Genetically modified cells sensitive to Clostridium neurotoxin

Abstract

Cells genetically engineered to be highly sensitive to Clostridium neurotoxins, e.g., botulinum neurotoxin and tetanus neurotoxin, or modified or recombinant versions thereof, are described herein. Methods for producing such genetically engineered cells and methods for using such cells to test the activity of modified or recombinant Clostridium neurotoxins are described herein.

Description

Technology Field

[0001] The present invention generally relates to genetically engineered cells having increased sensitivity to Clostridium neurotoxins, such as botulinum neurotoxin and tetanus neurotoxin. The present invention also relates to a method for producing such cells and a method for using such cells to test the activity of polypeptides derived from such neurotoxins, such as modified and recombinant versions of such Clostridium neurotoxins. Background Technology

[0002] Anaerobic Gram-positive bacterium Clostridium botulinum ( Clostridium botulinum ) produces various different types of neurotoxins, including botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT).

[0003] BoNT is the most potent toxin known, with a median lethal dose (LD50) in mice depending on the serotype. 50 The value is in the range of 0.5 to 5 ng / kg. BoNT is adsorbed to the gastrointestinal tract and, after entering the general circulation, binds to the presynaptic membrane of cholinergic nerve terminals to prevent the release of the neurotransmitter acetylcholine.

[0004] BoNT is widely known for its ability to induce flaccid muscle paralysis. Due to the aforementioned muscle-relaxing properties, BoNT has been used in various medical and cosmetic procedures, including the treatment of glabellar lines or hyperkinetic facial lines, headaches, hemifacial spasms, bladder hyperactivity, hyperhidrosis, nasolabial folds, cervical dysplasia, blepharospasm, and spasticity.

[0005] Currently, there are at least eight distinct classes of BoNT, 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 similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and this classification by serotype correlates with percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on percentage sequence identity with amino acids.

[0006] Different serotypes of BoNT differ among affected animal species in terms of the severity and duration of induced paralysis. For example, BoNT / A is the most lethal of all known biological substances and is 500 times more potent than BoNT / B in rats in terms of paralysis. Additionally, in mice, the duration of paralysis after BoNT / A injection is 10 times longer than after BoNT / E injection.

[0007] In nature, Clostridium neurotoxin is synthesized as a short-chain polypeptide that is modified post-translationally by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleaving occurs at a specific cleavage site, often referred to as the activation site, located between cysteine ​​residues that provide the inter-chain disulfide bonds. The active form of the toxin is this bichain form. The two chains are called a heavy chain (H-chain) with a molecular weight of approximately 100 kDa and a 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 component in the exposed loop region. After the targeting moiety binds to its target neuron and the bound toxin is internalized into the cell via the endosome, the translocation domain translocates the L-chain across the endosome membrane into the cytoplasm.

[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 (Reference [Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. ] See reference). The acronym SNARE stands for the term soluble NSF attachment receptor ( S oluble N SF A ttachment R It originates from the receptor, where NSF is the N-ethylmaleimide-sensitive factor ( N -ethylmaleimide- S sensitive F It means actor). The protease domain has zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins.

[0009] Through their respective protease domains, various different Clostridium neurotoxins cleave different SNARE proteins. BoNT / B, BoNT / D, BoNT / F, BoNT / G, and TeNT cleave synaptobrevin, known as the vesicle-associated membrane protein (VAMP). BoNT / A, BoNT / C, and BoNT / E cleave a 25 kDa (SNAP-25) synaptosome-associated protein. BoNT / C cleaves syntaxin.

[0010] SNARE proteins associate with the membranes of secretory vesicles or cell membranes and facilitate the exocytosis of molecules by mediating the fusion of secretory vesicles and cell membranes, thereby allowing the contents of the vesicles to be released outside the cell. The cleavage of these SNARE proteins inhibits this exocytosis, thereby suppressing the release of neurotransmitters from these neurons. As a result, striated muscles are paralyzed and sweat gland secretion ceases.

[0011] Therefore, once delivered to the desired target cells, Clostridium neurotoxin can inhibit cellular secretion from the target cells.

[0012] Modifying Clostridium neurotoxin to alter its properties is known in the relevant art. Modifications may include amino acid modifications, such as the addition, deletion, and / or substitution of amino acid(s), and / or chemical modifications, such as the addition of phosphates or carbohydrates or the formation of disulfide bonds. Modifications may also involve the rearrangement of Clostridium neurotoxin components, for example, flanking protease components into translocation and targeting components.

[0013] Also, Clostridia ( ClostridiaIt is known in the art to produce recombinant Clostridium neurotoxins that are genetically identical to the neurotoxin from ), or differ from the wild-type Clostridium neurotoxin in that they contain additional amino acids, fewer amino acids, or different amino acids, or have components arranged in a different order than that arranged in the wild-type Clostridium neurotoxin. These recombinant Clostridium neurotoxins may also be chemically modified as described above.

[0014] However, differences between modified and recombinant Clostridium neurotoxins and their wild-type counterparts can affect the desired SNARE protein-cleaving properties of the neurotoxins. Therefore, it may be important to determine whether these differences improve, reduce, or eliminate this activity.

[0015] Various conventional assays are available to enable a person skilled in the art to confirm whether these modified or recombinant Clostridium neurotoxins possess the desired activity of cleaving targeted SNARE proteins. These assays involve testing for the presence of products generated from the cleavage of SNARE proteins. For example, the presence of cleavage products can be detected by contacting cells with the modified or recombinant neurotoxin, lysing the cells, and analyzing them by SDS-PAGE. Alternatively, cleavage products can be detected by contacting the cell lysate with an antibody.

[0016] Although natural cells can be used in this assay, they may have only limited sensitivity to Clostridium neurotoxin; therefore, the assay typically requires the use of high concentrations of these cells. Additionally, it is preferable to use clonal stable cell lines for this assay.

[0017] Therefore, there is a need for genetically engineered cells with increased sensitivity to Clostridium neurotoxin for use in this assay.

[0018] The present invention relates to cells genetically engineered to partially express or overexpress a receptor for Clostridium neurotoxin, or a variant or fragment thereof. Such receptors may be protein receptors or gangliosides. Preferably, (in the context of the cell itself or any method or use involving the cell described herein) the cell described herein comprises an exogenous nucleic acid that provides expression or overexpression (preferably overexpression) of the receptor and / or ganglioside, wherein said exogenous nucleic acid is under the control of a constitutive and / or inducible promoter (preferably a constitutive promoter).

[0019] The present invention also relates in part to a method for producing such cells. The method comprises the step of introducing into a cell a nucleic acid encoding a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin; and / or an enzyme of the ganglioside synthesis pathway, or a variant or fragment thereof having catalytic activity of such enzyme.

[0020] The present invention also relates to a assay for determining the activity of partially modified or recombinant neurotoxins. The method comprises the steps of contacting the aforementioned cells with the modified or recombinant neurotoxin under conditions and for a period such that the protease domain of the wild-type Clostridium neurotoxin can cleave an indicator protein in the cells, and determining the presence of a product generated from the cleavage of such indicator proteins.

[0021] The present invention encompasses a method for testing / evaluating the activity of a batch of Clostridium neurotoxin for therapeutic / cosmetic use. This method is advantageously useful for monitoring toxin activity during storage and tracking activity over time. An additional benefit is the ability to determine optimal storage conditions (e.g., without degrading activity levels). The method is particularly advantageous for characterizing the activity of recombinant Clostridium neurotoxin (e.g., cell binding / SNARE cleaving ability).

[0022] One aspect of the present invention provides an in vitro method for characterizing the activity of a Clostridium neurotoxin (preferably BoNT) formulation or identifying a Clostridium neurotoxin (preferably BoNT) formulation for therapeutic (and / or cosmetic) use, the method comprising the following steps:

[0023] a. A step of providing a cell (e.g., a genetically engineered cell) having an exogenous nucleic acid that provides expression or overexpression of a receptor and / or ganglioside (preferably a receptor) having binding affinity for Clostridium neurotoxin, and an exogenous nucleic acid that provides expression or overexpression of an indicator protein (preferably an indicator protein containing a SNARE domain) capable of being cleaved by Clostridium neurotoxin; preferably, wherein the cell does not express the receptor and / or ganglioside (preferably a receptor) in the absence of said exogenous nucleic acid;

[0024] b. A step of contacting the above cells with a Clostridium neurotoxin formulation;

[0025] c. a step of comparing the cleavage level of an indicator protein after contact with a Clostridium neurotoxin formulation (e.g., administration) with the cleavage level prior to contact with a Clostridium neurotoxin formulation; and

[0026] d. (i) confirming that the Clostridium neurotoxin formulation is suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein increases after contact, or (ii) confirming the presence of activity if the level of cleavage of the indicator protein increases after contact; or

[0027] e. (i) confirming that the Clostridium neurotoxin formulation is unsuitable for therapeutic (and / or cosmetic) use if the cleavage level of the indicator protein after contact does not increase, or (ii) confirming the absence of activity if the cleavage level of the indicator protein after contact does not increase.

[0028] Another aspect of the present invention provides an in vitro method for characterizing the activity of a Clostridium neurotoxin (preferably BoNT) formulation or identifying a Clostridium neurotoxin (preferably BoNT) formulation suitable for therapeutic (and / or cosmetic) use, the method comprising the following steps:

[0029] a. A step of processing a cell (e.g., genetically modified cell) to incorporate an exogenous nucleic acid that provides expression or overexpression of a receptor and / or ganglioside (preferably a receptor) having binding affinity for Clostridium neurotoxin, and an exogenous nucleic acid that provides expression or overexpression of an indicator protein (preferably an indicator protein containing a SNARE domain) capable of being cleaved by Clostridium neurotoxin; preferably, wherein the cell does not express the receptor and / or ganglioside (preferably a receptor) in the absence of said exogenous nucleic acid;

[0030] b. A step of contacting the above cells with a Clostridium neurotoxin formulation;

[0031] c. a step of comparing the cleavage level of an indicator protein after contact with a Clostridium neurotoxin formulation (e.g., administration) with the cleavage level prior to contact with a Clostridium neurotoxin formulation; and

[0032] d. (i) confirming that the Clostridium neurotoxin is suitable for therapeutic (and / or cosmetic) use if the cleavage level of the indicator protein increases after contact, or (ii) confirming the presence of activity if the cleavage level of the indicator protein increases after contact; or

[0033] e. (i) confirming that the Clostridium neurotoxin formulation is unsuitable for therapeutic (and / or cosmetic) use if the cleavage level of the indicator protein after contact does not increase, or (ii) confirming the absence of activity if the cleavage level of the indicator protein after contact does not increase.

[0034] Another aspect of the present invention provides an in vitro method for characterizing the activity of a Clostridium neurotoxin (preferably BoNT) formulation or identifying a Clostridium neurotoxin (preferably BoNT) formulation suitable for therapeutic (and / or cosmetic) use, the method comprising the following steps:

[0035] a. A step of providing a cell (e.g., a genetically engineered cell) having an exogenous nucleic acid that provides expression or overexpression of a receptor and / or ganglioside (preferably a receptor) having binding affinity for Clostridium neurotoxin, and an exogenous nucleic acid that provides expression or overexpression of an indicator protein (preferably an indicator protein containing a SNARE domain) capable of being cleaved by Clostridium neurotoxin; preferably, wherein the cell does not express the receptor and / or ganglioside (preferably a receptor) in the absence of said exogenous nucleic acid;

[0036] b. A step of contacting the above cells with a Clostridium neurotoxin formulation;

[0037] c. a step of comparing the cleavage level of an indicator protein after contact with a Clostridium neurotoxin formulation (e.g., administration) with the cleavage level after contact with a Clostridium neurotoxin formulation; and

[0038] d. (i) confirming that the Clostridium neurotoxin formulation is suitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is increased or equal to the level of cleavage after contact with the control Clostridium neurotoxin formulation, or (ii) confirming the presence of activity if the level of cleavage of the indicator protein after contact is increased or equal to the level of cleavage after contact with the control Clostridium neurotoxin formulation; or

[0039] e. (i) confirming that the Clostridium neurotoxin formulation is unsuitable for therapeutic (and / or cosmetic) use if the level of cleavage of the indicator protein after contact is not increased or is not the same as the level of cleavage after contact with the control Clostridium neurotoxin formulation, or (ii) confirming the absence of activity if the level of cleavage of the indicator protein after contact is not increased or is not the same as the level of cleavage after contact with the control Clostridium neurotoxin formulation.

