitutions, and the encoded protein is a variant that retains the ability to bind to the wild-type protein receptor or a Clostridial neurotoxin.

[0088] SV2C is the most sensitive to BoNT / A. As such, in certain embodiments where sensitivity to BoNT / A is desired, cells are genetically modified to express or overexpress SV2C or its variant or fragment that can bind to BoNT / A. However, SV2C does not bind to BoNT / E, it instead binds to SV2A and SV2B. As such, in certain embodiments where sensitivity to BoNT / E is desired, cells are genetically modified to express or overexpress SV2A and / or SV2B or its variant or fragment that can bind to BoNT / E.

[0089] The present invention contemplates that cells can be modified to express or overexpress two or more protein receptors, two or more enzymes of the ganglioside synthesis pathway, or protein receptor(s) and enzyme(s) of the ganglioside synthesis pathway. For example, cells can be modified to express or overexpress SV2A and SV2C. Such cells can have increased sensitivity to, for example, BoNT / A and BoNT / E. Cells can also be modified to express or overexpress GD3 synthase and SV2A and / or SV2C.

[0090] Moreover, it is known that chimeric receptors can bind to neurotoxins.For example, chimeric receptors that comprise the domain of the above protein receptors that bind to neurotoxins (for example, the fourth luminal domain of SV2) fused to the transmembrane domain of another receptor, for example, the LDL receptor, are known to bind to BoNT and allow its internalization into cells.Therefore, the present invention also contemplates modifying cells to express such chimeric receptors.

[0091] The cell used in the present invention can be any cell, prokaryotic or eukaryotic, that can express ganglioside and / or protein receptor as described above.Examples of such cells include neuronal cells, neuroendocrine cells (e.g., PC12), embryonic kidney cells (e.g., HEK293 cells), breast cancer cells (e.g., MC7), neuroblastoma cells (e.g., Neuro2a (N2a), M17, IMR-32, N18 and LA-N-2 cells) and neuroblastoma-glioma hybrid cells (e.g., NG108 cells).In certain embodiments, the cell is a neuroblastoma or neuroblastoma-glioma cell.In certain embodiments, the cell is an NG108, M17 or IMR-32 cell.In certain embodiments, the cell is an NG108 cell.

[0092] Cells modified to express or overexpress Clostridial toxin receptors can be further selected for increased sensitivity using directed evolution. In this process, cells are exposed to Clostridial neurotoxins and cells that exhibit sensitivity to lower concentrations of Clostridial neurotoxins compared to other cells (e.g., as determined by exhibiting cleavage of an indicator protein therein) are selected. These cells are expected to have greater sensitivity to Clostridial neurotoxins than typical cells. This process can be repeated with increasingly lower concentrations of Clostridial neurotoxins, and cells that exhibit sensitivity to the lower concentrations are selected.

[0093] Cells selected in each round have increasing susceptibility to clostridial neurotoxins.

[0094] As previously discussed, the cells of the invention can be used in assays to determine the activity of a polypeptide (e.g., a modified or recombinant Clostridial neurotoxin). Such assays involve contacting a cell with the polypeptide and testing for the presence of a product(s) resulting from cleavage of a SNARE protein in the cell.

[0095] The term "contacting" as used herein refers to bringing a cell and a Clostridial neurotoxin into physical proximity to allow for physical and / or chemical interaction. Contacting is performed under conditions and for a time sufficient to allow interaction of the polypeptide with a protein (e.g., a SNARE protein) that is susceptible to proteolysis by wild-type Clostridial neurotoxin.

[0096] In certain embodiments, such contacting may be by culturing cells in a medium containing the polypeptide. The polypeptide is usually present in the medium at a concentration of 0.0001 nM to 10,000 nM, 0.0001 to 1,000 nM, 0.0001 to 100 nM, 0.0001 to 1 nM, 0.0001 to 0.1 nM, 0.0001 to 0.01 nM, or 0.0001 to 0.001 nM. Such culturing may be, for example, for 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 40 hours or more, or 48 hours or more.

[0097] In certain other embodiments, such contacting can be by transfecting (eg, transient transfection) the cell with an exogenous nucleic acid encoding the polypeptide.

[0098] To enable use in such assays, the cells contain proteins that are susceptible to proteolysis by wild-type Clostridial neurotoxins. These proteins are referred to herein as "indicator protein(s)." The indicator protein may be endogenous (e.g., endogenous SNARE proteins) or the cells may be genetically modified to express or overexpress the indicator protein.

[0099] As discussed above, SNARE proteins, such as SNAP-25, synaptobrevin and syntaxin, are known to be susceptible to proteolysis by clostridial neurotoxins. For example, BoNT / A, BoNT / C and BoNT / E are known to cleave SNAP-25, BoNT / C is also known to cleave syntaxin, and other BoNT serotypes and TeNT are known to cleave synaptobrevin. Thus, the present invention contemplates that such indicator proteins may comprise the amino acid sequence of such SNARE proteins. The present invention also contemplates that indicator proteins may alternatively comprise the amino acid sequence of a variant or fragment of such SNARE proteins, provided that the variant or fragment is susceptible to proteolysis by wild-type clostridial neurotoxins. In certain embodiments, the variant may have at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the SNARE protein. A portion of an indicator protein having the amino acid sequence of a SNARE protein, or a variant or fragment thereof, is referred to herein as the "SNARE domain" of the indicator protein.

[0100] The term "susceptible to proteolysis" means that the protein is proteolytically cleavable by the protease component of a wild-type Clostridial neurotoxin. In other words, such a protein contains a protease recognition and cleavage site, allowing it to be recognized and cleaved by the protease component of a wild-type Clostridial neurotoxin.

[0101] In certain embodiments, the indicator protein is labeled. For example, US 8,940,482 to Oyler et al. describes a cell-based assay for evaluating the activity of clostridial neurotoxins, in which cells are modified to express a labeled fusion protein comprising a fluorescent protein domain fused to SNAP-25. The fluorescent protein domain is C-terminal to the SNAP-25 domain and becomes part of the resulting C-terminal fragment after cleavage of SNAP-25 by clostridial neurotoxins. In the assay described by Oyler, the full-length fusion protein is not readily degraded in cells, but the resulting C-terminal fragment results in the degradation of the fluorescent protein. This is due to the presence in SNAP-25 of a residue that serves as a degron at the N-terminus of the resulting fragment only when exposed by cleavage. Such an "N-degron" is tagged by ubiquitin ligase, and thus the fragment is targeted by the proteasome for degradation.

[0102] Thus, the present invention contemplates embodiments such as those described in Oyler, in which cells are modified to express a labeled indicator protein that is not readily degraded in full-length form. In such embodiments, cleavage results in a labeled fragment that is readily degraded in the cell (e.g., due to the presence of an N-degron). The indicator protein is labeled with a moiety that forms a readily degraded fragment, and the label is degraded along with the fragment. In such embodiments, the ability of the polypeptide to cleave a SNARE protein in a cell can be determined by the presence (or lack thereof) of a signal from the label after the cell has contacted the polypeptide.

[0103] In certain such embodiments, the indicator protein also contains a label on a portion of the indicator protein that, after cleavage, forms a fragment that is not as readily degraded as the other fragment. For example, the indicator protein can be a fusion protein that contains two labels and a SNARE domain with the label adjacent to the SNARE domain. In embodiments in which the full-length indicator protein is not readily degraded in cells, but after its cleavage, one of the resulting fragments is readily degraded, cleavage of the SNARE protein can be determined by comparing the signal obtained from the label on the readily degradable fragment with the signal from the label on the less readily degradable fragment. For example, in embodiments such as those in Oyler, where the C-terminal fragment resulting from cleavage is readily degradable but the N-terminal fragment is not readily degradable, cleavage can be determined by comparing the signal obtained from the label on the C-terminal fragment with the signal from the label on the N-terminal fragment. In such embodiments, labels can be selected that emit fluorescent signals that are more clearly distinguishable from one another (e.g., red and green or red and cyan).

[0104] The term "label" as used herein means a detectable marker, including, for example, a radioactive label, an antibody and / or a fluorescent label. The amount of test substrate and / or cleavage product can be determined by, for example, an autoradiographic or spectroscopic method, including a method based on the energy resonance transfer between at least two labels, such as a FRET assay (discussed further below). Alternatively, immunological methods such as Western blot or ELISA can be used for detection.

[0105] Examples of labels that can be used in the practice of the present invention include radioisotopes, fluorescent labels, phosphorescent labels, luminescent labels, and compounds that can bind to labeled binding partners. Examples of fluorescent labels include yellow fluorescent protein (YFP), blue fluorescent protein (BFP), green fluorescent protein (GFP), such as NeonGreen, red fluorescent protein (RFP), such as mScarlet, cyan fluorescent protein (CFP) and its fluorescent mutants. Examples of luminescent labels include luminescent proteins, luciferase, such as firefly luciferase, Renilla and Gaussia luciferase, chemiluminescent compounds and electrochemiluminescent (ECL) compounds. In the embodiment discussed above, where the N-terminal label and the C-terminal label are selected so that the signals emitted are more easily distinguishable from each other, examples of such label pairs can include RFP and GFP and RFP and CFP. For example, RFP, such as mScarlet, can serve as N-terminal label, and GFP or CFP, such as NeonGreen, can serve as C-terminal label.

