Compositions and methods for characterizing botulinum neurotoxins

Abstract

Form-specific cell based assays for the characterization of Clostridia botulinum neurotoxins and genetically modified cells utilized in such assays are described. Such assays and genetically modified cells can discriminate between complexed and non-complexed forms of botulinum neurotoxin having the same serotype, for example serotype E. Methods are also provided for generating and identifying cell lines that can be utilized in such cell based assays.

Description

Attny Dkt No. 102320.0037PCTCOMPOSITIONS AND METHODS FOR CHARACTERIZING BOTULINUM NEUROTOXINSThis application claims the benefit of United States Provisional Patent Application Serial No. 63 / 650,083 filed on May 21, 2024 and of United States Provisional Patent Application Serial No. 63 / 646,083 filed on May 13, 2024. These and all other referenced extrinsic materials are incorporated herein by reference in their entirety. Where a definition or use of a term in a reference that is incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein is deemed to be controlling.This application references a sequence identification file entitled “BioMadison_102320_0037PCT_SEQID”, which is 3 KB in size and was created on April 21, 2025. The contents of this sequence identification file are incorporated by reference in their entirety.Field of the Invention

[0001] The field of the invention is cell based assays for proteases, in particular botulinum neurotoxins.Background

[0002] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum, and are among the most potent toxins known. These toxins are a well-recognized source of food poisoning, often resulting in serious harm or even death of the victims. Due to their lethality at very small doses these neurotoxins are also considered to be potential bioweapons.Attny Dkt No. 102320.0037PCT

[0005] Despite their high toxicity, botulinum neurotoxins have clinical utility in alleviating the symptoms of diseases and conditions associated with muscle contraction (c.g., strabismus). Increasingly, botulinum neurotoxins have been used to treat conditions such as chronic migraine and hyperhidrosis. Botulinum neurotoxin has also been used for cosmetic purposes, such as in reducing the appearance of facial lines and wrinkles associated with normal aging.

[0006] Most pharmaceutical formulations utilize botulinum neurotoxin serotype A, which has a prolonged duration of action that reduces the frequency of re-treatment. Recently, however, there has been an increasing interest in formulations that utilize botulinum neurotoxin serotype E. This has been driven by concerns over the potential for development of neutralizing antibodies directed to botulinum neurotoxin serotype A in individuals receiving repeated long-term treatment as well as a desire for treatment modalities that would benefit from botulinum neurotoxin serotype E’s shorter duration of action.

[0007] There are seven structurally related botulinum neurotoxins or serotypes (A through G). Each of these neurotoxins is composed of a heavy chain (=100 KD) and a light chain (=50 KD), and have distinct amino acid sequences and performance characteristics. The heavy chain is responsible for localization and internalization of the neurotoxin by motor neurons and other susceptible cells (Fischer et al, J Biol Chem, 283(7):3997-4003, 2007). Once internalized, the light chain is released from the heavy chain and enters the cytoplasm. The light chain has a proteolytic activity of exquisite specificity, selectively cleaving proteins mediating release of synaptosomes from the cell and thereby preventing the release of neurotransmitter compounds.

[0008] Botulinum neurotoxin serotype E is found in Clostridia botulinum as a protein complex, where the heavy and light chains are covalently linked and complexed with neurotoxin associated proteins (NAPs) (Avilar, Dermatologic Surgery, 50:9S (supplement), 2024). For example, United States Patent Application Publication No. 2009 / 0149393 (to Singh and Zhang) documents the presence of NAPs having molecular weights of 118, 80, 65, 40, and 18 kDa in botulinum neurotoxin serotype E as it occurs in Clostridium botulinum. These NAPs are believed to stabilize the neurotoxin, protect it from the acidic environment of the digestive tract, and facilitate transport across the intestinal barrier. It is also possible that such NAPs increase the toxicity of this neurotoxin / NAP complex relative to the neurotoxin alone.Attny Dkt No. 102320.0037PCT

[0009] Such botulinum neurotoxins can occur in a test sample as the linked heavy and light chain without associated NAPs. Such non-complcxcd neurotoxins can be a product of extensive processing of materials obtained from Clostridium botulinum, where such processing removes NAPs from the neurotoxin / NAP complex. Such non-complexed neurotoxins can also result from expression of the neurotoxin in a genetically engineered cell that does not express the NAPs. Such genetically modified cells can have the advantage of more readily scalable culture conditions than those of the Clostridium botulinum. Distinction between complexed and noncomplexed forms of botulinum neurotoxins can provide insights into the origin of neurotoxin found in a sample, as non-complexed neurotoxin is unlikely to be of natural origin.

[0010] Botulinum neurotoxins are generally characterized using a mouse lethality assay, an immunoassay, or a cell-based assay utilizing cultured cells that can internalize the neurotoxin. Users of mouse lethality assays are facing increasing pressure to seek alternative methods, driven by cost and ethical concerns regarding such animal testing. Immunoassays directed to the neurotoxin may not be suitable substitutes in some applications, however, as they do not provide cell membrane binding, internalization, and processing steps that are central to intoxication. In addition, a neurotoxin-directed immunoassay typically provides an estimate of mass rather than activity. Cell-based assays can measure proteolytic activity of a neurotoxin while providing such membrane binding, internalization. To date, however, such assays do not provide differentiation between complexed and non-complexed forms of botulinum neurotoxin.