[0040] The control Clostridium neurotoxin formulation is a formulation of Clostridium neurotoxin (i.e., a batch of Clostridium neurotoxin) known to have cleavage activity and / or to be suitable for therapeutic (and / or cosmetic) use (in other words, the control is a positive control). Preferably, the (test) Clostridium neurotoxin formulation and the control neurotoxin formulation are Clostridium neurotoxin formulations of the same type / serotype, and, for example, if the (test) Clostridium neurotoxin formulation is a BoNT / E formulation, the control Clostridium neurotoxin is preferably also a BoNT / E formulation.

[0041] Another aspect of the present invention provides a method for manipulating cells suitable for use in an assay to characterize the activity of a Clostridium neurotoxin (preferably BoNT) formulation or to identify a Clostridium neurotoxin (preferably BoNT) formulation suitable for therapeutic (and / or cosmetic) use, the method comprising the following steps:

[0042] Step of processing cells to incorporate the following:

[0043] (i) an exogenous nucleic acid that provides expression or overexpression of a receptor and / or ganglioside having binding affinity for Clostridium neurotoxin, preferably wherein the cell does not express a receptor and / or ganglioside (preferably a receptor) in the absence of said exogenous nucleic acid; and

[0044] (ii) an exogenous nucleic acid that provides expression or overexpression of an indicator protein capable of being cleavable by Clostridium neurotoxin (preferably, an indicator protein containing a SNARE domain).

[0045] The following are optional embodiments of any aspect of the present invention as described herein.

[0046] In one embodiment, the cell does not normally express a receptor and / or ganglioside (preferably a receptor, preferably the receptor is SV2A). In other words, in a preferred embodiment, the cell does not express a receptor and / or ganglioside (preferably a receptor, preferably the receptor is SV2A) in the absence of the exogenous nucleic acid.

[0047] In one embodiment, the cell is a cell type different from the natural target of Clostridium neurotoxin. In other words, in one embodiment, the cell is not the natural target of Clostridium 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.

[0048] In one embodiment, the cell is not a nerve cell (e.g., a neuron).

[0049] Advantageously, by providing the expression or overexpression of receptors and / or gangliosides having binding affinity for Clostridium neurotoxin (e.g., where low cleavage activity may be falsely detected due to the low affinity of Clostridium neurotoxin for binding and translocation to the cells used in the assay rather than the low activity of the protease domain), the method and cells of the present invention allow for reduced false-negative results. Furthermore, the present invention provides a broader spectrum of cell types that can be used in such assays, thereby reducing dependence on neurons that may be difficult to culture in vitro (e.g., those naturally expressing sufficient levels of receptors / gangliosides). These advantages are provided in the context of cell-based assays (which enable the characterization of binding, translocation, and protease activity), unlike cell-free systems (which characterize only protease activity).

[0050] The term "when the cleavage level of the indicator protein after contact does not increase" means that there is substantially no increase in the cleavage level. In relation to the term "when the cleavage level of the indicator protein after contact does not increase," the term "substantially" as used herein preferably means that there is no statistically significant increase. Such (non-substantially) increase may be 30%, 25%, 20%, 15%, 10%, 5%, or less than 1%, preferably less than 20%. Such (non-substantially) increase may be 5%, 2%, 1%, or less than 0.5%, preferably less than 0.1%. More preferably, the term "when the cleavage level of the indicator protein after contact does not increase" as used herein means that the cleavage level of the indicator protein after contact is not reduced at all (i.e., the increase in the cleavage level is 0%).

[0051] The level of receptors and / or gangliosides (preferably receptors) expressed in the cells described herein is preferably greater than the level of receptors and / or gangliosides (preferably receptors) expressed in natural targets for Clostridium neurotoxin (e.g., nerve cells). For example, the level of receptors and / or gangliosides (preferably receptors) expressed in the cells described herein may be ≥ 10%, ≥ 20%, ≥ 30%, ≥ 40%, ≥ 50%, ≥ 60%, ≥ 70%, ≥ 80%, ≥ 90%, or ≥ 100% compared to the level of receptors and / or gangliosides (preferably receptors) expressed in natural targets for Clostridium neurotoxin (e.g., nerve cells).

[0052] The level of receptor and / or ganglioside (preferably receptor) expressed in the cells described herein may preferably be greater than the level of receptor and / or ganglioside (preferably receptor) expressed in cells lacking the 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% compared to the level of receptor and / or ganglioside (preferably receptor) expressed in cells lacking the exogenous nucleic acid (e.g., otherwise equivalent cells).

[0053] In one embodiment, the term “overexpression” used in connection with any aspect or embodiment described herein preferably means ≥ 10%, ≥ 20%, ≥ 30%, ≥ 40%, ≥ 50%, ≥ 60%, ≥ 70%, ≥ 80%, ≥ 90%, or ≥ 100% expression relative to the level of receptor and / or ganglioside (preferably receptor) expressed in natural targets (e.g., nerve cells) for Clostridium neurotoxin. In one embodiment, the term “overexpression” used in connection with any aspect or embodiment described herein preferably means ≥ 10%, ≥ 20%, ≥ 30%, ≥ 40%, ≥ 50%, ≥ 60%, ≥ 70%, ≥ 80%, ≥ 90%, or ≥ 100% expression relative to the level of receptor and / or ganglioside (preferably receptor) expressed in cells lacking the exogenous nucleic acid (e.g., otherwise equivalent cells).

[0054] In one embodiment, the Clostridium neurotoxin is BoNT / A (or BoNT / AH CC It may be a BoNT containing a domain, and preferably the receptor may be SV2A, SV2B and / or SV2C (preferably SV2A).

[0055] Additionally or alternatively, Clostridium neurotoxin is BoNT / B (or BoNT / BH CC It may be a BoNT containing a domain, and preferably the receptor may be Syt-I and / or Syt-II.

[0056] Additionally or alternatively, Clostridium neurotoxin is BoNT / E (or BoNT / EH CC It may be a BoNT containing a domain, and preferably the receptor may be SV2A and / or SV2B (preferably SV2A).

[0057] Additionally or alternatively, Clostridium neurotoxin is BoNT / G (or BoNT / GH CC It may be a BoNT containing a domain, and preferably the receptor may be Syt-I and / or Syt-II.

[0058] For additional information on suitable receptors / gangliosides, refer to the literature incorporated herein by reference [Binz and Rummel (Journal of Neurochemistry, Volume 109, Issue 6, June 2009, Pages 1584-1595)]. Brief explanation of the drawing

[0059] Figure 1A shows Western blots using anti-SNAP-25 antibody after treating N2a cells with BoNT / A for 8 hours at 0.1 nM, 1 nM, or 10 nM, or for 24 hours at 1 nM, 0.1 nM, or 0.01 nM. The presence of a lower band indicates the presence of cleavage products. Figure 1B shows Western blots using anti-SNAP-25 antibody after treating M17 cells with BoNT / A for 8 hours at 0.1 nM, 1 nM, or 10 nM, or for 24 hours at 1 nM, 0.1 nM, or 0.01 nM. The presence of the lower band indicates the presence of cleavage products. Figure 1C shows Western blots using anti-SNAP-25 antibody after treating IMR-32 cells with BoNT / A for 8 hours at 0.1 nM, 1 nM, or 10 nM, or for 24 hours at 1 nM, 0.1 nM, or 0.01 nM. The presence of the lower band indicates the presence of cleavage products. Figure 1D shows Western blots using anti-SNAP-25 antibody after treating NG108 cells with BoNT / A for 8 hours at 0.1 nM, 1 nM, or 10 nM, or for 24 hours at 1 nM, 0.1 nM, or 0.01 nM. The presence of the lower band indicates the presence of cleavage products. Figure 2A shows a fluorescence micrograph of NG108 cells 1 day after transfection with a plasmid containing the mScarlet-SNAP25-GeNluc construct. Figure 2B shows a fluorescence micrograph of M17 cells 1 day after transfection with a plasmid containing the mScarlet-SNAP25-GeNluc construct. Figure 3 shows a fluorescence micrograph of puromycin-resistant N108 cells stably transfected with plasma containing the mScarlet-SNAP25-GeNluc construct. Figure 4 is a bar graph showing the average cell count per HPF for green-fluorescent cells after treatment with 0, 0.1 nM or 1 nM BoNT / A. Figure 5A shows a scatter plot of flow cytometry data for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct, where the x-axis represents granularity / complexity and the y-axis represents cell size. Figure 5B shows a histogram of emission fluorescence intensity measured at 525 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 5C shows a histogram of emission fluorescence intensity measured at 585 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 5D shows a histogram of emission fluorescence intensity measured at 617 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 5E shows a histogram of emission fluorescence intensity measured at 665 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 5F shows a histogram of emission fluorescence intensity measured at 785 nm for NG108 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 5G shows a scatter plot of flow cytometry data for 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 shows a scatter plot of flow cytometry data for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct, where the x-axis represents granularity / complexity and the y-axis represents cell size. Figure 6B shows a histogram of emission fluorescence intensity measured at 525 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 6C shows a histogram of emission fluorescence intensity measured at 585 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 6D shows a histogram of emission fluorescence intensity measured at 617 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 6E shows a histogram of emission fluorescence intensity measured at 665 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 6F shows a histogram of emission fluorescence intensity measured at 785 nm for M17 cells stably transfected with the mScarlet-SNAP-25-GeNluc construct. Figure 6G shows a scatter plot of flow cytometry data for 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 shows a histogram of emission fluorescence intensity measured at 525 nm for control NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct. Figure 7B shows a histogram of emission fluorescence intensity measured at 525 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 0.1 nM BoNT / A. Figure 7C shows a histogram of emission fluorescence intensity measured at 525 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 1.0 nM BoNT / A. Figure 8A shows a histogram of emission fluorescence intensity measured at 785 nm for control NG108 cells transfected with the mScarlet-SNAP25-GeNluc indicator construct. Figure 8B shows a histogram of emission fluorescence intensity measured at 785 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 0.1 nM BoNT / A. Figure 8C shows a histogram of emission fluorescence intensity measured at 785 nm for NG108 cells transfected with the mScarlet-SNAP-25-GeNluc indicator construct and treated with 1.0 nM BoNT / A. Figure 9 shows Western blots 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 shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and not treated with toxin. Figure 10B shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT / A for 72 hours. Figure 10C shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 1 nM BoNT / A for 72 hours. Figure 10D shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 0.1 nM BoNT / A for 72 hours. Figure 10E shows flow cytometry data for NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT / E for 72 hours. Figure 10F shows a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and not treated with toxin. Figure 10G shows a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 10 nM BoNT / A for 72 hours. Figure 10H shows 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 shows a fluorescence micrograph of NG108 cells transfected with the mScarlet-SNAP25-GeNluc construct and treated with 0.1 nM BoNT / A for 72 hours. Figure 10J shows 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 shows flow cytometry data for wild-type NG108 cells. Figure 11B shows flow cytometry data for genetically engineered NG108 cells that were screened for high expression of the indicator protein and sensitivity to 1,000 pM of BoNT / A and were not further treated with BoNT / A. Figure 11C shows flow cytometry data for genetically engineered NG108 cells screened for high expression of indicator protein and sensitivity to 1,000 pM BoNT / A and treated with 100 pM BoNT / A for 48 hours. Figure 11D shows flow cytometry data for genetically engineered NG108 cells screened for high expression of indicator protein and sensitivity to 1,000 pM BoNT / A and treated with 100 pM BoNT / A for 96 hours. Figure 11E shows flow cytometry data for genetically engineered NG108 cells that are selected for high expression of the indicator protein but not for sensitivity to BoNT / A and are not further treated with BoNT / A. Figure 11F shows flow cytometry data for genetically engineered NG108 cells treated with 100 pM BoNT / A for 96 hours, which are screened for high expression of the indicator protein but not for sensitivity to BoNT / A. Figure 12A shows flow cytometry data for genetically engineered NG108 cells that were screened for high expression of indicator protein and sensitivity to 100 pM of BoNT / A and were not further treated with BoNT / A. Figure 12B shows flow cytometry data for genetically engineered NG108 cells screened for high expression of indicator protein and sensitivity to 100 pM BoNT / A and treated with 100 pM BoNT / A for 96 hours. Figure 13A is a plot of the percentage amount of cleaved indicator protein after treatment of NG108 cells genetically engineered to express indicator protein and SV2A or SV2C with various concentrations of BoNT / A for various times. Figure 13B is a plot of the percentage amount of cleaved indicator protein after treatment of NG108 cells genetically engineered to express indicator protein and SV2A or SV2C with various concentrations of BoNT / E for various times. Specific details for implementing the invention

[0060] It should be understood that the present invention is not limited to the embodiments described herein. In fact, many variations, modifications, and substitutions will be apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used to practice the invention.