[0106] In certain embodiments, the label is a protein label, such as an antibody, a fluorescent protein, a luminescent protein, and a luciferase.

[0107] As used herein, "N-terminal label" refers to a label located on a portion of an indicator protein, whether or not it is a protein, that is N-terminal to the clostridial neurotoxin cleavage site, and "C-terminal label" refers to a label located on a portion of an indicator protein, whether or not it is a protein, that is C-terminal to the clostridial neurotoxin cleavage site. The label does not have to be N-terminal or C-terminal of the indicator protein to be called a terminal or C-terminal label. Rather, these terms refer to the location of the label relative to the clostridial neurotoxin cleavage site. In certain embodiments of the invention, RFP, e.g., mScarlet, is used as the N-terminal label, and GFP, e.g., NeonGreen or CFP, is used as the C-terminal label.

[0108] Another assay is the fluorescence resonance energy transfer (FRET) assay. In such an assay, the indicator protein contains a donor label on one side of the cleavage site and an acceptor label on the other side. The donor label absorbs energy and then transfers it to the acceptor label. The energy transfer results in a reduction in the fluorescence intensity of the donor chromophore and an increase in the emission intensity of the acceptor chromophore. Cleavage of the substrate results in less successful energy transfer. Thus, the success of cleavage can be determined based on the reduction in the ability of this transfer to occur. In such an embodiment, YFP and CFP can pair as a FRET pair, as can RFP and GFP.

[0109] In a certain embodiment of the present invention, the indicator protein is a fusion protein that comprises a SNARE domain.The fusion protein can also comprise additional domains, such as a labeling domain.The labeling domain can have the amino acid sequence of a protein label.One example of such a fusion protein comprises an N-terminal labeling domain, such as the amino acid sequence of mScarlet, an SNARE domain, such as the amino acid sequence of SNAP-25, and a C-terminal labeling domain, such as the amino acid sequence of NeonGreen.

[0110] The fusion protein may also include other domains, such as a selection marker (discussed further below). In such embodiments, the selection marker domain may be separated from the fusion protein portion containing the remaining domains (e.g., the SNARE domain and the labeling domain(s)) by a linker that can be cleaved post-translationally to allow separation of the selection marker and the remainder of the indicator protein. The linker may, for example, be self-cleaving (e.g., 2A self-cleaving peptide).

[0111] As discussed above, cells can be modified to express or overexpress indicator proteins. Those skilled in the art will know which nucleic acids can be used to enable such expression and how to modify such cells to express such indicator proteins. One example of such a nucleic acid is SEQ ID NO:1, which expresses a fusion protein with mScarlet as N-terminal label, SNAP-25 as SNARE domain, NeonGreen as C-terminal label, luciferase as additional label domain, puromycin-N-acetyltransferase as selection marker, and 2A self-cleaving peptide. Another example of such a nucleic acid is SEQ ID NO:2, which expresses a fusion protein with mScarlet as N-terminal label, SNAP-25 as SNARE domain, CFP as C-terminal label, luciferase as additional label domain, puromycin-N-acetyltransferase as selection marker, and 2A self-cleaving peptide.

[0112] The present invention also relates, in part, to a method for producing the genetically modified cell described above.The method comprises introducing an exogenous nucleic acid encoding a protein of interest into the cell.The protein of interest can be, for example, a Clostridial neurotoxin receptor or its variant or fragment that has the ability to bind to Clostridial neurotoxin, an enzyme of ganglioside synthesis pathway or its variant or fragment that has the catalytic activity of such enzyme, and / or an indicator protein.

[0113] In certain embodiments, the method includes transforming the cell with a nucleic acid encoding the protein of interest. Such transformation may be by transfection.

[0114] In certain embodiments, the nucleic acid encodes a fusion protein that includes two or more domains, each domain having the amino acid sequence of a protein of interest or other components of the fusion protein. For example, the nucleic acid may encode a fusion protein that includes the amino acid sequence of a protein receptor (e.g., SV2A or SV2C) and the amino acid sequence of an enzyme of a ganglioside synthesis pathway (e.g., GD3 synthase). In another example, the nucleic acid may encode a fusion protein that includes the amino acid sequence of a protein receptor, the amino acid sequence of an enzyme of a ganglioside synthesis pathway, and the amino acid sequence of a selection marker. In yet another example, the nucleic acid may encode a fusion protein that includes the amino acid sequence of a protein receptor, the amino acid sequence of an enzyme of a ganglioside synthesis pathway, the amino acid sequence of an indicator protein, and the amino acid sequence of a selection marker.

[0115] In such embodiments, the domains may be separated from one another by a linker, which may, for example, be enzymatically cleavable in the cell or may contain a self-cleaving peptide (e.g., the 2A self-cleaving peptide) to allow the individual domains to form separate proteins in the cell.

[0116] The nucleic acid may optionally include a regulatory element. The term "regulatory element" as used herein refers to a regulatory element of gene expression, including transcription and translation, and includes elements such as TATA box, promoter, enhancer, ribosome binding site, Shine-Dalgarno sequence, IRES region, polyadenylation signal, terminal cap structure, etc. Regulatory elements may include one or more heterologous regulatory elements or one or more homologous regulatory elements. A "homologous regulatory element" is a regulatory element of the wild-type cell from which the nucleic acid molecule is derived, which is involved in regulating gene expression of the nucleic acid molecule or polypeptide in the wild-type cell. A "heterologous regulatory element" is a regulatory element that is not involved in regulating gene expression of the nucleic acid molecule or polypeptide in the wild-type cell. Regulatory elements of inducible expression, such as an inducible promoter, may also be used.

[0117] The nucleic acid molecule may be, for example, hnRNA, mRNA, RNA, DNA, PNA, LNA and / or modified nucleic acid molecule. The nucleic acid molecule may be circular, linear, integrated into genome or episomal. Also included are concatemers that code for fusion proteins that contain 3, 4, 5, 6, 7, 8, 9 or 10 polypeptides. Furthermore, the nucleic acid molecule may contain a signal sequence for intracellular transport, for example, a sequence that codes for a signal for transport into an intracellular compartment or for transport across a cell membrane.

[0118] Nucleic acids can be designed to provide high levels of expression in a host cell. Methods for designing nucleic acid molecules to increase protein expression in a host cell are known in the art and include reducing the frequency (occurrence) of "slow codons" in the encoding nucleic acid sequence.

[0119] The nucleic acid can be introduced using any means known in the art, for example, contained in a vector (e.g., a plasmid) that is used to introduce the nucleic acid into the cell.

[0120] Any vector known in the art to allow expression of nucleic acid in cells can be used. The vector can be suitable for in vitro and / or in vivo expression of the protein of interest. The vector can be for transient and / or stable gene expression. The vector can further comprise regulatory elements and / or selection markers. The vector can be, for example, artificial or of viral, phage or bacterial origin. Examples of vectors for use in the present invention include adenoviral vectors, vaccinia vectors, SV-40 viral vectors, retroviral vectors, lambda-derivatives and plasmids. Examples of plasmids for use in the present invention include plasmids with pD2500 or pcDNA3.1 backbone.

[0121] Methods for using vectors to introduce nucleic acids into cells are known in the art, see Laura Bonetta, "The Inside Scoop-Evaluating Gene Delivery Methods," Nature Methods 2: 875-883 (2005).

[0122] The host cell may contain an inducer of expression of the protein of interest. Such an inducer of expression may be a nucleic acid molecule or a chemical entity, including a polypeptide or a small chemical entity. The inducer of expression may, for example, increase the transcription or translation of the nucleic acid molecule encoding the protein of interest. The inducer may, for example, be expressed by recombinant means known to those skilled in the art. Alternatively, the inducer may be isolated from the cell, for example, a Clostridium cell.

[0123] In certain embodiments, successfully transformed cells can be determined by determining the presence of a selectable marker. In such embodiments, the vector containing the exogenous nucleic acid encoding the desired protein may also contain nucleic acid encoding a selectable marker.

[0124] In certain embodiments, the selection marker is a detectable tag. Examples of such tags include His-tag, GST-tag, Strep-tag and SBP-tag. The tag can be expressed as part of a fusion protein that also contains the protein of interest. In such embodiments, the tag can be adjacent to one or more protease cleavage sites or self-cleaving peptides. This allows the tag to be cleaved from the protein after translation.

[0125] In certain other embodiments, the selection marker confers resistance to antibiotics. Examples of such selection markers include puromycin-N-acetyltransferase (resistance to puromycin), aminoglycoside 3'f3-phosphotransferase (resistance to G418), blasticidin S deaminase (resistance to blasticidin S) and hygromycin B phosphotransferase (resistance to hygromycin B). Thus, successful transformation of the cells can be determined by exposing the cells to the relevant antibiotic.