[0011] Thus, there is still a need for methods that can distinguish between complexed and non- complexed forms of botulinum neurotoxins.Summary of The Invention

[0012] The inventive subject matter provides compositions and methods utilized in characterizing botulinum neurotoxins (BoNTs). Surprisingly, Inventors have found that neuroblastoma cell lines that have been genetically modified to produce a reporting peptide or reporting construct that is sensitive to a botulinum neurotoxin can demonstrate serotype specificity that is not exhibited by the parental cell line, and / or can in at least some serotypes differentiate between holotoxin (i.e., the neurotoxin in its natural, complexed form) and non- complexed neurotoxin.Attny Dkt No. 102320.0037PCT

[0013] Embodiments include a cell produced by genetic modification of a parental neuronal or neuronal-derived cell line, where the genetically modified cell a reporting construct. Such a cell can be more than 100-fold more selective for (e.g., sensitive to) intoxication by a non-complexed botulinum neurotoxin serotype E (NC-BoNT / E) (e.g., a botulinum neurotoxin serotype E without a neurotoxin associated protein (NAP)) than by a complexed botulinum neurotoxin serotype E (C-BoNT / E) (e.g., botulinum neurotoxin serotype E complexed with one or more NAPs). The parental neuronal or neuronal-derived cell line can be a human neuronal or neuronal-derived cell line or human neuroblastoma cell line. In some embodiments the parental neuronal or neuronal- derived cell line is LA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC- 19, M4b, CNh, G4b, HT-22, or PC 12.

[0014] Embodiments include a method of selectively quantifying NC-BoNT / E in a sample, by initially obtaining a first measurement from a cell as described above at a first wavelength and a second measurement is from the cell at a second wavelength. This first measurement is divided by the second measurement to calculate a first emission ratio. The cell is then contacted with a sample that is thought to contain a botulinum neurotoxin serotype E. After a suitable period of time (e.g., 24, 48, 72, or 96 hours), a third measurement from the cell is made at the first wavelength and a fourth measurement is from the cell at the second wavelength. The third measurement is divided by the fourth measurement to obtain a second emission ratio. A decrease or reduction of the second emission ratio relative to the first emission ratio is indicative of the presence of NC-BoNT / E. In some embodiments the first emission ratio is not distinguishable from the second emission ratio in the absence of NC-BoNT / E and when C- BoNT / E is present in the sample at a concentration of up to 0.5 nM. Suitable samples include, but are not limited to, a human tissue, an animal tissue, a food, a beverage, and a pharmaceutical preparation. Such human or animal tissue can be blood, serum, or plasma.

[0015] Embodiments include a method of improving form specificity of a cell-based assay for botulinum neurotoxin serotype E, by transfecting cells of a neuronal-derived cell line to generate two or more genetically modified cell lines expressing a reporting construct that is a substrate of for botulinum neurotoxin serotype E. Cells of the two or more genetically modified cells with NC-BoNT / E at a first concentration; and cells that are responsive to the neurotoxin are selected to generate a first subset of genetically modified cells responsive to NC-BoNT / E. Cells of thisAttny Dkt No. 102320.0037PCT first subset of cells are contacted with C-BoNT / E at a second concentration that is equal to or greater than (c.g., exceeds) the first concentration, and a second subset of genetically modified cells that are not responsive to C-BoNT / E are selected. Such cells of the second subset of genetically modified cells include cells with improved selectivity for NC-BoNT / E. Suitable neuronal-derived cells lines include, but are not limited to, a human neuronal, human neuronal- derived, or human neuroblastoma cell line. In some embodiments a suitable neuronal-derived cell line can be LA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC- 19, M4b, CNh, G4b, HT-22, or PC1217.

[0016] Genetically modified cells described above and as utilized in methods as described above can include a reporting construct, which can be expressed in the cell via stable or transient transfection. Such a reporting construct can include a membrane binding portion, a linking portion that includes a cleavage site sensitive to the botulinum neurotoxin protease activity, a reporting portion that includes a labile reporter and coupled to the membrane binding portion via the linking portion. Cleavage of the cleavage site results in release of the labile reporter into the genetically modified cell’s cytoplasm, where the labile reporter is subject to proteolysis. The labile reporter can be a fluorophore, which can be a fluorescent peptide. The reporting construct can also include a reference reporter that is coupled to the membrane binding portion, such that the reference reporter remains coupled to the membrane binding portion if the reporting construct is cleaved by the neurotoxin. Such a reference reporter can be a second fluorophore.

[0017] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.Brief Description of The Drawings

[0018] FIG. 1 : FIG. 1 provides a graphical depicting typical growth of A and B genetically modified cell lines.

[0019] FIG. 2: FIG. 2 provides a histogram depicting typical viability of A and B genetically modified cell lines.Attny Dkt No. 102320.0037PCT

[0020] FIG. 3 : FIG. 3 provides a graphical depicting typical growth of cells of the C genetically modified cell line.