[0061] In describing the present invention, it is understood that where a range of values ​​is provided in relation to an embodiment, each intervening value is encompassed within the embodiment.

[0062] As used herein, a “variant” of a protein or polypeptide refers to a protein or polypeptide having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the amino acid sequence of a reference protein or polypeptide.

[0063] As used herein, “sequence identity” refers to the identity between a reference amino acid or nucleotide sequence and a query amino acid or nucleotide sequence, wherein the sequences are aligned to obtain the highest sequence match, which can be calculated using a method coded in a computer program such as, for example, BLASTP, BLASTN, FASTA (Altschul 1990, J. Mol. Biol. 215:403).

[0064] As used herein, "fragment" of a protein or polypeptide refers to a truncated form of a protein or polypeptide or a truncated form of a variant of a protein or polypeptide.

[0065] The present invention relates to a genetically engineered cell having partially increased sensitivity to Clostridium neurotoxin.

[0066] Clostridium neurotoxin is a neurotoxin naturally produced by the bacterium Clostridium botulinum.

[0067] In certain embodiments of the present invention, the Clostridium neurotoxin is botulinum neurotoxin (BoNT) or tetanus neurotoxin (TeNT). As used herein, the terms “Clostridium neurotoxin,” “BoNT,” and “TeNT” each refer to wild-type Clostridium neurotoxins, including those produced by strains other than Clostridium botulinum, as well as modified and recombinant Clostridium neurotoxins.

[0068] Modified Clostridium neurotoxin may contain one or more modifications compared to wild-type Clostridium neurotoxin, including amino acid modifications and / or chemical modifications. Amino acid modifications include the deletion, substitution, or addition of one or more amino acid residues. Chemical modifications include modifications made to one or more amino acid residues, such as the addition of phosphates or carbohydrates or the formation of disulfide bonds.

[0069] In certain embodiments, modifications may be made to alter the properties of Clostridium neurotoxin. Modifications to Clostridium neurotoxin may increase or decrease its biological activity.

[0070] The biological activity of Clostridium neurotoxin encompasses at least three distinct activities: the first activity is the "proteolytic activity" present in the protease component of the neurotoxin, which plays a role in hydrolyzing the 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 involved in transporting the neurotoxin across the endosome membrane into the cytoplasm. The third activity is the "receptor binding activity" present in the targeting component of the neurotoxin, which is involved in the binding of the neurotoxin to receptors on target cells.

[0071] In certain embodiments, the modification of the neurotoxin may involve the terminal cleavage component(s) of the Clostridium neurotoxin while still retaining the activity of the component(s) of the Clostridium neurotoxin. For example, the neurotoxin may be modified to include only a portion of the protease component required for proteolytic activity, only a portion of the translocation component required for translocation activity, and / or only a portion of the targeting component required for receptor binding activity.

[0072] Clostridium neurotoxin is initially produced as an inactive single-chain polypeptide and is positioned in its active bichain form following cleavage of the neurotoxin at its active site. This cleavage generates a bichain protein having a heavy chain (H-chain) containing translocation and targeting components and a light chain (L-chain) containing protease components.

[0073] In certain embodiments, the biological activity of Clostridium neurotoxin is modified by altering the active site of the neurotoxin. By doing so, the ability of the neurotoxin to be activated may be increased, decreased, or maintained at the same level. In certain embodiments, the biological activity of Clostridium neurotoxin is increased or triggered by modifying the active site so that it is more easily cleaved to activate the neurotoxin. In embodiments where activation is required only in a specific environment or cell, the active site may be modified so that it is cleaved only by a protease present in that environment or cell. In certain other environments, the biological activity of the neurotoxin is decreased or deactivated by modifying the active site so that it is less easily cleaved.

[0074] In certain embodiments, the biological activity of the Clostridium neurotoxin is modified by modifying the protease component of the neurotoxin. By doing so, the proteolytic activity of the neurotoxin may be increased, decreased, or maintained at the same level. In certain embodiments, the protease component may be replaced with a protease component from a different Clostridium neurotoxin or its variant or fragment. For example, BoNT / A may be modified by replacing its protease component with the protease component of BoNT / E.

[0075] In certain embodiments, the biological activity of the Clostridium neurotoxin is modified by altering the potential component of the neurotoxin. By doing so, the potential activity of the neurotoxin may be increased, decreased, or maintained at the same level. In certain embodiments, the potential component may be replaced with a potential component from a different Clostridium neurotoxin or its variant or fragment. For example, BoNT / A may be modified by replacing its potential component with the potential component of BoNT / E.

[0076] In certain embodiments, the biological activity of the Clostridium neurotoxin is modified by modifying the targeting component of the neurotoxin. By doing so, the targeting ability of the neurotoxin may be increased, decreased, or maintained at the same level. In certain embodiments, the targeting component may be replaced with a targeting component from a different Clostridium neurotoxin or its variant or fragment. For example, BoNT / A may be modified by replacing the targeting component with the targeting component of BoNT / E. In other specific embodiments, the targeting component may be replaced with a non-Clostridium polypeptide, for example, an antibody.

[0077] In addition, modification may involve the rearrangement of Clostridium neurotoxin components, for example, flanking protease components into translocation and targeting components.

[0078] Recombinant Clostridium neurotoxins are produced by genetic engineering. They may be genetically identical to wild-type Clostridium neurotoxins or differ from wild-type Clostridium neurotoxins in that they contain additional amino acids, fewer amino acids, or different amino acids. For example, a recombinant Clostridium neurotoxin that mirrors any of the aforementioned modified Clostridium neurotoxins may be produced. Recombinant Clostridium neurotoxins may also have components arranged in a different order than those in wild-type Clostridium neurotoxins. Recombinant Clostridium neurotoxins may also be chemically modified as described above.

[0079] In certain embodiments, the modified or recombinant Clostridium neurotoxin is a polypeptide having an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity with wild-type Clostridium neurotoxin, e.g., BoNT or TeNT of serotypes A, B, C, D, E, F, G, or H.

[0080] A series of programs based on various algorithms are available to the ordinary skilled person for comparing different sequences. In this regard, the Needleman and Wunsch or Smith and Waterman algorithms provide particularly reliable results. To perform sequence alignment and calculate the sequence identity values ​​cited herein, the commercially available program DNASTAR Lasergene MegAlign version 7.1.0, based on the Clustal W algorithm, was used for 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 (this will always be used as the standard setting for sequence alignment unless otherwise specified).

[0081] The BoNT / A serotype is divided into at least six sub-serotypes (also known as subtypes) of BoNT / A1 to BoNT / A6 that share at least 84% and up to 98% amino acid sequence identity. BoNT / A proteins within a given subtype share higher amino acid percentage sequence identity.

[0082] Clostridium neurotoxin targets neurons by binding to receptors. Receptors for Clostridium neurotoxin include protein receptors and plasma membrane gangliosides.

[0083] Gangliosides are oligoglycosylceramides derived from lactosylceramides and containing sialic acid residues such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), or 3-deoxy-D-glycero-D-galactonulosonic acid (KDN). Gangliosides are present and concentrated on the cell surface, with two hydrocarbon chains of the ceramide moiety embedded in the plasma membrane and the oligosaccharide located on the extracellular surface, providing recognition sites for extracellular molecules or the surface of adjacent cells. Gangliosides also specifically bind to viruses and bacterial toxins such as Clostridium neurotoxin.

[0084] Gangliosides are defined by a nomenclature where M, D, T, and Q refer to mono-, di-, tri-, and tetrasialogangliosides, respectively, and numbers 1, 2, 3, etc., refer to the migration order of gangliosides in thin-layer chromatography. For example, the migration order of monosialogangliosides is GM3 > GM2 > GM1. Additional terms such as GM1a, GD1b, etc., are added to indicate variations within the basic structure. Glycosphingolipids having 0, 1, 2, and 3 sialic acid residues connected to an internal galactose unit are called sialo- (or 0-), a-, b-, and c-series gangliosides, respectively, and gangliosides having sialic acid residues connected to an internal N-galactosamine residue are classified as a-series gangliosides. The pathways for the biosynthesis of the O-, α-, β-, and c-series gangliosides are, for example, [Ledeen et al., Trends in Biochemical SciencesAs exemplified in [ , 40: 407-418 (2015)], it involves the sequential activity of sialyltransferase and glycosyltransferase. Further sialation of each series at different positions on the carbohydrate chain can yield an increasingly complex and heterogeneous range of products, such as α-series gangliosides having sialic acid residues connected to internal N-acetylgalactosamine residues. Gangliosides are delivered to the outer lobe of the plasma membrane by a transport system involving vesicle formation.

[0085] 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.

[0086] Clostridium neurotoxin possesses two independent binding sites in its HCC domain for ganglioside 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 the "E(Q) ... H(K) ... SXWY ... G" motif, whereas BoNT / C and BoNT / D exhibit two independent ganglioside-binding sites (Lam et al., Progress in Biophysics and Molecular Biology , 117:225-231 (2015)). While most BoNTs bind only to gangliosides having a 2,3-linked N-acetylneuraminic acid residue (denoted as Siia5) attached to Gal4 of the oligosaccharide core, the corresponding ganglioside-binding pocket of TeNT can also bind to GM1a, a ganglioside lacking the Siia5 sugar residue. BoNT / D was found to bind to GM1a and GD1a. Reference [Kroken et al., Journal of Biological ChemistryRefer to , 286:26828-26837 (2011). Combining data derived from ganglioside-deficient mice with data derived from biochemical assays, BoNT / A, BoNT / E, BoNT / F, and BoNT / G exhibit 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 disiallyl motif found in GD1b, GT1b, and GQ1b. Therefore, abundant complex polysialo-gangliosides such as GD1a, GD1b, and GT1b appear to be essential for the specific accumulation of all BoNT serotypes and TeNT on neuronal cell surfaces as the first stage of toxicity. [Rummel, Andreas, "Double receptor anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity," Botulinum Neurotoxins, See Springer Berlin Heidelberg (2012) 61-90.

[0087] In the above regard, in a specific embodiment of the present invention, a cell is genetically engineered to express or overexpress a ganglioside. In a specific embodiment, a cell is genetically engineered to express or overexpress GM1a, GD1a, GD1b, GT1b, and / or GQ1b. In a specific embodiment, a cell is engineered to express or overexpress GD1a, GD1b, and / or GT1b. In a specific embodiment, a cell is engineered to express or overexpress GD1b and / or GT1b.

[0088] Gangliosides are synthesized starting from ceramides. From ceramides, one pathway forms glucosylceramide (GlcCer) by the addition of a glucose unit by glucosylceramide synthase. Subsequently, β1,4-galactosyltransferase I (GalT-I) catalyzes the addition of a galactose unit to GlcCer to form lactosylceramide (LacCer). From LacCer, GalNAc-transferase (GalNAcT) can form GA1 by adding N-acetylgalactosamine, or GM3 synthase can form GM3 by adding sialic acid. From GM3, GD3 can be formed by the addition of sialic acid by GD3 synthase. GT3 can be formed from GD3 by the addition of additional sialic acid by GT3 synthase. In a separate pathway, a galactose unit is added to LacCer by galactosylceramide synthase to form galactosylceramide (GalCer). Subsequently, an additional carbohydrate group is added by GM4 synthase to form GM4. Then, GM3, GD3, and GT3 can be modified to form more complex gangliosides of the "a," "b," or "c" series, respectively. These 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.

[0089] Accordingly, the cells of the present invention can be engineered to express or overexpress a desired ganglioside by being engineered to express or overexpress an enzyme of a biosynthetic pathway that induces a ganglioside. For example, the cells can be engineered to contain exogenous nucleic acids encoding these enzymes (i.e., by transfection). Accordingly, in certain embodiments, the cells were engineered 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.

[0090] A person skilled in the art will understand that variants or fragments of these enzymes that retain the desired catalytic activity may also play a role in the synthesis of the ganglioside of interest. Accordingly, in certain embodiments, cells have been genetically engineered to express or overexpress a variant or fragment of an enzyme of the ganglioside synthesis pathway that retains the ability of the corresponding enzyme. For example, in certain embodiments, cells were genetically engineered 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 galactose 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 galactose to LacCer, and / or a variant of GM4 synthase capable of adding a carbohydrate group to GalCer.