[0126] In certain embodiments, successful transformation of cells genetically engineered to express or overexpress a protein receptor that binds to gangliosides and / or Clostridial neurotoxins can be determined by contacting such cells with a Clostridial neurotoxin and determining whether cleavage of an indicator protein occurs therein.

[0127] The present invention further relates to the use of the genetically modified cells described above in assays for determining the biological activity of a polypeptide, such as a modified or recombinant Clostridial neurotoxin.

[0128] The biological activity of such polypeptides can be determined by a variety of tests, all of which are known to those skilled in the art.

[0129] As previously discussed, the assay involves contacting a cell with a polypeptide under conditions and for a period of time that will allow the protease domain of a wild-type Clostridial neurotoxin to cleave an indicator protein in the cell, and determining the presence of a product resulting from cleavage of the indicator protein. The indicator protein may be endogenous to the cell (e.g., an endogenous SNARE protein) or may be an exogenous indicator protein of the type previously described.

[0130] Such assays also typically include determining the extent of conversion of the indicator protein to its cleavage product(s). Observation of one or more cleavage product(s) produced after contacting the polypeptide with the indicator protein, or observation of an increase in the amount of cleavage product(s), indicates proteolytic activity of the polypeptide.

[0131] The determining step may include comparing the full-length indicator protein and the cleavage product(s). The comparing may include determining the amount of full-length indicator protein and / or the amount of one or more cleavage product(s) and may include calculating the ratio of full-length indicator protein and cleavage product(s). Furthermore, an assay for determining proteolytic activity may include comparing the cleavage product(s) that appears after contact of the assayed polypeptide with the indicator protein with a control. The control may be, for example, the cleavage product(s) that appears after contact of a Clostridial neurotoxin known to be capable of cleaving the same indicator protein.

[0132] In certain embodiments, after contacting the cells with the polypeptide, the cells can be lysed and analyzed by gel electrophoresis and Western blotting.For example, an anti-SNAP-25 antibody that binds to the N-terminus of SNAP-25 can be used in Western blot to determine the presence of full-length SNAP-25 and cleaved SNAP-25 (which will migrate in a separate band from full-length SNAP-25).

[0133] Methods and techniques used to lyse host cells, such as bacterial cells, are known in the art, and examples include sonication or the use of a French press.

[0134] In certain embodiments, the full-length indicator protein is not readily degraded in cells, but after its cleavage, one of the resulting fragments is readily degraded, which may be due, for example, to the presence of a residue at the N-terminus of the resulting fragment that serves as a degron only when exposed by cleavage.

[0135] In such an embodiment, the indicator protein may be labeled on a portion thereof that is more easily degraded after cleavage. The label should be selected such that when degradation of the fragment occurs, the label is also degraded. In such an embodiment, whether cleavage occurs can be determined based on measuring the signal from the label.

[0136] The signal received can be compared to a control.

[0137] In certain such embodiments, where the separate fragment formed after cleavage is not similarly readily degradable, the indicator protein may also contain a label on the portion of the indicator protein that forms the fragment after cleavage. Thus, in such embodiments, whether cleavage occurs can be determined by comparing the signal from the label on the more readily degradable fragment with the signal from the label on the less readily degradable fragment that serves as a control.

[0138] The signal(s) from the label(s) can be analyzed using fluorescence-activated cell sorting (FACS). For example, in an embodiment where the full-length indicator protein and its N-terminal fragment formed after cleavage are not easily degradable in cells, but the C-terminal fragment obtained from cleavage is easily degradable, and the N-terminal label is mScarlet and the C-terminal label is NeonGreen, FACS analysis of cells after successful cleavage shows lower green emission compared to red emission. In contrast, if no cleavage occurs, red and green fluorescence should be equally widespread.

[0139] Alternatively, fluorescent micrographs of the cells can be taken. In embodiments such as those described above, successful cleavage will result in less green fluorescence being emitted in the cells compared to control cells not exposed to the protease. In contrast, red fluorescence should remain the same as in the control.

[0140] Also, in certain embodiments, the assay can be a FRET assay.As previously discussed, in such an assay, the indicator protein comprises an N-terminal label and a C-terminal label, one label being a donor label and the other being an acceptor label.The energy transfer between the donor label and the acceptor label leads to a reduction in the fluorescence intensity of the donor label and an increase in the emission intensity of the acceptor label.The success of such transfer depends on the labels remaining in close proximity.The cleavage of the indicator protein tends to make these labels more distant, and therefore such transfer is less successful.The success of cleavage can therefore be determined based on the reduction in the ability of energy transfer to occur.

[0141] In addition to the above, any other means known in the art for analyzing fluorescence from an indicator protein to determine whether cleavage has occurred can be used in the practice of the present invention.

[0142] In certain embodiments, a polypeptide is considered proteolytically active if 20% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the indicator protein is converted to cleavage product(s) in less than 1 minute, less than 5 minutes, less than 20 minutes, less than 40 minutes, less than 60 minutes, or less than 120 minutes.

[0143] Cleavage can be measured at intervals to follow catalytic activity over time.

[0144] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein.

[0145] EXAMPLES

[0146] Example 1 - Selection of optimal parent cell lines for the generation of indicator cell lines

[0147] With the aim of selecting the optimal parental cell line for the development of stably transfected cell lines, Neuro2A (N2a, ATCC CCL-131), BE(2)-M17 (M17, ATCC CRL-2267), IMR-32 (ATCC CCL-127), and NG108-15 [108CC15] (ATCC HB-12317) cells were studied.

[0148] Upon delivery, the cells were allowed to recover and grow. The cell stocks were then frozen and stored in liquid nitrogen. Once sufficient vials of cell stocks had been made, the cells were assayed for their sensitivity to BoNT / A.

[0149] Cells were cultured for 8 or 24 hours in medium containing BoNT / A (Metabiologics, Inc.). Cells cultured for 8 hours were cultured in medium containing 0.1 nM, 1 nM, or 10 nM BoNT / A. Cells cultured for 24 hours were cultured in medium containing 1 nM, 0.1 nM, or 0.01 nM BoNT / A.

[0150] Cleavage of endogenous SNAP-25 was analyzed by Western blot using anti-SNAP-25 antibody (Sigma no. S9684) using standard protocols (Figure 1). NG108 cells showed greater sensitivity to BoNT / A than the other cells, with N2a cells being the least sensitive. Therefore, the NG108 cell line was selected as the primary candidate for the development of a stably transfected indicator cell line. M17 and IMR-32 cell lines showed similar sensitivity to BoNT / A at the concentrations and times tested. M17 was selected as a backup for NG108 due to ease and familiarity of culture.

[0151] Example 2 - Transfection of cells with plasmids containing indicator constructs

[0152] The sensitivity of NG108 and M17 cells to puromycin (InvivoGen No. ANT-PR) and G418 (VWR No. 97064-358) was determined. Cells were grown to approximately 50% confluency and then cultured with various concentrations of puromycin and G418. Both cell lines showed similar sensitivity to puromycin and G418.

[0153] A plasmid (pD2500, Atum) was modified to contain nucleic acid sequences encoding puromycin-N-acetyltransferase (PuroR), a chimeric protein, and a 2A self-cleaving peptide. In the expressed product, the 2A self-cleaving peptide was located between PuroR and the chimeric protein. The chimeric protein contained SNAP-25 adjacent to the N- and C-terminal fluorescent proteins and luciferase (located at the C-terminus). PuroR conferred resistance to puromycin. In addition to the fluorescence-based measurement of degradation promoted by the fluorescent protein, luciferase allowed for luminescence measurement of degradation. The N-terminal fluorescent protein was mScarlet, and the C-terminal fluorescent protein was either NeonGreen, green fluorescent protein, or cyan fluorescent protein (CFP). The plasmid insert containing nucleic acid encoding PuroR, the 2A self-cleaving peptide, and the construct containing mScarlet, SNAP-25, NeonGreen, and luciferase (mScarlet-SNAP25-GeNluc) had the nucleotide sequence of SEQ ID NO:1. The plasmid insert containing nucleic acid encoding PuroR, the 2A self-cleaving peptide and a construct containing mScarlet, SNAP-25, CFP and luciferase (mScarlet-SNAP25-CyanNluc) had the nucleotide sequence of SEQ ID NO:2.

[0154] NeonGreen was chosen for its excitation / emission spectrum and intensity, and in the event that NeonGreen was not sufficiently degraded upon cleavage of the indicator protein, CFP was chosen as a backup due to previous data showing that it is rapidly degraded upon cleavage of the indicator protein.

[0155] NG108 and M17 cells were transfected with plasmids containing either the mScarlet-SNAP25-GeNluc or mScarlet-SNAP25-CyanNluc constructs. Transfections were performed with Lipofectamine 3000 (ThermoFisher) or polyethylenimine using standard protocols.