[0091] In certain embodiments, the variant is a protein having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of an enzyme of a biosynthetic pathway that induces a ganglioside and maintains the desired catalytic activity of such enzyme. In these specific embodiments, the variant is a protein having an amino acid sequence having 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 maintaining the desired catalytic activity of such enzyme.

[0092] The fragment may 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.

[0093] Test methods that can be used to determine whether a variant or fragment possesses the desired catalytic activity are known in the relevant art. For example, a person skilled in the art will be aware of test methods that can be used to determine whether a variant or fragment of GD3 synthase has the ability to add sialic acid to GM3.

[0094] A person skilled in the art will understand that the enzyme mentioned above may also be coded by a nucleic acid different from the exogenous nucleic acid mentioned above by means of a conservative substitution known in the art. A person skilled in the art will also understand that a variant of the enzyme may be coded by a nucleic acid having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, for example, a nucleic acid encoding a wild-type enzyme. Accordingly, the present invention also considers a cell genetically engineered to contain a nucleic acid encoding one of the enzymes mentioned above and / or an exogenous nucleic acid having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a nucleic acid different from the wild-type nucleic acid encoding such enzyme only by means of a conservative substitution, wherein the encoded protein is a wild-type enzyme or a variant that retains the catalytic activity of the wild-type enzyme.

[0095] In certain embodiments, cells are engineered to express or overexpress an enzyme that catalyzes what is determined to be the rate-limiting step in the biosynthesis of a desired ganglioside, or a variant or fragment thereof having the desired catalytic activity of such enzyme. For example, GD3 synthase is an enzyme that catalyzes the rate-limiting step in the biosynthesis of "b" series gangliosides, particularly in the addition of sialic acid to GM3. Thus, in embodiments where the expression or overexpression of GD1b, GT1b, and / or GQ1b is preferred, cells are engineered to express or overexpress GD3 synthase or a variant or fragment thereof having the ability to add sialic acid to GM3.

[0096] For example, cells may be transfected with a nucleic acid encoding GD3 synthase or a nucleic acid as described above having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with such nucleic acid, for example, a nucleic acid encoding a variant thereof that retains the catalytic activity of GD3 synthase or a nucleic acid different from wild-type GD3 synthase only in conservative substitutions.

[0097] The binding of specific Clostridium 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), while BoNT / E can bind only to the SV2A and SV2B isoforms. BoNT / B and BoNT / G bind to both isoforms (I and II) of synaptotagmin. Synaptotagmin and SV2 are localized in synaptic vesicles and are exposed to the extracellular space when the vesicles fuse with the presynaptic membrane. During this period, Clostridium neurotoxins bind to their protein receptors.

[0098] Accordingly, the cells of the present invention can be engineered to express or overexpress a desired protein receptor, e.g., SV2 (e.g., SV2A, SV2B, and SV2C) or synaptotagmin (e.g., synaptotagmin I and synaptotagmin II). For example, the cells can be engineered to contain exogenous nucleic acids encoding these protein receptors (e.g., by transfection).

[0099] The present invention also considers proteins that differ from these protein receptors but still retain the ability to bind to Clostridium neurotoxin. These proteins may be variants or fragments of these protein receptors that retain the ability of the receptor to bind to Clostridium neurotoxin. Accordingly, in certain embodiments, cells were engineered to express or overexpress a variant or fragment of SV2 that binds to BoNT / A, BoNT / D, BoNT / E, BoNT / F, and / or TeNT. Additionally, in certain embodiments, cells were engineered to express or overexpress a variant or fragment of synaptotagmin that binds to BoNT / B and / or BoNT / G.

[0100] In certain embodiments, the variant is a protein having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of a protein receptor that binds to Clostridium neurotoxin. In these specific embodiments, the variant is a protein having an amino acid sequence having 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)).

[0101] The fragment may 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.

[0102] 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 a 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 these specific embodiments, the variant or fragment may comprise a fourth luminal domain of wild-type SV2, for example, a fourth luminal domain of SV2A (SEQ ID: 11), a fourth luminal domain of SV2B (SEQ ID: 12), or a fourth luminal domain of SV2C (SEQ ID: 13).

[0103] Assay methods that can be used to determine whether a variant or fragment possesses the desired Clostridium neurotoxin binding activity are known in the relevant art. For example, a person skilled in the art will know that an assay method can be used to determine whether a variant or fragment of SV2C has the ability to bind to BoNT / A.

[0104] A person skilled in the art will understand that the enzyme mentioned above may also be coded by a nucleic acid different from the exogenous nucleic acid mentioned above by means of a conserved substitution known in the art. A person skilled in the art will also understand that a variant of a protein receptor may be coded by a nucleic acid having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with, for example, a nucleic acid encoding a wild-type protein receptor. Accordingly, the present invention also considers a cell genetically engineered to contain a nucleic acid encoding that of the protein receptor mentioned above and / or an exogenous nucleic acid having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a nucleic acid different from the wild-type nucleic acid encoding such protein receptor only by means of a conserved substitution, wherein the coded protein is a variant that retains the ability to bind to the wild-type protein receptor or Clostridium neurotoxin.

[0105] SV2C is most sensitive to BoNT / A. Thus, in certain embodiments where sensitivity to BoNT / A is required, cells are genetically engineered to express or overexpress SV2C or its variants or fragments capable of binding to BoNT / A. However, SV2C does not bind to BoNT / E but instead binds to SV2A and SV2B. Thus, in certain embodiments where sensitivity to BoNT / E is required, cells are genetically engineered to express or overexpress SV2A and / or SV2B, or its variants or fragments capable of binding to BoNT / E.

[0106] The present invention considers that cells may be engineered 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 may be engineered to express or overexpress SV2A and SV2C. Such cells may have increased sensitivity to, for example, BoNT / A and BoNT / E. Additionally, cells may be engineered to express or overexpress GD3 synthase and SV2A and / or SV2C.

[0107] Furthermore, it is known that chimeric receptors can bind to neurotoxins. For example, it is known that a chimeric receptor comprising a domain of the aforementioned protein receptor that binds to a neurotoxin (e.g., the fourth luminal domain of SV2) fused to a transmembrane domain of another receptor, such as the LDL receptor, binds to BoNT and allows for internalization into the cell. Accordingly, the present invention also considers manipulating cells to express such chimeric receptors.

[0108] The cells used in the present invention may be any prokaryotic or eukaryotic cell capable of expressing ganglioside and / or protein receptors 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.

[0109] Cells engineered to express or overexpress the Clostridium toxin receptor can be further selected for increased sensitivity using directed evolution. In this process, cells are exposed to Clostridium neurotoxin, and cells exhibiting sensitivity to lower concentrations of Clostridium neurotoxin are selected compared to other cells (e.g., determined by showing cleavage of the indicator protein at lower concentrations of Clostridium neurotoxin). These cells are expected to have greater sensitivity to Clostridium neurotoxin overall than the cells. This process can be repeated with even lower concentrations of Clostridium neurotoxin using the selected cells that exhibit sensitivity to lower concentrations.

[0110] Cells selected in each round will have increased sensitivity to Clostridium neurotoxin.

[0111] As previously discussed, the cells of the present invention can be used in an assay to determine the activity of a polypeptide (e.g., modified or recombinant Clostridium neurotoxin). Such an assay involves the steps of contacting the cells with the polypeptide and testing for the presence of product(s) generated from the cleavage of the SNARE protein in the cells.

[0112] As used herein, the term “contact” refers to bringing a cell and a Clostridium neurotoxin into physical proximity to allow for physical and / or chemical interactions. Contact is performed under conditions and for a period of time sufficient to allow for interactions between a polypeptide and a protein susceptible to proteolysis by wild-type Clostridium neurotoxin (e.g., SNARE proteins).

[0113] In certain embodiments, such contact may be achieved by culturing cells in a medium containing polypeptides. The polypeptides are typically present in the medium at concentrations of 0.0001 nM to 10,000 nM, 0.0001 to 1,000 nM, 0.0001 to 100 nM, 0.0001 to 10 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 cultivation may be, for example, 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.

[0114] In other specific embodiments, such contact may be achieved by transfecting the cell with an exogenous nucleic acid encoding a polypeptide (e.g., transient transfection).

[0115] For use in this assay, cells contain proteins that are susceptible to proteolysis by wild-type Clostridium neurotoxin. These proteins will be referred to herein as “indicator protein(s).” The indicator proteins may be endogenous (e.g., endogenous SNARE proteins), or cells may be genetically engineered to express or overexpress the indicator proteins.

[0116] As discussed above, SNARE proteins such as SNAP-25, synaptobrevin, and syntaxin are known to be susceptible to proteolysis by Clostridium neurotoxin. 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. Accordingly, the present invention considers that such indicator proteins may contain the amino acid sequences of these SNARE proteins. The present invention also considers that if a variant or fragment is susceptible to proteolysis by wild-type Clostridium neurotoxin, the indicator protein may instead contain the amino acid sequences of the variant or fragment of such SNARE protein. 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, will be referred to herein as the "SNARE domain" of the indicator protein.

[0117] The term "susceptible to proteolysis" means that the protein can be proteolytically cleaved by the protease component of wild-type Clostridium neurotoxin. In other words, such proteins contain protease recognition and cleavage sites that enable them to be recognized and cleaved by the protease component of wild-type Clostridium neurotoxin.

[0118] In certain embodiments, the indicator protein is labeled. For example, U.S. Patent No. 8,940,482 by Oyler et al. describes a cell-based assay for evaluating the activity of Clostridium neurotoxin, in which cells are engineered to express a labeled fusion protein containing a fluorescent protein domain fused to SNAP-25. The fluorescent protein domain is the C-terminus of the SNAP-25 domain and becomes part of the C-terminal fragment generated after cleavage of SNAP-25 by Clostridium neurotoxin. In the assay described by Oyler, the full-length fusion protein is not readily degraded in the cell, but the generated C-terminal fragment subsequently leads to the degradation of the fluorescent protein. This is because a residue is present in SNAP-25 that acts as a degron only when exposed at the N-terminus of the fragment generated by cleavage. Since this "N-degron" is tagged by a ubiquitin ligase, the fragment is targeted by the proteasome for degradation.

[0119] Accordingly, the present invention considers an embodiment such as that described in Euler, in which a cell is engineered to express a labeled indicator protein that is not easily degraded in its full-length form. In this embodiment, cleavage produces a labeled fragment that is easily degraded in the cell (e.g., due to the presence of an N-degron). The indicator protein is labeled on the portion forming the easily degraded fragment, and the label is degraded along with the fragment. In this embodiment, the ability of the polypeptide to cleave the SNARE protein in the cell may be determined by the presence (or absence thereof) of a signal from the label after contact between the cell and the polypeptide.

[0120] In these specific embodiments, the indicator protein also includes a label on a portion of the indicator protein that forms a fragment that is not as easily degraded as the other fragment after cleavage. For example, the indicator protein may be a fusion protein comprising two labels and a SNARE domain having a label flanking the SNARE domain. In an embodiment where the full-length indicator protein is not easily degraded in the cell, but one of the fragments generated after its cleavage is easily degraded, the cleavage of the SNARE protein may be determined by comparing a signal obtained from a label on an easily degradable fragment with a signal from a label on a less easily degradable fragment. For example, in an embodiment such as Euler, where the C-terminal fragment generated from the cleavage is easily degradable but the N-terminal fragment is not, the cleavage may be determined by comparing a signal obtained from a label on a C-terminal fragment with a signal from a label on an N-terminal fragment. In these embodiments, labels that emit fluorescent signals that can be more clearly distinguished from one another (e.g., red and green or red and cyan) may be selected.

[0121] As used herein, the term “label” means a detectable marker and includes, for example, radioactive labels, antibodies, and / or fluorescent labels. The amount of test substrate and / or cleavage product may be determined by autoradiographic or spectroscopic methods including methods based on energy resonance transfer between at least two labels, such as, for example, the FRET assay (discussed further below). Alternatively, immunological methods such as Western blot or ELISA may be used for detection.

[0122] Examples of labels that may be used in the practice of the present invention include radioisotopes; fluorescent labels; phosphorescent labels; luminescent labels; and compounds capable of binding to a labeled binding partner. Examples of fluorescent labels include yellow fluorescent protein (YFP); blue fluorescent protein (BFP); green fluorescent protein such as NeonGreen (GFP); red fluorescent protein such as mScarlet (RFP); cyan fluorescent protein (CFP); and their fluorescent mutants. Examples of luminescent labels include photoproteins; firefly luciferase, renilla ( Renilla ) and Gausia( Gaussia ) Includes luciferases such as luciferase; chemiluminescent compounds; and electrochemiluminescent (ECL) compounds. In embodiments as discussed above, where N-terminal labels and C-terminal labels are selected so that the emitted signals can be more easily distinguished from one another, examples of such label pairs may include RFP and GFP and RFP and CFP. For example, an RFP such as mScarlet may serve as an N-terminal label, and a GFP such as NeonGreen or a CFP may serve as a C-terminal label.

[0123] In certain embodiments, the label is a protein label such as an antibody, fluorescent protein, photoprotein, and luciferase.

[0124] As used herein, “N-terminal marker” means a marker located on an indicator protein portion that is N-terminus relative to a Clostridium neurotoxin cleavage site, whether or not it is a protein, and “C-terminal marker” means a marker located on an indicator protein portion that is C-terminus relative to a Clostridium neurotoxin cleavage site, whether or not it is a protein. A marker does not need to be located at the N-terminus or C-terminus of an indicator protein to be called an N-terminal or C-terminal marker. Rather, these terms refer to the location of the marker relative to the Clostridium neurotoxin cleavage site. In certain embodiments of the invention, an RFP such as mScarlet is used as an N-terminal marker, and a GFP such as NeonGreen or a CFP is used as a C-terminal marker.

[0125] Another assay is the fluorescence resonance energy transfer (FRET) assay. In this assay, the indicator protein includes a donor label on one side of the cleavage site and a recipient label on the other side. The donor label absorbs energy and subsequently transfers it to the recipient label. Energy transfer results in a decrease in the fluorescence intensity of the donor chromophore and an increase in the emission intensity of the recipient chromophore. Cleavage of the substrate results in less successful energy transfer. Therefore, successful cleavage can be determined based on the reduced ability of this transfer to occur. In this embodiment, YFP and CFP can form a pair as a FRET pair, such as RFP and GFP.

[0126] In a specific embodiment of the present invention, the indicator protein is a fusion protein comprising a SNARE domain. The fusion protein may also include additional domains, such as a label domain. The label domain may have an amino acid sequence of a protein label. Examples of such fusion proteins include an N-terminal label domain such as an amino acid sequence for mScarlet; a SNARE domain such as an amino acid sequence for SNAP-25; and a C-terminal label domain such as an amino acid sequence for NeonGreen.

[0127] The fusion protein may also include other domains, such as a screening marker (discussed further below). In these embodiments, the screening marker domain may be separated from the portion of the fusion protein containing the remaining domains (e.g., SNARE domain and label domain(s)) by a linker that can be cleaved to allow separation of the screening marker and the remaining indicator protein after translation. The linker may be, for example, a self-cleavage (e.g., a 2A self-cleavage peptide).

[0128] As previously discussed, cells can be engineered to express or overexpress indicator proteins. A person skilled in the art will know which nucleic acids can be used to allow such expression and how to engineer these cells to express these indicator proteins. An example of such nucleic acid is sequence identification number: 1, which expresses a fusion protein having mScarlet as an N-terminal label, SNAP-25 as a SNARE domain, NeonGreen as a C-terminal label, luciferase as an additional label domain, puromycin-N-acetyltransferase as a screening marker, and a 2A self-cleaving peptide. Another example of such nucleic acid is sequence identification number: 2, which expresses a fusion protein having mScarlet as an N-terminal marker, SNAP-25 as a SNARE domain, CFP as a C-terminal marker, luciferase as an additional marker domain, puromycin-N-acetyltransferase as a screening marker, and a 2A self-cleaving peptide.

[0129] The present invention also relates in part to a method for producing the aforementioned genetically engineered cells. The method comprises the step of introducing an exogenous nucleic acid encoding a protein of interest into a cell. The protein of interest may be, for example, a Clostridium neurotoxin receptor or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin; an enzyme of the ganglioside synthesis pathway or a variant or fragment thereof having catalytic activity of such enzyme; and / or an indicator protein.

[0130] In certain embodiments, the method involves the step of transforming a cell with a nucleic acid encoding a protein of interest. This transformation may be by transfection.

[0131] In certain embodiments, the nucleic acid codes for a fusion protein comprising two or more domains, each domain having an amino acid sequence for the protein of interest or another component of the fusion protein. For example, the nucleic acid may code for a fusion protein comprising an amino acid sequence for a protein receptor (e.g., SV2A or SV2C) and an amino acid sequence for an enzyme of the ganglioside synthesis pathway (e.g., GD3 synthase). In another example, the nucleic acid may code for a fusion protein comprising an amino acid sequence for a protein receptor, an amino acid sequence for an enzyme of the ganglioside synthesis pathway, and an amino acid sequence for a screening marker. In yet another example, the nucleic acid may code for a fusion protein comprising an amino acid sequence for a protein receptor, an amino acid sequence for an enzyme of the ganglioside synthesis pathway, an amino acid sequence for an indicator protein, and an amino acid sequence for a screening marker.

[0132] In this embodiment, the domains can be separated from each other by a linker. The linker can be cleaved, for example, by an enzyme in a cell, or can contain a self-cleaving peptide (e.g., 2A self-cleaving peptide) to allow the individual domains to form separate proteins in the cell.

[0133] Nucleic acids may optionally contain regulatory elements. As used herein, the term "regulatory element" refers to a regulatory element of gene expression, including transcription and translation, and includes elements such as TATA boxes, promoters, enhancers, ribosome binding sites, Shine-Dalgarno sequences, IRES regions, polyadenylation signals, terminal capping structures, etc. A regulatory element may include one or more heterologous regulatory elements or one or more homologous regulatory elements. A "homologous regulatory element" is a regulatory element from the wild-type cell from which the nucleic acid molecule is derived, and which is involved in the regulation of 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 the regulation of gene expression of the nucleic acid molecule or polypeptide in the wild-type cell. Regulatory elements for inducible expression, such as inducible promoters, may also be used.

[0134] The nucleic acid molecules may be, for example, hnRNA, mRNA, RNA, DNA, PNA, LNA, and / or modified nucleic acid molecules. The nucleic acid molecules may be circular, linear, or incorporated into a genome or episome. Additionally, concatemers encoding fusion proteins containing 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides are included. Furthermore, the nucleic acid molecules may contain sequences encoding signal sequences for intracellular transport, such as signals for transport into intracellular compartments or transport across the cell membrane.

[0135] Nucleic acids can be designed to provide high levels of expression in host cells. Methods for designing nucleic acid molecules to increase protein expression in host cells are known in the relevant art and include reducing the frequency (number of occurrences) of "slow codons" in coding nucleic acid sequences.

[0136] Nucleic acids may be introduced using any means known in the relevant art. For example, they may be included in a vector (e.g., a plasmid) used to introduce the nucleic acids into a cell.

[0137] Any vector known in the art that allows for the expression of nucleic acids in cells may be used. The vector may be suitable for the in vitro and / or in vivo expression of the protein of interest. The vector may be a vector for transient and / or stable gene expression. The vector may additionally include regulatory elements and / or screening markers. The vector may be, for example, artificial, or of viral, phage, or bacterial origin. Examples of vectors for use in the present invention include adenovirus vectors, vaccinia vectors, SV-40 virus vectors, retrovirus vectors, λ-derivatives, and plasmids. Examples of plasmids for use in the present invention include plasmids having a pD2500 or pcDNA3.1 backbone.

[0138] Methods using vectors to introduce nucleic acids into cells are known in the relevant technical field. Reference [Laura Bonetta, "The Inside Scoop-Evaluating Gene Delivery Methods," Nature Refer to Methods 2: 875-883 (2005).

[0139] The host cell may contain an expression inducer of the protein of interest. Such an expression inducer may be a nucleic acid molecule or a chemical entity comprising a polypeptide or a small chemical entity. The expression inducer may, for example, increase the transcription or translation of the nucleic acid molecule encoding the protein of interest. The inducer may be expressed, for example, by recombinant means known to a person skilled in the art. Alternatively, the inducer may be isolated from cells, for example, Clostridium cells.

[0140] In certain embodiments, successfully transformed cells can be determined by determining the presence of a selection marker. In these embodiments, a vector containing an exogenous nucleic acid encoding a desired protein may also contain a nucleic acid encoding a selection marker.

[0141] In certain embodiments, the screening marker is a detectable tag. Examples of such tags include His tag, GST tag, Strep tag, and SBP tag. The tag may be expressed as part of a fusion protein that also includes the protein of interest. In these embodiments, the tag may be flanked by one or more protease cleavage sites or self-cleavage peptides. This allows the tag to be cleaved from the post-translational protein.

[0142] In other specific embodiments, screening markers confer resistance to antibiotics. Examples of such screening 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 cells can be determined by exposing the cells to the relevant antibiotics.

[0143] In certain embodiments, the successful transformation of cells genetically engineered to express or overexpress a protein receptor that binds to gangliosides and / or Clostridium neurotoxin can be determined by bringing these cells into contact with Clostridium neurotoxin and determining whether the indicator protein within them is cleaved.

[0144] The present invention further relates to the use of the aforementioned genetically engineered cells in an assay for determining the biological activity of polypeptides, for example, modified or recombinant Clostridium neurotoxins.

[0145] The biological activity of these polypeptides can be measured by various tests, all of which are known to those skilled in the art.

[0146] As previously discussed, the assay involves the steps of contacting cells with a polypeptide under conditions and for a period that allow the protease domain of wild-type Clostridium neurotoxin to cleave the indicator protein in the cells, and determining the presence of a product generated from the cleavage of the indicator protein. The indicator protein may be endogenous to the cells (e.g., endogenous SNARE proteins) or may be an exogenous indicator protein of the type previously described.

[0147] This assay also typically involves a step of determining the degree of conversion of the indicator protein into cleavage product(s). Observation of one or more cleavage product(s) generated after contacting the polypeptide with the indicator protein, or observation of an increase in the amount of cleavage product(s), indicates the proteolytic activity of the polypeptide.

[0148] The determination step may involve comparing the full-length indicator protein with the cleavage product(s). The comparison may involve determining the amount of the full-length indicator protein and / or the amount of one or more cleavage product(s), and may also involve calculating the ratio of the full-length indicator protein to the cleavage product(s). Additionally, the assay for determining proteolytic activity may include a step of comparing the cleavage product(s) that appear after contact between the polypeptide to be tested, the indicator protein, and the control with the control. The control may be, for example, the cleavage product(s) that appear after contact with a Clostridium neurotoxin known to be capable of cleaving the same indicator protein.

[0149] In certain embodiments, after contacting 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 blotting to determine the presence of full-length SNAP-25 and cleaved SNAP-25 (which moves to a band separate from full-length SNAP-25).

[0150] Methods and techniques used to lyse host cells, such as bacterial cells, are known in the relevant art. Examples include sonication or the use of a French press.

[0151] In certain embodiments, the full-length indicator protein is not easily degraded in the cell, but one of the fragments generated after its cleavage is easily degraded. This may be attributed, for example, to the presence of a residue at the N-terminus of the generated fragment that acts as a degron only when exposed by cleavage.

[0152] In this embodiment, the indicator protein may be labeled on a portion that is more easily degraded after cleavage. The label should be selected so that when the degradation of the fragment occurs, the label is also degraded. In this embodiment, whether cleavage occurs may be determined based on signal measurement from the label.

[0153] The received signal can be compared with the control group.

[0154] In this particular embodiment, where another fragment formed after cleavage is not easily degraded, the indicator protein may also include a label on a portion of the indicator protein that forms the corresponding fragment after cleavage. Accordingly, in this embodiment, whether cleavage occurs can be determined by comparing a signal from a label on a more easily degradable fragment with a signal from a label on a less easily degradable fragment acting as a control.

[0155] The signal(s) of 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 within the cell, but the C-terminal fragment generated from cleavage is easily degradable, and the N-terminal label is mScarlet and the C-terminal label is NeonGreen, FACS analysis of the cells after successful cleavage will show lower green emission compared to red emission. In contrast, if cleavage does not occur, red and green fluorescence will be equally dominant.

[0156] Alternatively, fluorescence micrographs of the cells can be taken. In an embodiment such as the one described above, successful cleavage will result in less green fluorescence emitted from the cells compared to control cells not exposed to protease. In contrast, red fluorescence will remain the same as the control.