[0156] 24 hours after transfection, cells were analyzed by fluorescence microscopy to confirm the efficiency of transfection and the correct expression of the indicator protein (Figure 2A-B, an example of cells expressing indicator proteins containing NeonGreen is shown). Due to overexpression, much of the indicator protein was cytosolic. The red and green colors, representing the N- and C-termini of the indicator proteins, respectively, were easily detected, indicating that both termini were colocalized. High transient expression was observed in both cell types, with a transfection efficiency of >70%.

[0157] Once we had verified that the cells were efficiently transfected and that the indicator protein was correctly expressed, we either selected the transfected cells with 2.5 μg / ml puromycin or "shocked" them with an initial high dose of 20 μg / ml puromycin for 1 day and then cultured them in 5-10 μg / ml puromycin. Both treatments yielded a pool of fluorescent, puromycin-resistant cells.

[0158] Concurrently, several further transfections were performed with both indicator constructs and selection for puromycin resistance was performed to obtain further pools of fluorescent puromycin-resistant cells. This ultimately resulted in approximately 6 independent pools of fluorescent puromycin-resistant NG108 cells and 2 independent pools of puromycin-resistant fluorescent M17 cells. Pools were expanded and stocks were frozen and tested for thaw viability.

[0159] Puromycin-resistant cells were analyzed to confirm stable transfection of the indicator construct (Figure 3). In NG108 cells stably transfected with the indicator construct containing NeonGreen, both mScarlet (red) and NeonGreen (green) colocalized, indicating that full-length intact protein was made and distributed within the cells. Furthermore, fluorescence was seen primarily at the cell membrane, indicating that the protein was correctly localized (due to the presence of SNAP-25).

[0160] [Example 3] - Confirmation of cleavage of indicator protein

[0161] A plasmid (pcDNA3.1) was modified to contain SEQ ID NO:3, which encodes the BoNT / A light chain, CFP and an N-terminal SBP tag. The nucleic acid encoding the BoNT / A light chain was synthesized using DNA2.0 (Atum).

[0162] Cells from Example 2 stably transfected with the indicator constructs (mScarlet-SNAP25-GeNluc or mScarlet-SNAP25-CyanNluc) were transiently transfected with an expression vector containing the CFP-BoNT / A construct. At 24 and 48 hours after transfection, numerous red cells, but not green or cyan, were demonstrated, indicating that the indicator protein was cleaved and the C-terminal fragment was rapidly degraded.

[0163] [Example 4] - Confirmation of cleavage of indicator protein

[0164] NG108 cells from Example 2 stably transfected with mScarlet-SNAP25-GeNluc indicator construct were plated (20-30K cells per well) in 96-well optical plates (ThermoFisher #165305) in complete DMEM medium (Corning #50-013-PB) and allowed to adhere for 4 hours. The medium was then changed to Neurobasal Plus (ThermoFisher #A35829) and the cells were cultured for an additional 20 hours, after which the medium was changed to Neurobasal Plus medium containing BoNT / A at 0 (control), 0.1 or 1.0 nM. The cells were cultured for an additional 24 hours. The cells were then trypsinized, washed once with medium, and plated at approximately 2 x 10 cells in DMEM / FBS medium or DPBS with 10 units of Benzonase / ml. 6 The cells were then analyzed on a SY3200 (Sony Biotechnologies) cell sorter using the appropriate lasers / filters for NeonGreen and mScarlet.

[0165] Figure 4 shows the number of green cells per HPF of NG108 cells expressing mScarlet-SNAP25-GeNluc after 24 hours of treatment with BoNT / A. A pool of cells treated with 1 nM BoNT / A showed an approximately 25% reduction in green positive cells per HPF.

[0166] [Example 5] - Confirmation of cleavage of indicator protein

[0167] Representative NG108 and M17 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were trypsinized, washed once with medium, and cultured at approximately 2×10 in DMEM / FBS medium or DPBS with 10 units of Benzonase / ml. 6The cells were then analyzed on a SY3200 (Sony Biotechnologies) cell sorter using the appropriate lasers / filters for NeonGreen and mScarlet.

[0168] NG108 cells were excited at 488 nM to directly excite NeonGreen while only minimally exciting mScarlet. Fluorescence emission was measured at various wavelengths. Additionally, side and forward scatter light intensities were measured to identify subpopulations of cells. Figure 5A represents a scatter plot showing side scatter (SS) on the x-axis and forward scatter (FS) on the y-axis, with the distribution illustrating the variation in cell granularity / complexity (SS) and cell size (FS). Figure 5B represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 525 nM (FITC filter). Figure 5C represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 585 nM (PE filter). Figure 5D represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 617 nM (PE-Texas Red filter). Figure 5E represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 665 nM (7AAD filter). Figure 5F represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 785 nM (PE-Cy7 filter). Figure 5G represents a scatter plot showing the cellular emission fluorescence of cells measured at 665 nM (7AAD filter) on the x-axis and side scatter (SS) on the y-axis. The histogram shows two separate peaks of fluorescence, the less fluorescent peak representing non-expressing cells and the more fluorescent peak representing cells expressing the indicator protein. At all wavelengths of emission reading, the fluorescence remains high. This includes the case where emission was measured at 785 nM (Figure 5F), where most of the light emitted is due to FRET, thus confirming FRET between NeonGreen and mScarlet. The percentage proportion of highly fluorescent cells in the N108 pool ranged from 61% to 93%.

[0169] M17 cells were excited at 488 nM to directly excite NeonGreen while only minimally exciting mScarlet. Fluorescence emission was measured at various wavelengths. Additionally, side and forward scatter light intensities were measured to identify subpopulations of cells. Figure 6A represents a scatter plot showing side scatter (SS) on the x-axis and forward scatter (FS) on the y-axis, with the distribution illustrating the variation in cell granularity / complexity (SS) and cell size (FS). Figure 6B represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 525 nM (FITC filter). Figure 6C represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 585 nM (PE filter). Figure 6D represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 617 nM (PE-Texas Red filter). Figure 6E represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 665 nM (7AAD filter). Figure 6F represents a histogram showing the cellular distribution of emission fluorescence intensity measured at 785 nM (PE-Cy7 filter). Figure 6G represents a scatter plot showing the cellular emission fluorescence of cells measured at 665 nM (7AAD filter) on the x-axis and side scatter (SS) on the y-axis. The histogram shows two separate peaks of fluorescence, the less fluorescent peak representing non-expressing cells and the more fluorescent peak representing cells expressing the indicator protein. At all wavelengths of emission reading, the fluorescence remains high. This includes the case where emission is measured at 785 nM (Figure 6F), where most of the emitted light is due to FRET, thus confirming FRET between NeonGreen and mScarlet. The percentage proportion of highly fluorescent cells in the M17 pool ranged from 14% to 24%, i.e., the proportion of cells expressing the fluorescent protein in the M17 cell pool was smaller compared to the NG108 cell pool.

[0170] NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were treated with BoNT / A at 0 (control), 0.1 or 1.0 nM as described in Example 4.

[0171] Fluorescence from a pool of stably transfected NG108 cells was measured using a SY3200 (Sony Biotechnologies) analyzer. Cells were excited at 488 nM to directly excite NeonGreen with minimal excitation of mScarlet. Fluorescence emission was measured at 530 nM (FITC filter) to detect NeonGreen fluorescence but not mScarlet fluorescence. Figure 7A represents a histogram showing the distribution of emission fluorescence intensity from untreated (control) cells measured at 525 nM. Figure 7B represents a histogram showing the distribution of emission fluorescence intensity from cells treated with 0.1 nM BoNT / A measured at 525 nM. Figure 7C represents a histogram showing the distribution of emission fluorescence intensity from cells treated with 1 nM BoNT / A measured at 525 nM. NeonGreen fluorescence was reduced by approximately 15% (mean fluorescence of cells in gate R2) in the cell pool after treatment with 1 nM BoNT / A (FIG. 7C) compared to the untreated control (FIG. 7A), suggesting that the resulting NeonGreen-containing C-terminal fragment was degraded after cleavage.

[0172] Flow cytometric determination of loss of FRET emission was also performed. Fluorescence from a pool of stably transfected NG108 cells was measured using a SY3200 (Sony Biotechnologies) analyzer. Cells were excited at 488 nM to directly excite NeonGreen with minimal excitation of mScarlet. Fluorescence emission was measured at 785 nM (Cy7 filter), which detects mScarlet fluorescence but not NeonGreen fluorescence. Figure 8A represents a histogram showing the distribution of emission fluorescence intensity from untreated (control) cells measured at 785 nM. Figure 8B represents a histogram showing the distribution of emission fluorescence intensity from cells treated with 0.1 nM BoNT / A measured at 785 nM. Figure 8C represents a histogram showing the distribution of emission fluorescence intensity from cells treated with 1 nM BoNT / A measured at 785 nM. FRET intensity decreased by 16% (mean fluorescence of cells in gate R6) after treatment with 1 nM BoNT / A (FIG. 8C) compared to untreated controls (FIG. 8A), consistent with NeonGreen degradation.