[0157] Additionally, in certain embodiments, the assay may be a FRET assay. As previously discussed, in this assay, the indicator protein comprises an N-terminal label and a C-terminal label, one of which is a donor label and the other is a recipient label. Energy transfer between the donor label and the recipient label results in a decrease in the fluorescence intensity of the donor label and an increase in the emission intensity of the recipient label. The success of this transfer depends on the labels remaining nearby. Cleaving the indicator protein tends to cause these labels to move further apart, making this transfer less successful. Therefore, successful cleavage can be determined based on the reduced ability of energy transfer to occur.

[0158] In addition to the above, any other means known in the relevant art field for analyzing fluorescence from an indicator protein to determine whether cleavage has occurred may be used in the practice of the present invention.

[0159] In certain embodiments, 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 into 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, the polypeptide is considered to be proteolytically active.

[0160] To track catalytic activity over time, cleavage can be measured at intervals.

[0161] All references cited herein are incorporated herein by reference in connection with the entire disclosure and the disclosures specifically mentioned in this specification.

[0162] Examples

[0163] Example 1 - Selection of Optimal Parent Cell Line for Indicator Cell Line Generation

[0164] 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 for the purpose of selecting the optimal parental cell line for the development of a stable transfected cell line.

[0165] Upon delivery, the cells were restored and grown. Subsequently, the cell stock was frozen and stored in liquid nitrogen. Once sufficient vials of cell stock were prepared, the cells were tested for sensitivity to BoNT / A.

[0166] Cells were cultured for 8 or 24 hours in a medium containing BoNT / A (Metabiologics, Inc.). Cells cultured for 8 hours were cultured in a medium containing 0.1 nM, 1 nM, or 10 nM BoNT / A. Cells cultured for 24 hours were cultured in a medium containing 1 nM, 0.1 nM, or 0.01 nM BoNT / A.

[0167] The cleavage of endogenous SNAP-25 was analyzed by Western blot using the anti-SNAP-25 antibody (Sigma # S9684) according to a standard protocol (Fig. 1). NG108 cells exhibited greater sensitivity to BoNT / A than other cells, while N2a cells were 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 exhibited similar sensitivity to BoNT / A at tested concentrations and times. Due to its ease of culture and familiarity, M17 was selected as a backup for NG108.

[0168] Example 2 - Transfection of cells with a plasmid containing an indicator construct

[0169] The sensitivity of NG108 and M17 cells to puromycin (InvivoGen #ANT-PR) and G418 (VWR #97064-358) was determined. Cells were grown to ~50% confluence and then cultured with various concentrations of puromycin and G418. Both cell lines exhibited similar sensitivity to puromycin and G418.

[0170] The plasmid (pD2500; Atum) was engineered to contain a nucleic acid sequence encoding puromycin-N-acetyltransferase (PuroR), a chimeric protein, and a 2A self-cleaving peptide. In the expressed product, the 2A self-cleaving peptide was positioned between PuroR and the chimeric protein. The chimeric protein contained SNAP-25 flanked between N-terminal and C-terminal fluorescent proteins and luciferase (positioned at the C-terminal). PuroR conferred resistance to puromycin. Luciferase allowed for luminescence measurements of degradation in addition to fluorescence-based measurements of degradation facilitated by the fluorescent proteins. The N-terminal fluorescent protein was mScarlet, and the C-terminal fluorescent protein was either NeonGreen, a green fluorescent protein, or CFP, a cyan fluorescent protein. A plasmid insert containing nucleic acid encoding PuroR, a 2A self-cleaving peptide, and a construct (mScarlet-SNAP25-GeNluc) containing mScarlet, SNAP-25, NeonGreen, and luciferase had the nucleotide sequence of Sequence Identification No. 1. A plasmid insert containing nucleic acid encoding PuroR, a 2A self-cleaving peptide, and a construct (mScarlet-SNAP25-CyanNluc) containing mScarlet, SNAP-25, CFP, and luciferase had the nucleotide sequence of Sequence Identification No. 2.

[0171] NeonGreen was selected due to its excitation / emission spectrum and intensity. If NeonGreen does not degrade well upon cleavage of the indicator protein, CFP was selected as a backup based on previous data indicating rapid degradation upon cleavage of the indicator protein.

[0172] NG108 cells and M17 cells were transfected with plasmids containing the mScarlet-SNAP25-GeNluc construct or the mScarlet-SNAP25-CyanNluc construct. Transfection with Lipofectamine 3000 (ThermoFisher) or polyethyleneimine was performed using a standard protocol.

[0173] 24 hours after transfection, cells were analyzed by fluorescence microscopy to confirm the transfection efficiency and the accurate expression of the indicator protein (Fig. 2 AB, showing an example of cells expressing an indicator protein containing NeonGreen). Due to overexpression, a large amount of the indicator protein was present in the cytosol. Red and green, representing the N- and C-terminal ends of the indicator protein, respectively, were easily detected, indicating that the terminal ends were co-localized. High transient expression was observed in both cell types with a transfection efficiency of >70%.

[0174] Once it was confirmed that the cells were efficiently transfected and the indicator protein was accurately expressed, the transfected cells were selected with 2.5 μg / ml puromycin or "shocked" with an initial high dose of 20 μg / ml puromycin for 1 day, followed by incubation in 5-10 μg / ml puromycin. Both of the above treatments produced a pool of fluorescent puromycin-resistant cells.

[0175] Simultaneously, multiple additional transfections were performed into both indicator constructs, and screening for puromycin resistance was conducted to generate an additional pool of fluorescent puromycin-resistant cells. This ultimately produced approximately six independent pools of fluorescent puromycin-resistant NG108 cells and two independent pools of puromycin-resistant fluorescent M17 cells. The pools were expanded, stocks were frozen, and thaw viability was tested.

[0176] Stable transfection of the indicator construct was confirmed by analyzing puromycin-resistant cells (Fig. 3). In NG108 cells stably transfected with an indicator construct containing NeonGreen, both mScarlet (red) and NeonGreen (green) were co-localized. This indicated that full-length intact proteins were produced and distributed within the cells. Additionally, fluorescence was predominantly observed on the cell membrane, indicating that the proteins were accurately localized (due to the presence of SNAP-25).

[0177] Example 3 - Confirmation of Indicator Protein Cleavage

[0178] The plasmid (pcDNA3.1) was engineered to contain sequence identification number: 3 encoding the BoNT / A light chain, CFP, and N-terminal SBP tag. The nucleic acid encoding the BoNT / A light chain was synthesized using DNA2.0 (ATEM).

[0179] Cells of Example 2, stably transfected with the indicator construct (mScarlet-SNAP25-GeNluc or mScarlet-SNAP25-CyanNluc), were transiently transfected with an expression vector containing the CFP-BoNT / A construct. At 24 and 48 after transfection, many red cells appeared that were not green or cyan, indicating that the indicator protein was cleaved and the C-terminal fragment was rapidly degraded.

[0180] Example 4 - Confirmation of Indicator Protein Cleavage

[0181] NG108 cells from Example 2, stably transfected with the mScarlet-SNAP25-GeNluc indicator construct, were plated in complete DMEM medium (Corning #50-013-PB) onto 96-well optical plates (Thermo Fisher #165305) (20–30K cells per well) and allowed to adhere for 4 hours. Subsequently, the medium was replaced with Neurobasal Plus (Thermo Fisher #A35829), and the cells were cultured for an additional 20 hours; afterward, the medium was replaced with Neurobasal Plus medium containing 0 (control), 0.1, or 1.0 nM of BoNT / A. The cells were cultured for an additional 24 hours. Subsequently, the cells were trypsinized, washed once in the medium, and ~2 x 10⁶ 6 The cells were resuspended in DMEM / FBS medium or DPBS containing 10 units of benzonase / ml at a rate of 10 cells / ml. Subsequently, the cells were analyzed on a SY3200 (Sony Biotechnologies) cell classifier using a laser / filter suitable for NeonGreen and mScarlet.

[0182] Figure 4 shows the number of green cells per HPF for mScarlet-SNAP25-GeNluc-expressing NG108 cells after treatment with BoNT / A for 24 hours. An approximate 25% reduction in green-positive cells per HPF was observed in the cell pool treated with 1 nM BoNT / A.

[0183] Example 5 - Confirmation of Indicator Protein Cleavage

[0184] Representative NG108 and M17 cells from Example 2, stably transfected with the mScarlet-SNAP25-GeNluc indicator construct, were trypsinized, washed once in medium, and ~2x10 6The cells were resuspended in DMEM / FBS medium or DPBS containing 10 units of benzonase / ml at a rate of 10 cells / ml. Subsequently, the cells were analyzed on a SY3200 (Sony Biotechnologies) cell classifier using a laser / filter suitable for NeonGreen and mScarlet.

[0185] NG108 cells were excited at 488 nm, with NeonGreen directly exciting them and mScarlet minimally exciting them. Fluorescence emission was measured at different wavelengths. Additionally, cell subpopulations were identified by measuring lateral- and forward-scattered light intensities. Figure 5A shows a scatter plot representing lateral scattering (SS) on the x-axis and forward scattering (FS) on the y-axis; the distribution illustrates variations in cell granularity / complexity (SS) and cell size (FS). Figure 5B shows a histogram representing the cell distribution of emitted fluorescence intensity measured at 525 nm (FITC filter). Figure 5C shows a histogram representing the cell distribution of emitted fluorescence intensity measured at 585 nm (PE filter). Figure 5D shows a histogram representing the cell distribution of emitted fluorescence intensity measured at 617 nm (PE-Texas Red filter). Figure 5E shows a histogram representing the cellular distribution of emission fluorescence intensity measured at 665 nm (7AAD filter). Figure 5F shows a histogram representing the cellular distribution of emission fluorescence intensity measured at 785 nm (PE-Cy7 filter). Figure 5G shows a scatter plot representing the cellular emission fluorescence of cells measured at 665 nm (7AAD filter) on the x-axis and side-scattering (SS) on the y-axis. The histogram exhibits two distinct fluorescence peaks: a less fluorescent peak representing non-expressing cells and a more fluorescent peak representing cells expressing the indicator protein. At all wavelengths of the emission readings, fluorescence remains high. This was confirmed when emission was measured at 785 nm (Figure 5F), and since most of the emitted light is attributed to FRET, it corroborates the FRET between NeonGreen and mScarlet. The percentage fraction of high-fluorescence cells in the N108 pool ranged from 61% to 93%.

[0186] M17 cells were excited at 488 nm, with NeonGreen directly exciting them and mScarlet minimally exciting them. Fluorescence emission was measured at different wavelengths. Additionally, cell subpopulations were identified by measuring lateral- and forward-scattered light intensities. Figure 6A shows a scatter plot representing lateral scattering (SS) on the x-axis and forward scattering (FS) on the y-axis; the distribution illustrates variations in cell granularity / complexity (SS) and cell size (FS). Figure 6B shows a histogram representing the cellular distribution of emitted fluorescence intensity measured at 525 nm (FITC filter). Figure 6C shows a histogram representing the cellular distribution of emitted fluorescence intensity measured at 585 nm (PE filter). Figure 6D shows a histogram representing the cellular distribution of emitted fluorescence intensity measured at 617 nm (PE-Texas Red filter). Figure 6E shows a histogram representing the cellular distribution of emission fluorescence intensity measured at 665 nm (7AAD filter). Figure 6F shows a histogram representing the cellular distribution of emission fluorescence intensity measured at 785 nm (PE-Cy7 filter). Figure 6G shows a scatter plot representing 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 distinct fluorescence peaks: a less fluorescent peak representing non-expressing cells and a more fluorescent peak representing cells expressing the indicator protein. At all wavelengths of the emission readings, fluorescence remains high. This is included when emission was measured at 785 nm (Figure 6F), and since most of the emitted light is attributed to FRET, it confirms the FRET between NeonGreen and mScarlet. The percentage fraction of highly fluorescent cells in the M17 pool ranged from 14% to 24%, meaning that the fraction of cells expressing fluorescent proteins in the M17 cell pool was smaller compared to the NG108 cell pool.

[0187] NG108 cells from Example 2, stably transfected with the mScarlet-SNAP25-GeNluc indicator construct, were treated with 0 (control), 0.1, or 1.0 nM of BoNT / A in the manner described in Example 4.

[0188] The fluorescence of 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 and minimally excite mScarlet. Fluorescence emission was measured at 530 nm (FITC filter), which detects NeonGreen fluorescence but not mScarlet fluorescence. Figure 7A shows a histogram representing the distribution of emitted fluorescence intensity from untreated (control) cells measured at 525 nm. Figure 7B shows a histogram representing the distribution of emitted fluorescence intensity from cells treated with 0.1 nM BoNT / A measured at 525 nm. Figure 7C shows a histogram representing the distribution of emitted fluorescence intensity from cells treated with 1 nM BoNT / A measured at 525 nm. The fluorescence of NeonGreen decreased by about 15% in the cell pool after treatment with 1 nM BoNT / A (Fig. 7C) compared to the untreated control (Fig. 7A) (average fluorescence of cells at gate R2), which suggests that the generated NeonGreen-containing C-terminal fragment is degraded after cleavage.

[0189] Flow cytometry determination of FRET emission loss was also performed. Fluorescence of 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 and minimally excite mScarlet. Fluorescence emission was measured at 785 nm (Cy7 filter), which detects mScarlet fluorescence but not NeonGreen fluorescence. Figure 8A shows a histogram representing the distribution of emitted fluorescence intensity from untreated (control) cells measured at 785 nm. Figure 8B shows a histogram representing the distribution of emitted fluorescence intensity from cells treated with 0.1 nM BoNT / A measured at 785 nm. Figure 8C shows a histogram representing the distribution of emitted fluorescence intensity from cells treated with 1 nM BoNT / A measured at 785 nm. FRET intensity decreased by 16% after treatment with 1 nM BoNT / A (Fig. 8C) compared to the untreated control (Fig. 8A) (average fluorescence of cells at gate R6), which is consistent with NeonGreen degradation.

[0190] Example 6 - Cutting Confirmation

[0191] Western blots were performed on NG108 cells from Example 2 stably transfected with the mScarlet-SNAP25-GeNluc indicator construct and treated without toxin (control), with 1 nM or 8 nM BoNT / A or 1 nM, 10 nM, or 100 nM BoNT / E in the manner described in Example 4 (Fig. 9).

[0192] Cell lines were tested for cleavage of SNAP-25 (endogenous or exogenous) using standard blotting techniques and a rabbit anti-SNAP25 primary antibody.

[0193] Cells were lysed using M-PER reagent (Thermo Fisher #78501) according to the manufacturer's recommendations. The lysate was purified at 15 kg for 10 minutes, and 10 μl of the sample was run on a NuPage 12% Bis-Tris Gel (Thermo Fisher #NP0341BOX) in MOPS buffer (Thermo Fisher #NP0001) at 200 V. Proteins were delivered to a PVDF membrane (Thermo Fisher #LC2005) using an XCell II blotting system and the NuPage delivery protocol. The generated blot was blocked in 1% BSA / 0.05% Tween 20 / PBS, primary antibody 1:3,000 anti-SNAP-25, secondary antibody 1:5,000 alkaline phosphatase-conjugated goat anti-rabbit (Thermo Fisher #31340), and developed on NBT / BCIP substrate (Thermo Fisher #34042). The developed blot was scanned, densitometrics were calculated using ImageJ, and plotted in MS Excel.

[0194] Indicator proteins were detected in all lysates. No distinct cleavage products were detected in the control samples. Cells treated with BoNT / A or BoNT / E produced cleavage products, which increased with higher doses. Interestingly, cells appear to be as sensitive to BoNT / E as they are to BoNT / A.

[0195] Example 7 - Transfection into a receptor construct

[0196] The plasmid (pD2500; Atom) was engineered to contain the receptor construct GD3-SV2C-Syt and a nucleic acid (Sequence Identification No.: 4) encoding aminoglycoside 3'-phosphotransferase (Neo). The nucleic acid expressed a fusion protein containing GD3 synthase, SV2C, syntaxin, and Neo, with their respective domains separated by a 2A self-cleavage peptide. Syntaxin was engineered into a fusion protein for use with other isoforms of BoNT. Neo conferred resistance to G418.

[0197] NG108 cells from Example 2, stably transfected with the mScarlet-SNAP25-GeNluc indicator construct, were grown to ~60% confluence in T75 flasks. Subsequently, they were transfected overnight with the receptor construct-containing plasmid (2 μg / ml) in 5 ml OptiMem / polyethyleneimine according to standard protocols. In the morning, the cells were washed 1X with fresh, full DMEM medium and cultured in full DMEM medium for an additional 24 to 48 hours. The medium was then replaced with full DMEM medium containing 500 μg / ml G418, and the cells were cultured for an additional 1 to 2 weeks, switching the medium / G418 as needed. Cells were observed to begin dying on day 3 after G418 addition and continued for ~1 week (~60% cell death). After ~2 weeks, the remaining cells were G418 resistant.

[0198] Example 8 - Induced Evolution of Cells

[0199] Cells from Example 7 were sorted twice to isolate the cells having the highest fluorescence and, therefore, the highest indicator protein expression.

[0200] Example 9 - Induced Evolution of Cells

[0201] Cells selected for high expression of the indicator protein in Example 8 were treated with BoNT / A at 0.1 nM, 1 nM, or 10 nM, or with 10 nM BoNT / E for 72 hours in the manner described in Example 4. After treatment, the cells were washed three times, triedpsinized, resuspended in fresh medium, and sorted. Screening of the cells revealed a clear dose-sensitive response to BoNT / A (Fig. 10A). Fluorescence microscopy results also showed that the cells exhibited a decrease in green fluorescence and maintained the same level of red fluorescence depending on the treatment dose (Fig. 10B). It was noted that the results clearly showed an increase in sensitivity to BoNT / A, but no similar increase was observed for BoNT / E.

[0202] Cells sensitive to 1,000 pM (1 nM) of BoNT / A were selected.

[0203] Example 10 - Induced Evolution of Cells

[0204] Wild-type NG108 cells (i.e., not transfected with reporter or receptor constructs) were classified (Fig. 11A). These cells do not exhibit red fluorescence as well as green fluorescence.

[0205] Cells from Example 9 sensitive to 1,000 pM (1 nM) BoNT / A were expanded and treated with 100 pM BoNT / A or untreated (control) in the manner previously discussed. After treatment for 48 or 96 hours, the cells were washed three times, triedpsinated, resuspended in fresh medium, and sorted. Figures 11B-D show flow cytometry data for the control group, cells treated with 100 pM BoNT / A for 48 hours, and cells treated with 100 pM BoNT / A for 96 hours, respectively.

[0206] Cells from Example 8, which were classified twice for high expression of the indicator construct but not classified in Example 9 for sensitivity to 1,000 pM BoNT / A, were treated with 100 pM BoNT / A or untreated for 96 hours in the manner previously discussed (control group). Figures 11E-F show flow cytometry data for the control group and cells treated with 100 pM BoNT / A for 96 hours, respectively.

[0207] In Figures 11A-F, roughly circular gates highlight cells that do not exhibit either red or green fluorescence (i.e., cells that do not express the indicator protein), roughly oval gates highlight cells that exhibit both red and green fluorescence (i.e., cells expressing the indicator protein with uncleaved indicator protein), and quadrilateral gates highlight cells that exhibit red fluorescence but relatively reduced green fluorescence (i.e., cells expressing the indicator protein with cleaved indicator protein).

[0208] Cells previously screened for sensitivity to 1 nM BoNT / A (Fig. 11D) showed much greater sensitivity (greater cleavage) to 100 pM BoNT / A than cells not screened in this way (Fig. 11F).

[0209] After 96 hours of treatment, cells sensitive to 100 pM of BoNT / A (>2 log more sensitive than wild-type NG108) were selected.

[0210] Example 11 - Induced Evolution of Cells

[0211] After 96 hours of treatment, cells from Example 10 sensitive to 100 pM BoNT / A were expanded and treated with 10 pM BoNT / A for 96 hours or untreated (control) in the manner previously discussed. After treatment, the cells were washed three times, triedpsin-treated, resuspended in fresh medium, and sorted.

[0212] Figures 12A-B show flow cytometry data for the control group and cells treated with 10 pM BoNT / A, respectively. There was a noticeable change in the fluorescence of the treated cells, although not as dramatic as that seen at higher concentrations of toxin.

[0213] Example 12 - Receptor construct for BoNT / E

[0214] To confer sensitivity 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 (sequence identification number: 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 the HiFi kit (New England Biolabs), an oligonucleotide from IDT, and an SV2A sequence synthesized by GeneArt.

[0215] These cells and cells from Example 7 expressing the GD3-SV2C-Syt receptor construct were cultured in media containing 0 (control) BoNT / A, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, 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 an anti-SNAP-25 Western blot was performed using an anti-SNAP-25 antibody. Densitometric data from the blot were plotted as the percentage of cleaved SNAP-25 (Fig. 13). Compared to cells expressing SV2C, the sensitivity of cells expressing SV2A to both BoNT / A and BoNT / E was significantly increased, confirming that SV2A is necessary for the conferral of BoNT / E sensitivity.

[0216] Sequence list

[0217] Explanation of the hierarchy

[0218] Sequence Identification Number: 1 is a nucleotide sequence of nucleic acid encoding a fusion protein containing N-terminal mScarlet label, SNAP-25, C-terminal NeonGreen label, and C-terminal luciferase.

[0219] Sequence Identification Number: 2 is a nucleotide sequence of nucleic acid encoding a fusion protein containing N-terminal mScarlet label, SNAP-25, C-terminal CFP label, and C-terminal luciferase.

[0220] Sequence Identification Number: 3 is a nucleotide sequence of nucleic acid encoding a fusion protein containing the light chains of N-terminal CFP and BoNT / A.

[0221] Sequence Identification Number: 4is a nucleotide sequence of nucleic acid encoding a fusion protein containing domains having amino acid sequences for GD3 synthase, SV2C, syntaxin, and aminoglycoside 3'-phosphortransferase. In the fusion protein, each domain is separated from each other by a 2A self-cleaving peptide.

[0222] Sequence Identification Number: 5 is a nucleotide sequence of nucleic acid encoding a fusion protein containing domains having amino acid sequences for GD3 synthase, SV2A, syntaxin, and aminoglycoside 3'-phosphortransferase. In the fusion protein, each domain is separated from each other by a 2A self-cleaving peptide.

[0223] Sequence Identification Number: 6 This is the amino acid sequence for GD3 synthase.

[0224] Sequence Identification Number: 7 is the amino acid sequence for the 2A self-cleaving peptide coded by sequence identification numbers: 4 and 5.

[0225] Sequence Identification Number: 8 is the amino acid sequence for SV2A.

[0226] Sequence Identification Number: 9 is the amino acid sequence for SV2B.

[0227] Sequence Identification Number: 10 is the amino acid sequence for SV2C.

[0228] Sequence Identification Number: 11 is the amino acid sequence for the fourth luminal domain of SV2A.

[0229] Sequence Identification Number: 12 is the amino acid sequence for the fourth luminal domain of SV2B.

[0230] Sequence Identification Number: 13 is the amino acid sequence for the 4th luminal domain of SV2C.

[0231] Sequence Identification Number: 14 is the amino acid sequence for synaptotagmin I.

[0232] Sequence Identification Number: 15is the amino acid sequence for synaptotagmin II.

[0233] Sequence Identification Number: 16 This is the amino acid sequence for MScarlet.

[0234] Sequence Identification Number: 17 is the amino acid sequence of NeonGreen.

[0235] Sequence Identification Number: 18 is the amino acid sequence for CFP.

[0236] Sequence Identification Number: 19 is the amino acid sequence for SNAP-25.

[0237] Sequence Identification Number: 20 is the amino acid sequence for aminoglycoside 3'-phosphortransferase (Neo).

[0238] Sequence Identification Number: 21 is the amino acid sequence for puromycin-N-acetyltransferase (PuroR).

[0239] Sequence Identification Number: 22 is the amino acid sequence for luciferase.