[0173] [Example 6] - Confirmation of cleavage

[0174] Western blots were performed on NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated with no toxin (control), 1 nM or 8 nM BoNT / A, or 1 nM, 10 nM or 100 nM BoNT / E as described in Example 4 (Figure 9).

[0175] Cell lines were tested for cleavage of SNAP-25 (either endogenous or exogenous) using standard blotting techniques and a rabbit anti-SNAP25 primary antibody.

[0176] Cells were lysed using M-PER Reagent (ThermoFisher #78501) according to the manufacturer's recommendations. Lysates were cleared at 15 kg for 10 min and 10 μl samples were run on NuPage 12% Bis-Tris gels (ThermoFisher #NP0341BOX) in MOPS buffer (ThermoFisher #NP0001) at 200 V. Proteins were transferred to PVDF membranes (ThermoFisher #LC2005) using the XCell II blotting system and the Nu-Page transfer protocol. The resulting blots were blocked in 1% BSA / 0.05% Tween20 / PBS, primary antibody 1:3,000 anti-SNAP-25, secondary antibody 1:5,000 alkaline phosphatase conjugated goat anti-rabbit (ThermoFisher #31340), and developed in NBT / BCIP substrate (ThermoFisher #34042). Developed blots were scanned and densitometrically calculated using ImageJ and plotted in MS Excel.

[0177] The indicator protein was detected in all lysates. No obvious cleavage products were detected in control samples. Cells treated with either BoNT / A or BoNT / E produced cleavage products, which increased when higher doses were used. Interestingly, the cells appeared to be nearly as sensitive to BoNT / E as they were to BoNT / A.

[0178] Example 7 - Transfection with receptor constructs

[0179] A plasmid (pD2500, Atum) was modified to contain the receptor construct GD3-SV2C-Syt and a nucleic acid (SEQ ID NO: 4) encoding aminoglycoside 3'-phosphotransferase (Neo). The nucleic acid expresses a fusion protein containing GD3 synthase, SV2C and syntaxin and Neo, with each domain separated from the other by a 2A self-cleaving peptide. Syntaxin was modified into the fusion protein for use with other isoforms of BoNT. Neo confers resistance to G418.

[0180] NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were grown to approximately 60% confluency in a T75 flask. They were then transfected overnight with a plasmid (2 μg / ml) containing the receptor construct in 5 ml of OptiMem / polyethyleneimine according to standard protocols. In the morning, the cells were washed once with fresh complete DMEM medium and cultured for an additional 24-48 hours in complete DMEM medium. The medium was then changed to complete DMEM medium with 500 μg / ml G418 and the cells were cultured for an additional 1-2 weeks, with medium / G418 changes as required. Cells were observed to begin dying by 3 days after G418 addition and continued for approximately 1 week (approximately 60% cell death). Cells remaining after approximately 2 weeks were G418 resistant.

[0181] Example 8 - Directed evolution of cells

[0182] The cells from Example 7 were subjected to two rounds of sorting to isolate cells that had the highest fluorescence and therefore the highest expression of the indicator protein.

[0183] Example 9 - Directed evolution of cells

[0184] The cells selected for high expression of the indicator protein in Example 8 were treated with 0.1 nM, 1 nM or 10 nM BoNT / A or 10 nM BoNT / E for 72 hours as described in Example 4. After treatment, the cells were washed three times, trypsinized, resuspended in fresh medium and sorted. Screening of the cells showed a clear dose-sensitive response to BoNT / A (Figure 10A). Fluorescence microscopy showed that the cells also decreased in green fluorescence with the dose of treatment while maintaining the same level of red fluorescence (Figure 10B). It was noted that the results clearly showed an increase in BoNT / A sensitivity, but no similar increase was seen for BoNT / E.

[0185] Cells that were sensitive to 1,000 pM (1 nM) BoNT / A were selected.

[0186] Example 10 - Directed evolution of cells

[0187] Wild-type NG108 cells (i.e., not transfected with reporter or receptor constructs) were selected (FIG. 11A). These cells show neither green nor red fluorescence.

[0188] Cells from Example 9 that were sensitive to 1,000 pM (1 nM) BoNT / A were expanded and treated with 100 pM BoNT / A as previously discussed, or not (control). After 48 or 96 hours of treatment, cells were washed three times, trypsinized, resuspended in fresh medium, and sorted. Figures 11B-D show flow cytometry data for control, cells treated with 100 pM BoNT / A for 48 hours, and cells treated with 100 pM BoNT / A for 96 hours, respectively.

[0189] Cells from Example 8 that were selected twice for high expression of the indicator construct but not for sensitivity to 1,000 pM BoNT / A in Example 9 were treated with 100 pM BoNT / A for 96 hours as previously discussed or were not treated (control). Figures 11E-F show flow cytometry data for cells treated with control and 100 pM BoNT / A for 96 hours, respectively.

[0190] In Figures 11A-F, the approximately circular gate highlights cells that exhibit neither red nor green fluorescence (i.e., cells that do not express the indicator protein), the approximately elliptical gate highlights cells that exhibit both red and green fluorescence (i.e., cells that express the indicator protein and in which the indicator protein has not been cleaved), and the quadrilateral gate highlights cells that exhibit red fluorescence but relatively reduced green fluorescence (i.e., cells that express the indicator protein but in which the indicator protein has been cleaved).

[0191] Cells previously selected for sensitivity to 1 nM BoNT / A (FIG. 11D) displayed significantly greater sensitivity (more cleavage) to 100 pM BoNT / A than cells that had not been so selected (FIG. 11F).

[0192] Cells that were sensitive to 100 pM BoNT / A after 96 hours of treatment (more than 2 logs more sensitive than wild type NG108) were selected.

[0193] Example 11 - Directed evolution of cells

[0194] Cells from Example 10 that were sensitive to 100 pM BoNT / A after 96 hours of treatment were expanded and treated with 10 pM BoNT / A for 96 hours as previously discussed, or not treated (control). After treatment, cells were washed three times, trypsinized, resuspended in fresh medium, and sorted.

[0195] 12A-B show flow cytometry data for control and 10 pM BoNT / A treated cells, respectively. Although not as dramatic as that seen with higher concentrations of toxin, there was a notable shift in fluorescence of treated cells.

[0196] Example 12 - Receptor construct for BoNT / E

[0197] To confer susceptibility to BoNT / E, NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct were transfected with a plasmid containing the GD3-SV2A-Syt receptor construct (SEQ ID NO:5) using the transfection procedure described in Example 2. This plasmid was constructed by modifying a plasmid containing the GD2-SV2C-Syt receptor construct using a HiFi kit (New England Biolabs), oligonucleotides from IDT, and the SV2A sequence synthesized by GeneArt.

[0198] These cells and cells from Example 7 expressing the GD3-SV2C-Syt receptor construct were cultured in medium containing either 0 (control), 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM BoNT / A or 0 nM (control) BoNT / A or 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0 nM (control) BoNT / E. Cells were treated for 16, 40, 64 or 88 hours. After treatment, cells were lysed and anti-SNAP-25 Western blots were performed using an anti-SNAP-25 antibody. Densitometry data from the blots were plotted as the percentage of cleaved SNAP-25 (Figure 13). There was a significant increase in the susceptibility of cells expressing SV2A to both BoNT / A and BoNT / E compared with cells expressing SV2C, confirming the requirement of SV2A to confer BoNT / E susceptibility.

[0199] Sequence Listing

[0200] Array Description

[0201] SEQ ID NO:1 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising an N-terminal mScarlet tag, SNAP-25, a C-terminal NeonGreen tag and a C-terminal luciferase.

[0202] SEQ ID NO:2 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising an N-terminal mScarlet tag, SNAP-25, a C-terminal CFP tag and a C-terminal luciferase.

[0203] SEQ ID NO:3 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising an N-terminal CFP and the light chain of BoNT / A.

[0204] SEQ ID NO:4 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising domains having the amino acid sequences of GD3 synthase, SV2C, syntaxin and aminoglycoside 3'-phosphotransferase, wherein each domain is separated from the other by a 2A self-cleaving peptide.

[0205] SEQ ID NO:5 is the nucleotide sequence of a nucleic acid encoding a fusion protein comprising domains having the amino acid sequences of GD3 synthase, SV2A, syntaxin and aminoglycoside 3'-phosphotransferase, wherein each domain is separated from the other by a 2A self-cleaving peptide.

[0206] SEQ ID NO:6 is the amino acid sequence of GD3 synthase.

[0207] SEQ ID NO:7 is the amino acid sequence of the 2A self-cleaving peptide encoded by SEQ ID NOs:4 and 5.

[0208] SEQ ID NO:8 is the amino acid sequence of SV2A.

[0209] SEQ ID NO: 9 is the amino acid sequence of SV2B.

[0210] SEQ ID NO:10 is the amino acid sequence of SV2C.

[0211] SEQ ID NO:11 is the amino acid sequence of the fourth luminal domain of SV2A.

[0212] SEQ ID NO: 12 is the amino acid sequence of the fourth luminal domain of SV2B.

[0213] SEQ ID NO: 13 is the amino acid sequence of the fourth luminal domain of SV2C.

[0214] SEQ ID NO:14 is the amino acid sequence of synaptotagmin I.

[0215] SEQ ID NO: 15 is the amino acid sequence of synaptotagmin II.

[0216] SEQ ID NO: 16 is the amino acid sequence of MScarlet.

[0217] SEQ ID NO: 17 is the amino acid sequence of NeonGreen.

[0218] SEQ ID NO: 18 is the amino acid sequence of CFP.

[0219] SEQ ID NO:19 is the amino acid sequence of SNAP-25.

[0220] SEQ ID NO:20 is the amino acid sequence of aminoglycoside 3'-phosphotransferase (Neo).

[0221] SEQ ID NO:21 is the amino acid sequence of puromycin-N-acetyltransferase (PuroR).

[0222] SEQ ID NO:22 is the amino acid sequence of luciferase.

[0223] SEQ ID NO:1

[0224] SEQ ID NO:2

[0225] SEQ ID NO:3

[0226] SEQ ID NO:4

[0227] SEQ ID NO:5

[0228] SEQ ID NO:6 MSPCGRARRQTSRGAMAVLAWKFPRTRLPMGASALCVVVLCWLYIFPVYRLPNEKEIVQGVLQQGTAWRRNQTAARAFRKQMEDCCDPAHLFAMTKMNSPMGKSMWYDGEFLYSFTIDNSTYSLFPQATPFQLPLKKCAVVGNGGILKKSGCGRQIDEANFVMRCNLPPLSSEYTKDV GSKSQLVTANPSIIRQRFQNLLWSRKTFVDNMKIYNHSYIYMPAFSMKTGTEPSLRVYYTLSDVGANQTVLFANPNFLRSIGKFWKSRGIHAKRLSTGLFLVSAALGLCEEVAIYGFWPFSVNMHEQPISHHYYDNVLPFSGFHAMPEEFLQLWYLHKIGALRMQLDPCEDTSLQPTS

[0229] SEQ ID NO:7 GSGATNFSLLKQAGDVEENPGP

[0230] SEQ ID NO:8 MEEGFRDRAAFIRGAKDIAKEVKKHAAKKVVKGLDRVQDEYSRRSYSRFEEEDDDDDFPAPSDGYYRGEGTQDEEEGGASSDATEGHDEDDEIYEGEYQGIPRAESGGKGERMADGAPLAGVRGGLSDGEGPPGGRGEAQRRKEREELAQQYEAILRECGHGRFQWTLYFVLGLALMADGVEVFV VGFVLPSAEKDMCLSDSNKGMLGLIVYLGMMVGAFLWGGLADRLGRRQCLLISLSVNSVFAFFSSFVQGYGTFLFCRLLSGVGIGGSIPIVFSYFSEFLAQEKRGEHLSWLCMFWMIGGVYAAAMAWAIIPHYGWSFQMGSAYQFHSWRVFVLVCAFPSVFAIGALTTQPESPRFFLENGKHDEAW MVLKQVHDTNMRAKGHPERVFSVTHIKTIHQEDELIEIQSDTGTWYQRWGVRALSLGGQVWGNFLSCFGPEYRRITLMMMGVWFTMSFSYYGLTVWFPDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCTFINTVF YNTDLFEYKFVNSRLINSTFLHNKEGCPLDVTGTGEGAYMVYFVSFLGTLAVLPGNIVSALLMDKIGRLRMLAGSSVMSCVSCFFLSFGNSESAMIALLCLFGGVSIASWNALDVLTVELYPSDKRTTAFGFLNALCKLAAVLGISIFTSFVGITKAAPILFASAALALGSSLALKLPETRGQVLQ

[0231] SEQ ID NO:9 MEDSYKDRTSLMKGAKDIAREVKKQTVKKVNQAVDRAQDEYTQRSYSRFQDEEDDDDYYPAGETYNGEANDDEGSSEATEGHDEDDEIYEGEYQGIPSMNQAKDSIVSVGQPKGDEYKDRRELESERRADEEELAQQYELIIQECGHGRFQWALFFVLGMALMADGVEVFVVGFVLPSAET DLCIPNSGSGWLGSIVYLGMMVGAFFWGGLADKVGRKQSLLICMSVNGFFAFLSSFVQGYGFFLFCRLLSGFGIGGAIPTVFSYFAEVLAREKRGEHLSWLCMFWMIGGIYASAMAWAIIPHYGWSFSMGSAYQFHSWRVFVIVCALPCVSSVVALTFMPESPRFLLEVGKHDEAWMILKLI HDTNMRARGQPEKVFTVNKIKTPKQIDELIEIESDTGTWYRRCFVRIRTELYGIWLTFMRCFNYPVRDNTIKLTIVWFTLSFGYYGLSVWFPDVIKPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNT DFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYSAYWIYFVNFLGTLAVLPGNIVSALLMDRIGRLTMLGGSMVLSGISCFFLWFGTSESMMIGMLCLYNGLTISAWNSLDVVTVELYPTDRRATGFGFLNALCKAAAVLGNLIFGSLVSITKSIPILLASTVLVCGGLVGLCLPDTRTQVLM

[0232] SEQ ID NO:10 MDDYKYQDNYGGYAPSDGYYRGNESNPEEDAQSDVTEGHDEEDEIYEGEYQGIPHPDDVKAKQAKMAPSRMDSLRGQTDLMAERLEDEEQLAHQYETIMDECGHGRFQWILFFVLGLALMADGVEVFVVSFALPSAEKDMCLSSSSKKGMLGMIVYLGMMAGAFILGGLAD KLGRKRVLSMSLAVNASFASLSSFVQGYGAFLFCRLISGIGIGGALPIVFAYFSEFLSREKRGEHLSWLGIFWMTGGLYASAMAWSIIPHYGWGFSMGTNYHFHSWRVFVIVCALPCTVSMVALKFMPESPRFLLEMGKHDEAWMILKQVHDTNMRAKGTPEKVFTVSNIK TPKQMDEFIEIQSSTGTWYQRWLVRFKTIFKQVWDNALYCVMGPYRMNTLILAVVWFAMAFSYYGLTVWFPDMIRYFQDEEYKSKMKVFFGEHVYGATINFTMENQIHQHGKLVNDKFTRMYFKHVLFEDTFDECYFEDVTSTDTYFKNCTIESTIFYNTDLYEHKFINC RFINSTFLEQKEGCHMDLEQDNDFLIYLVSFLGSLSVLPGNIISALLMDRIGRLKMIGGSMLISAVCCFFLFFGNSESAMIGWQCLFCGTSIAAWNALDVITVELYPTNQRATAFGILNGLCKFGAILGNTIFASFVGITKVVPILLAAASLVGGGLIALRLPETREQVLM

[0233] SEQ ID NO:11 FPDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCTFINTVFYNTDLFEYKFVNSRLINSTFLHNKEGCPLDVTGTGEGAY

[0234] SEQ ID NO:12 WFPDMIRYFQDEEYKSKMKVFFGEHVYGATINFTMENQIHQHGKLVNDKFTRMYFKHVLFEDTFFDECYFEDVTSTDTYFKNCTIESTIFYNTDLYEHKFINCRFINSTFLEQKEGCHMDLEQDNDFLIY

[0235] SEQ ID NO:13 WFPDVIKPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYSAY

[0236] SEQ ID NO:14 MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLPWALIAIAIVAVLLVLTCCFCICKKCLFKKKNKKKGKEKGGKNAINMK DVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKVFLLPDKKKKFETKVHRKTLNPVFNEQF TFKVPYSELGGKTLVMAVYDFDRFSKHDIIGEFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLKKMDVGGLSDPYVKIH LMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVVTVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEVDAMLAVKK

[0237] SEQ ID NO:15 MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLPPWALIAIAVVAGLLLLTCCFCICKKCCCKKKKNKKEKGKGMKN AMNMKDMKGGQDDDDAETGLTEGEGEGEEEKEPENLGKLQFSLDYDFQANQLTVGVLQAAELPALDMGGTSDPYVKVFLLPDKKKKYETKVHRKTLNPAFNETFT FKVPYQELGGKTLVMAIYDFDRFSKHDIIGEVKVPMNTVDLGQPIEEWRDLQGGEKEEPEKLGDICTSLRYVPTAGKLTVCILEAKNLKKMDVGGLSDPYVKIHL MQNGKRLKKKKTTVKKKTLNPYFNESFSFEIPFEQIQKVQVVVTVLDYDKLGKNEAIGKIFVGSNATGTELRHWSDMLANPRRPIAQWHSLKPEEEVDALLGKNK

[0238] SEQ ID NO:16 MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLED GTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK

[0239] SEQ ID NO:17 MASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFP ADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTGFEDFVGDWRQTAGYNLDQVLEQGGVSSLFQ

[0240] SEQ ID NO:18 LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGD TLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGL

[0241] SEQ ID NO:19 MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG

[0242] SEQ ID NO:20 MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAAGRDWLLLGEVPGQDLLSSHLAPAEKVSIMADAMRRLHTLDPATCP FDHQAKHRIERARTRMEAGLVDQDDLDEEHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF

[0243] SEQ ID NO:21 MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA

[0244] SEQ ID NO:22 HTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA [Explanation of symbols]

[0245] [Figure 4] Average Cell counts in HPF: Average cell counts in HPF Toxin concentration: Toxin concentration [Figures 5, 6, 7, 8] FS Lin: FS linear SS Log: SS logarithm Regi...: Area Count: Count %Hist: Histogram% Mean: Average FITC Log: FITC logarithm PE Log: PE logarithm PE-Texas Red Log: PE-Texas Red Log 7AAD Log: 7AAD logarithm PE-Cy7 Log: PE-Cy7 logarithm [Figure 7] Contr: Contrast [Figure 8] Control: [Figure 9] NG108 mScarlet-SNAP25-GeNluc 2a Puro R BoNT / A and / E cleavage experiment: NG108 mScarlet-SNAP25-GeNluc 2a Puro R BoNT / A and / E cleavage experiment Indicator with puro R: Indicator with Puro R Indicator without pro R: Indicator without Puro R Indicator N-terminal cleavage products BoNT / A: N-terminal cleavage products BoNT / A indicator Endogenous SNAP25 and cleavage product: Endogenous SNAP25 and cleavage product Indicator N-terminal cleavage products BoNT / E: N-terminal cleavage products BoNT / E indicator No Toxin: No toxins [Figure 10F-J] Merge: Overlay [Figure 13A] Percent SNAP25 Cleavage: Percentage of SNAP25 cleaved

Claims

1. (a) a Clostridial neurotoxin receptor or a variant or fragment thereof having the ability to bind to a Clostridial neurotoxin; (b) a ganglioside having binding affinity for a Clostridial neurotoxin; and (c) a cell that has been genetically modified to express or overexpress a labeled indicator protein that is cleavable by a Clostridial neurotoxin, said cell comprises an exogenous nucleic acid that provides said expression or overexpression of said Clostridial neurotoxin receptor or a variant or fragment thereof; The cell comprises an exogenous nucleic acid that provides expression or overexpression of an enzyme of the ganglioside synthesis pathway or a catalytically active variant or fragment thereof of such an enzyme; and The cells contain an exogenous nucleic acid that provides for expression or overexpression of a labeled indicator protein that is cleavable by a Clostridial neurotoxin.

2. In vitro methods of characterizing the activity of or identifying Clostridial neurotoxin preparations for therapeutic and / or cosmetic use, including: a. providing a cell having (i) an exogenous nucleic acid that provides expression or overexpression of a Clostridial neurotoxin receptor or a variant or fragment thereof having the ability to bind to a Clostridial neurotoxin; (ii) an exogenous nucleic acid that provides expression or overexpression of an enzyme of the synthetic pathway of a ganglioside or a variant or fragment thereof having the catalytic activity of such an enzyme, wherein the ganglioside has binding affinity for a Clostridial neurotoxin; and (iii) an exogenous nucleic acid that provides expression or overexpression of a labeled indicator protein cleavable by a Clostridial neurotoxin; b. contacting the cell with a Clostridial neurotoxin formulation; c. comparing the level of cleavage of the indicator protein after contact with the Clostridial neurotoxin formulation to the level of cleavage before contact with the Clostridial neurotoxin formulation; d. (i) identifying the Clostridial neurotoxin preparation as suitable for therapeutic and / or cosmetic use if the level of cleavage of the indicator protein after contact is increased, or (ii) identifying the presence of activity if the level of cleavage of the indicator protein after contact is increased; or e. (i) identifying the Clostridial neurotoxin preparation as not suitable for therapeutic and / or cosmetic use if the level of cleavage of the indicator protein after contact is not increased, or (ii) identifying the absence of activity if the level of cleavage of the indicator protein after contact is not increased.

3. In vitro methods for characterizing the activity of Clostridial neurotoxin preparations or identifying Clostridial neurotoxin preparations suitable for therapeutic and / or cosmetic use, including: a. providing a cell having (i) an exogenous nucleic acid that provides expression or overexpression of a Clostridial neurotoxin receptor or a variant or fragment thereof having the ability to bind to a Clostridial neurotoxin; (ii) an exogenous nucleic acid that provides expression or overexpression of an enzyme of the synthetic pathway of a ganglioside or a variant or fragment thereof having the catalytic activity of such an enzyme, wherein the ganglioside has binding affinity for a Clostridial neurotoxin; and (iii) an exogenous nucleic acid that provides expression or overexpression of a labeled indicator protein cleavable by a Clostridial neurotoxin; b. contacting the cell with a Clostridial neurotoxin formulation; c. comparing the level of cleavage of the indicator protein following contact with the Clostridial neurotoxin formulation to the level of cleavage following contact with a control Clostridial neurotoxin formulation; d. (i) identifying the Clostridial neurotoxin formulation as suitable for therapeutic and / or cosmetic use if the level of cleavage of the indicator protein after contact is increased or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin formulation, or (ii) identifying the presence of activity if the level of cleavage of the indicator protein after contact is increased or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin formulation; or e. (i) identifying the Clostridial neurotoxin preparation as not suitable for therapeutic and / or cosmetic use if the level of cleavage of the indicator protein after contact is not increased relative to or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin preparation, or (ii) identifying the absence of activity if the level of cleavage of the indicator protein after contact is not increased relative to or equivalent to the level of cleavage after contact with a control Clostridial neurotoxin preparation.

4. The cell of claim 1, wherein the cell overexpressing the receptor and ganglioside expresses more of the receptor and ganglioside compared to a natural target cell of the Clostridial neurotoxin.

5. 10. The cell of claim 1 or 4, wherein the cell that overexpresses the receptor and ganglioside expresses more of the receptor and ganglioside when compared to a cell lacking the exogenous nucleic acid.

6. The cell of any one of claims 1 or 4 to 5, wherein the cell is a neuronal cell, a non-neuronal cell, a neuroendocrine cell, an embryonic kidney cell, a breast cancer cell, a neuroblastoma cell or a neuroblastoma-glioma hybrid cell.

7. The cell of any one of claims 1 or 4 to 6, wherein the cell is a neuroblastoma cell or a neuroblastoma-glioma cell.

8. The cell of any one of claims 1 or 4 to 7, wherein the cell is a neuroblastoma-glioma cell.

9. The cell of any one of claims 1 or 4 to 8, wherein the cell is an NG108 cell.

10. The cell of any one of claims 1 or 4 to 9, wherein the cell has been genetically modified to overexpress a ganglioside.

11. 11. The cell of any one of claims 1 or 4 to 10, wherein the cell is genetically modified to express or overexpress GM1a, GD1a, GD1b, GT1b and / or GQ1b.

12. The cell of any one of claims 1 or 4 to 11, wherein the cell is genetically modified to express or overexpress GD1a, GD1b, and / or GT1b.

13. The cell of any one of claims 1 or 4 to 12, wherein the cell is genetically modified to express or overexpress GD1b and / or GT1b.

14. 14. The cell of any one of claims 1 or 4 to 13, wherein the cell is genetically modified to express or overexpress glucosylceramide synthase, GalT-I, GalNAcT, GM3 synthase, GD3 synthase, GT3 synthase, galactosylceramide synthase, GM4 synthase, GalT-II, ST-IV or ST-V or a catalytically active variant or fragment thereof of such enzymes.

15. 15. The cell of any one of claims 1 or 4 to 14, wherein the cell is genetically modified to express or overexpress GD3 synthase or a catalytically active variant or fragment thereof of GD3 synthase.

16. 16. The cell of any one of claims 1 or 4 to 15, wherein the cell is genetically modified to express or overexpress GD3 synthase.

17. 17. The cell of any one of claims 1 or 4 to 16, wherein the cell is genetically modified to overexpress a protein receptor or a variant or fragment thereof having the ability to bind to a Clostridial neurotoxin.

18. 18. The cell of any one of claims 1 or 4 to 17, wherein the cell is genetically modified to express or overexpress SV2 or synaptotagmin or a variant or fragment thereof that has the ability to bind to a Clostridial neurotoxin.

19. 20. The cell of any one of claims 1 or 4 to 18, wherein the cell is genetically modified to express or overexpress SV2 or a variant or fragment thereof that has the ability to bind to a Clostridial neurotoxin.

20. 20. The cell of any one of claims 1 or 4 to 19, wherein the cell is genetically modified to express or overexpress SV2A or SV2C or a variant or fragment thereof that has the ability to bind to a Clostridial neurotoxin.

21. 21. A cell according to any one of claims 1 or 4 to 20, wherein the cell has been genetically modified to express or overexpress the fourth luminal domain of SV2A or SV2C.

22. The cell of any one of claims 1 or 4 to 21, wherein the cell is genetically modified to express or overexpress an indicator protein.