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Claims

Claim 1 (a) a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin; (b) an enzyme of the ganglioside synthesis pathway having binding affinity for Clostridium neurotoxin, or a variant or fragment thereof having catalytic activity of such enzyme; and (c) a cell genetically engineered to express or overexpress an indicator protein comprising an amino acid sequence of a SNARE protein cleaved by Clostridium neurotoxin, wherein the cell comprises an exogenous nucleic acid providing said expression or overexpression of said Clostridium neurotoxin receptor, or a variant or fragment thereof, and the cell comprises an exogenous nucleic acid providing said expression or overexpression of an enzyme of the ganglioside synthesis pathway, or a variant or fragment thereof having catalytic activity of such enzyme, and the cell comprises an exogenous nucleic acid providing said expression or overexpression of an indicator protein comprising an amino acid sequence of a SNARE protein cleaved by Clostridium neurotoxin. Claim 2 A method in vitro for characterizing the activity of a Clostridium neurotoxin formulation or identifying a Clostridium neurotoxin formulation for therapeutic, cosmetic, or both, comprising the following steps: a. (i) an exogenous nucleic acid providing expression or overexpression of a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin; (ii) an enzyme of a ganglioside synthesis pathway having binding affinity for Clostridium neurotoxin, or an enzyme thereof having catalytic activity of such enzyme, or a variant or fragment thereof having catalytic activity of such enzyme; and (iii) providing a cell having the exogenous nucleic acid providing expression or overexpression of an indicator protein comprising an amino acid sequence of a SNARE protein cleavable by Clostridium neurotoxin; b. contacting the cell with a Clostridium neurotoxin formulation; c. A step of comparing the cleavage level of an indicator protein after contact with a Clostridium neurotoxin formulation with the cleavage level prior to contact with a Clostridium neurotoxin formulation; and d. (i) confirming that the Clostridium neurotoxin formulation is suitable for therapeutic, cosmetic, or both if the cleavage level of the indicator protein after contact increases, or (ii) confirming the presence of activity if the cleavage level of the indicator protein after contact increases; or e. (i) confirming that the Clostridium neurotoxin formulation is unsuitable for therapeutic, cosmetic, or both if the cleavage level of the indicator protein after contact does not increase, or (ii) confirming the absence of activity if the cleavage level of the indicator protein after contact does not increase. Claim 3 A method in vitro for characterizing the activity of a Clostridium neurotoxin formulation or identifying a Clostridium neurotoxin formulation suitable for therapeutic, cosmetic, or both, comprising the following steps: a. (i) an exogenous nucleic acid providing expression or overexpression of a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin; (ii) an enzyme of a ganglioside synthesis pathway having binding affinity for Clostridium neurotoxin, or an enzyme thereof having catalytic activity of such enzyme, or a variant or fragment thereof having catalytic activity of such enzyme; and (iii) providing a cell having the exogenous nucleic acid providing expression or overexpression of an indicator protein comprising an amino acid sequence of a SNARE protein cleavable by Clostridium neurotoxin; b. contacting the cell with a Clostridium neurotoxin formulation; c. A step of comparing the cleavage level of an indicator protein after contact with a Clostridium neurotoxin formulation with the cleavage level after contact with a control Clostridium neurotoxin formulation; and d. (i) confirming that the Clostridium neurotoxin formulation is suitable for therapeutic, cosmetic, or both if the cleavage level of the indicator protein after contact is increased or equal to the cleavage level after contact with the control Clostridium neurotoxin formulation, or (ii) confirming the presence of activity if the cleavage level of the indicator protein after contact is increased or equal to the cleavage level after contact with the control Clostridium neurotoxin formulation; or e.(i) confirming that the Clostridium neurotoxin formulation is unsuitable for therapeutic, cosmetic, or both if the level of cleavage of the indicator protein after contact is not increased or is not the same as the level of cleavage after contact with the control Clostridium neurotoxin formulation, or (ii) confirming the absence of activity if the level of cleavage of the indicator protein after contact is not increased or is not the same as the level of cleavage after contact with the control Clostridium neurotoxin formulation. Claim 4 In claim 1, the cell overexpressing the receptor and the enzyme of the ganglioside synthesis pathway is a cell that expresses the receptor and the enzyme more than the natural target of Clostridium neurotoxin. Claim 5 In claim 1, the cell overexpressing the receptor and the enzyme of the ganglioside synthesis pathway expresses the receptor and the enzyme more than the cell lacking exogenous nucleic acid. Claim 6 In paragraph 1, a cell that 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. Claim 7 In paragraph 1, a cell that is a neuroblastoma cell or a neuroblastoma-glioma cell. Claim 8 In paragraph 1, a cell that is a neuroblastoma-glioma cell. Claim 9 In paragraph 1, the cell is an NG108 cell. Claim 10 In claim 1, a cell genetically engineered to overexpress an enzyme of the ganglioside synthesis pathway. Claim 11 In claim 1, cells genetically engineered to express or overexpress GM1a, GD1a, GD1b, GT1b, and / or GQ1b. Claim 12 In claim 1, a cell genetically engineered to express or overexpress GD1a, GD1b, and / or GT1b. Claim 13 In claim 1, a cell genetically engineered to express or overexpress GD1b and / or GT1b. Claim 14 In claim 1, a cell genetically engineered 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 catalytic activity of such enzyme. Claim 15 In claim 1, a cell genetically engineered to express or overexpress GD3 synthase, or a variant or fragment thereof having catalytic activity of GD3 synthase. Claim 16 In claim 1, a cell genetically engineered to express or overexpress GD3 synthase. Claim 17 In claim 1, a cell genetically engineered to overexpress a protein receptor which is a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin. Claim 18 In claim 1, a cell genetically engineered to express or overexpress SV2 or synaptotagmin, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin. Claim 19 In claim 1, a cell genetically engineered to express or overexpress SV2, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin. Claim 20 In claim 1, a cell genetically engineered to express or overexpress SV2A or SV2C, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin. Claim 21 In claim 1, a cell genetically engineered to express or overexpress the fourth luminal domain of SV2A or SV2C. Claim 22 In claim 1, the indicator protein comprises an amino acid sequence of syntaxin, synaptobrevin, or SNAP-25, or a variant or fragment thereof susceptible to proteolysis by a protease component of wild-type Clostridium neurotoxin. Claim 23 In paragraph 1, the indicator protein is a cell that is a labeled indicator protein. Claim 24 In claim 1, the cell wherein the indicator protein comprises an N-terminal label and a C-terminal label. Claim 25 In claim 1, the cell in which the indicator protein comprises the amino acid sequence of a fluorescent protein label. Claim 26 In claim 1, the indicator protein is a cell comprising the amino acid sequence of mScarlet and the amino acid sequence of NeonGreen. Claim 27 In claim 1, the cell wherein the indicator protein comprises mScarlet as an N-terminal label and NeonGreen as a C-terminal label. Claim 28 A method for producing a cell according to any one of claims 1 and 4 to 27, comprising the step of introducing into a cell a nucleic acid encoding an indicator protein comprising an amino acid sequence of a SNARE protein cleavable by Clostridium neurotoxin; (i) a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin; (ii) an enzyme of a ganglioside synthesis pathway having binding affinity for Clostridium neurotoxin, or a variant or fragment thereof having catalytic activity of such enzyme; and (iii) an amino acid sequence of an indicator protein comprising an indicator protein having an amino acid sequence of a SNARE protein cleavable by Clostridium neurotoxin. Claim 29 In paragraph 28, a method in which nucleic acid is introduced by transfection. Claim 30 A assay for determining the activity of a modified or recombinant Clostridium neurotoxin, comprising the steps of: contacting a cell of any one of claims 1 and 4 to 27 with the modified or recombinant Clostridium neurotoxin under conditions and for a period such that the protease domain of the wild-type Clostridium neurotoxin can cleave an indicator protein in the cell; and determining the presence of a product generated from the cleavage of such indicator protein. Claim 31 In paragraph 30, a assay in which the full-length indicator protein is not easily degraded in the cell, but one of the fragments generated after its cleavage is easily degraded. Claim 32 A test method according to claim 30, wherein the indicator protein contains a C-terminal label, the full-length indicator protein is not easily degraded in the cell, but after its cleavage, the resulting C-terminal fragment is easily degraded, and the degradation of the C-terminal fragment leads to the degradation of the C-terminal label. Claim 33 A test method according to claim 30, wherein the indicator protein contains a C-terminal label, the full-length indicator protein is not easily degraded, but after cleavage therefrom, the generated C-terminal fragment is easily degraded, the degradation of the C-terminal fragment leads to degradation of the C-terminal label, and the cleavage of the indicator protein is determined by measuring the signal from the C-terminal label after contacting the cell with a modified or recombinant neurotoxin. Claim 34 In paragraph 30, a assay method in which, after such contact, cells are lysed, the resulting cell lysate is contacted with an antibody, and a Western blot is performed. Claim 35 A method according to claim 2 or 3 in which cells overexpressing the receptor and the enzyme of the ganglioside synthesis pathway express the receptor and the enzyme more than the natural target of Clostridium neurotoxin. Claim 36 A method according to claim 2 or 3, wherein a cell overexpressing the receptor and the enzyme of the ganglioside synthesis pathway expresses more of the receptor and the enzyme compared to a cell lacking exogenous nucleic acid. Claim 37 A method according to claim 2 or 3, 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. Claim 38 A method according to paragraph 2 or 3 in which the cell is a neuroblastoma cell or a neuroblastoma-glioma cell. Claim 39 A method according to paragraph 2 or 3 in which the cell is a neuroblastoma-glioma cell. Claim 40 A method according to paragraph 2 or 3 in which the cell is an NG108 cell. Claim 41 A method according to paragraph 2 or 3 in which the cell is genetically engineered to overexpress a ganglioside. Claim 42 The method of claim 2 or 3, wherein the cells are genetically engineered to express or overexpress GM1a, GD1a, GD1b, GT1b, and / or GQ1b. Claim 43 The method of claim 2 or 3, wherein the cell is genetically engineered to express or overexpress GD1a, GD1b, and / or GT1b. Claim 44 The method according to claim 2 or 3, wherein the cells are genetically engineered to express or overexpress GD1b and / or GT1b. Claim 45 A method according to claim 2 or 3, wherein the cell is genetically engineered 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 catalytic activity of such enzyme. Claim 46 A method according to claim 2 or 3, wherein the cell is genetically engineered to express or overexpress GD3 synthase, or a variant or fragment thereof having catalytic activity of GD3 synthase. Claim 47 A method according to paragraph 2 or 3 in which the cell is genetically engineered to express or overexpress GD3 synthase. Claim 48 A method according to claim 2 or 3, wherein the cell is genetically engineered to express or overexpress a protein receptor that is a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin. Claim 49 A method according to claim 2 or 3, wherein the cell is genetically engineered to express or overexpress a variant or fragment thereof having the ability to bind to SV2 or synaptotagmin, or Clostridium neurotoxin. Claim 50 A method according to claim 2 or 3, wherein the cell is genetically engineered to express or overexpress a variant or fragment thereof having the ability to bind to SV2 or Clostridium neurotoxin. Claim 51 A method according to claim 2 or 3, wherein the cell is genetically engineered to express or overexpress SV2A or SV2C, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin. Claim 52 A method according to claim 2 or 3, wherein the cell is genetically engineered to express or overexpress the fourth luminal domain of SV2A or SV2C. Claim 53 A method according to claim 2 or 3, wherein the indicator protein comprises an amino acid sequence of a variant or fragment thereof susceptible to proteolysis by syntaxin, synaptobrevin, or SNAP-25, or a protease component of wild-type Clostridium neurotoxin. Claim 54 A method according to paragraph 2 or 3, wherein the indicator protein is a labeled indicator protein. Claim 55 A method according to claim 2 or 3, wherein the indicator protein comprises an N-terminal label and a C-terminal label. Claim 56 A method according to claim 2 or 3, wherein the indicator protein comprises the amino acid sequence of a fluorescent protein label. Claim 57 A method according to claim 2 or 3, wherein the indicator protein comprises the amino acid sequence of mScarlet and the amino acid sequence of NeonGreen. Claim 58 A method according to claim 2 or 3, wherein the indicator protein comprises mScarlet as an N-terminal label and NeonGreen as a C-terminal label. Claim 59 A method for manipulating cells suitable for use in an assay to characterize the activity of a Clostridium neurotoxin formulation or to identify a Clostridium neurotoxin formulation suitable for therapeutic, cosmetic, or both, comprising the following steps: a. processing cells to incorporate: i. an exogenous nucleic acid providing expression or overexpression of a Clostridium neurotoxin receptor, or a variant or fragment thereof having the ability to bind to Clostridium neurotoxin; ii. an exogenous nucleic acid providing expression or overexpression of an enzyme of a ganglioside synthesis pathway having binding affinity to Clostridium neurotoxin, or a variant or fragment thereof having catalytic activity of such enzyme; and iii. an exogenous nucleic acid providing expression or overexpression of an indicator protein comprising an amino acid sequence of a SNARE protein capable of being cleaved by Clostridium neurotoxin. Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete

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