23. 23. The cell of any one of claims 1 or 4 to 22, wherein the cell has been genetically modified to express or overexpress an indicator protein comprising a SNARE domain.

24. 24. The cell of any one of claims 1 or 4 to 23, wherein the cell has been genetically modified to express or overexpress an indicator protein, the indicator protein comprising the amino acid sequence of syntaxin, synaptobrevin or SNAP-25, or a variant or fragment thereof that is susceptible to proteolysis by a protease component of a wild-type Clostridial neurotoxin.

25. The cell of any one of claims 1 or 4 to 24, wherein the cell has been genetically modified to express or overexpress a labeled indicator protein.

26. 26. The cell of any one of claims 1 or 4 to 25, wherein the cell is genetically modified to express or overexpress an indicator protein comprising an N-terminal tag and a C-terminal tag.

27. 27. The cell of any one of claims 1 or 4 to 26, wherein the cell is genetically modified to express or overexpress an indicator protein comprising an amino acid sequence of a fluorescent protein tag.

28. The cell according to any one of claims 1 or 4 to 27, wherein the cell is genetically modified to express or overexpress an indicator protein comprising the amino acid sequence of mScarlet and the amino acid sequence of NeonGreen.

29. 29. The cell of any one of claims 1 or 4 to 28, wherein the cell is genetically modified to express or overexpress an indicator protein comprising mScarlet as an N-terminal label and NeonGreen as a C-terminal label.

30. 30. A method for generating a cell according to any one of claims 1 or 4 to 29, comprising introducing into the cell: (i) a Clostridial neurotoxin receptor or a variant or fragment thereof having the ability to bind to a Clostridial neurotoxin; (ii) an enzyme in the synthetic pathway of a ganglioside or a variant or fragment thereof having the catalytic activity of such an enzyme, wherein the ganglioside has binding affinity for a Clostridial neurotoxin; and (iii) a nucleic acid encoding a labeled indicator protein cleavable by a Clostridial neurotoxin.

31. 31. The method of claim 30, wherein the nucleic acid is introduced by transfection.

32. An assay for determining the activity of a modified or recombinant Clostridial neurotoxin, the method comprising contacting a cell described in any one of claims 1 or 4 to 29 with a modified or recombinant Clostridial neurotoxin under conditions and for a period of time that will allow a protease domain of a wild-type Clostridial neurotoxin in the cell to cleave an indicator protein, and determining the presence of a product resulting from cleavage of such indicator protein.

33. 33. The assay of claim 32, wherein the full-length indicator protein is not readily degraded in cells, but after its cleavage, one of the resulting fragments is readily degraded.

34. 34. The assay of claim 32 or 33, wherein the indicator protein comprises a C-terminal label, and wherein the full-length indicator protein is not readily degraded in cells, but following its cleavage, the resulting C-terminal fragment is readily degraded, and degradation of the C-terminal fragment results in degradation of the C-terminal label.

35. The assay of any one of claims 32 to 34, wherein the indicator protein comprises a C-terminal label, and the full-length indicator protein is not readily degraded in cells, but following its cleavage, the resulting C-terminal fragment is readily degraded, and degradation of the C-terminal fragment results in degradation of the C-terminal label, and cleavage of the indicator protein is determined by measuring a signal from the C-terminal label after contacting cells with a modified or recombinant Clostridial neurotoxin.

36. 33. The assay of claim 32, wherein after such contacting, the cells are lysed, the resulting cell lysate is contacted with an antibody, and a Western blot is performed.

37. The method of claim 2 or 3, wherein the cells that overexpress the receptor and ganglioside express more of the receptor and ganglioside compared to natural target cells of the Clostridial neurotoxin.

38. The method of any one of claims 2, 3 or 37, wherein cells that overexpress the receptor and ganglioside express more of the receptor and ganglioside when compared to cells lacking the exogenous nucleic acid.

39. The method of any one of claims 2, 3 or 37 to 38, wherein the cell is a neuronal cell, a non-neuronal cell, a neuroendocrine cell, an embryonic kidney cell, a breast cancer cell, a neuroblastoma cell or a neuroblastoma-glioma hybrid cell.

40. The method of any one of claims 2, 3 or 37-39, wherein the cell is a neuroblastoma cell or a neuroblastoma-glioma cell.

41. The method of any one of claims 2, 3 or 37 to 40, wherein the cell is a neuroblastoma-glioma cell.

42. The method of any one of claims 2, 3 or 37 to 41, wherein the cell is an NG108 cell.

43. The method of any one of claims 2, 3 or 37-42, wherein the cells are genetically modified to overexpress gangliosides.

44. The method of any one of claims 2, 3 or 37 to 43, wherein the cells are genetically modified to express or overexpress GM1a, GD1a, GD1b, GT1b and / or GQ1b.

45. The method of any one of claims 2, 3 or 37-44, wherein the cells are genetically modified to express or overexpress GD1a, GD1b, and / or GT1b.

46. ​​The cell or method of any one of claims 2, 3 or 37-45, wherein the cell has been genetically modified to express or overexpress GD1b and / or GT1b.

47. The method of any one of claims 2, 3 or 37 to 46, wherein the cells have been genetically modified to express or overexpress glucosylceramide synthase, GalT-I, GalNAcT, GM3 synthase, GD3 synthase, GT3 synthase, galactosylceramide synthase, GM4 synthase, GalT-II, ST-IV or ST-V or a variant or fragment thereof having the catalytic activity of such enzymes.

48. The method of any one of claims 2, 3 or 37 to 47, wherein the cell has been genetically modified to express or overexpress GD3 synthase or a variant or fragment thereof having catalytic activity of GD3 synthase.

49. The method of any one of claims 2, 3 or 37-48, wherein the cells are genetically modified to express or overexpress GD3 synthase.

50. The method of any one of claims 2, 3 or 37 to 49, wherein the cell has been genetically modified to overexpress a protein receptor or a variant or fragment thereof having the ability to bind to a Clostridial neurotoxin.

51. The method of any one of claims 2, 3 or 37 to 50, wherein the cells have been genetically modified to express or overexpress SV2 or synaptotagmin or a variant or fragment thereof capable of binding to a Clostridial neurotoxin.

52. The method of any one of claims 2, 3 or 37 to 51, wherein the cells have been genetically modified to express or overexpress SV2 or a variant or fragment thereof capable of binding to a Clostridial neurotoxin.

53. The method of any one of claims 2, 3 or 37 to 52, wherein the cells are genetically modified to express or overexpress SV2A or SV2C or a variant or fragment thereof capable of binding to a Clostridial neurotoxin.

54. The method of any one of claims 2, 3 or 37 to 53, wherein the cells are genetically modified to express or overexpress the fourth luminal domain of SV2A or SV2C.

55. The method of any one of claims 2, 3 or 37 to 54, wherein the cells are genetically modified to express or overexpress an indicator protein.

56. The method of any one of claims 2, 3 or 37 to 55, wherein the cells have been genetically modified to express or overexpress an indicator protein comprising a SNARE domain.

57. The method of any one of claims 2, 3 or 37 to 56, wherein the cells have been genetically modified to express or overexpress an indicator protein, and the indicator protein comprises the amino acid sequence of syntaxin, synaptobrevin or SNAP-25, or a variant or fragment thereof that is susceptible to proteolysis by a protease component of a wild-type Clostridial neurotoxin.

58. The method of any one of claims 2, 3 or 37-57, wherein the cells have been genetically modified to express or overexpress a labeled indicator protein.

59. The method of any one of claims 2, 3 or 37 to 58, wherein the cells are genetically modified to express or overexpress an indicator protein comprising an N-terminal tag and a C-terminal tag.

60. The method of any one of claims 2, 3 or 37 to 59, wherein the cells have been genetically modified to express or overexpress an indicator protein comprising an amino acid sequence of a fluorescent protein tag.

61. A method described in any one of claims 2, 3, or 37 to 60, wherein the cells are genetically modified to express or overexpress an indicator protein comprising the amino acid sequence of mScarlet and the amino acid sequence of NeonGreen.

62. A method according to any one of claims 2, 3 or 37 to 61, wherein the cells have been genetically modified to express or overexpress an indicator protein comprising mScarlet as an N-terminal label and NeonGreen as a C-terminal label.

63. A method of modifying a cell suitable for use in an assay for characterizing the activity of a Clostridial neurotoxin preparation or for identifying a Clostridial neurotoxin preparation suitable for therapeutic and / or cosmetic use, the method comprising: a. Engineering cells to incorporate: i. an exogenous nucleic acid that provides expression or overexpression of a Clostridial neurotoxin receptor or a variant or fragment thereof that has the ability to bind to a Clostridial neurotoxin; ii. an exogenous nucleic acid that provides expression or overexpression of an enzyme of the synthetic pathway of a ganglioside or a catalytically active variant or fragment thereof of such an enzyme, wherein the ganglioside has binding affinity for a Clostridial neurotoxin; and iii. An exogenous nucleic acid that provides for expression or overexpression of a labeled indicator protein that is cleavable by a Clostridial neurotoxin.

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