[0021] FIG. 4: FIG. 4 provides a histogram depicting typical viability of cells of the C genetically modified cell line.

[0022] FIG. 5 : FIG. 5 provides a graphical depiction of typical comparative growth curves for A, B, and C genetically modified cell lines.

[0023] FIGs. 6A and 6B: FIG. 6A provides a graphical depiction of a typical response of genetically modified cell line C to non-complexed botulinum neurotoxin serotype E (NC- BoNT / E), shown as YFP / CFP normalized ratio. FIG. 6B provides a graphical depiction of results of a typical study in which cells of genetically modified cell line C are exposed to C- BoNT / E (panels A and B) and NC-BoNT / E (panel C) in side-by-side assays.

[0024] FIG. 7: FIG. 7 provides a graphical depiction of a typical comparison of sensitivity BoCell® and C genetically modified cell line response to BoNT / E holotoxin.

[0025] FIG. 8: FIG. 8 provides a flowchart of a representative method for generating neuronal- derived cell lines that are selectively responsive to non-complexed botulinum neurotoxin serotype E.

[0026] FIG.9 : FIG. 9 provides a flowchart of a representative method for generating neuronal- derived cell lines that are selectively responsive to complexed botulinum neurotoxin serotype E.

[0027] FIG. 10: FIG. 10 provides a flowchart of a representative method for generating neuronal-derived cell lines that are selectively responsive to non-complexed botulinum neurotoxin serotype E.

[0028] FIG. 11: FIG. 11 provides a flowchart of a representative method for generating neuronal-derived cell lines that are selectively responsive to complexed botulinum neurotoxin serotype E.Attny Dkt No. 102320.0037PCTDetailed Description

[0029] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0030] The present invention provides compositions and methods for differentiation between complexed and non-complexed botulinum neurotoxin serotype E (C-BoNT / E and NC-BoNT / E. respectively) using a cell based assay.

[0031] Within the context of this application, complexed botulinum neurotoxin serotype E (C- BoNT / E) refers to a protein complex comprising botulinum neurotoxin serotype E associated with one or more neurotoxin associated proteins (NAPs) associated with the neurotoxin as it is expressed in Clostridium botulinum. Such NAPs include, but are not limited to, peptides having molecular weights of about 118, 80, 65, 40, and 18 kDa and co-eluting with botulinum neurotoxin serotype E on size exclusion chromatography as described in United States Patent Application Serial No. 121247,124. Such C-BoNT / Es can be provided as complexes isolated from Clostridium botulinum, or can be provided by complexing a non-complexed botulinum neurotoxin serotype E (NC-BoNT / E) with one or more NAP and allowing a complex between the NAP(s) and the NC-BoNT / E to form. Such C-BoNT / E can be found in foods contaminated with C. botulinum, tissues and blood of individuals exposed to C. botulinum, in some pharmaceutical formulations used to treat diseases or conditions characterized by muscle contraction, and potentially in neurotoxin-based bioweapons.

[0032] Within the context of this application, non-complexed botulinum neurotoxin serotype E (NC-BoNT / E) refers to a botulinum neurotoxin serotype E that is not associated with a NAP. Such a NC-BoNT / E has a molecular weight of approximately 190 kD, and does not co-elute with a NAP on size exclusion chromatography. Such NC-BoNT / Es can be provided as recombinant proteins expressed in organisms other than Clostridium botulinum (e.g., E. coli, a yeast, etc.) such that NAPs that form part of the C-BoNT / E are not present during expression of the neurotoxin. Such recombinant organisms can have less stringent culture requirement than Clostridium botulinum and so be more suitable for pharmaceutical manufacture at scale.Attny Dkt No. 102320.0037PCTAlternatively, NC-BoNT / E can be provided by removal of NAPs from C-BoNT / E, for example by denaturation (c.g., by exposure to a chaotropc, pH shift, osmolarity shift, exposure to surfactants, etc.) followed by segregation of the neurotoxin from disassociated NAPs (e.g., by size exclusion chromatography, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, ultrafiltration, dialysis, etc.). Such NC-BoNT / E is typically man-made, and can be found in some pharmaceutical formulations used to treat diseases or conditions characterized by muscle contraction and potentially in neurotoxin-based bio weapons.

[0033] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0034] As used herein, and unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms "coupled to" and "coupled with" are used synonymously. In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention mayAttny Dkt No. 102320.0037PCT contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0035] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0036] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0037] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0038] Inventors have, surprisingly, found that genetically modified cells suitable for use in cellbased assays for botulinum neurotoxins can be insensitive to C-BoNT / E at relatively high concentrations (e.g., about 5 nM), but are sensitive to NC-BoNT / E at relatively low concentrations (e.g., about 10- fold, 100-fold, 1,000-fold or more lower than C-BoNT / E). Sensitivity can be defined as providing a response to the neurotoxin (e.g., a change in a signal from a neurotoxin-sensitive reporter, generation of peptides associated with the neurotoxin’sAttny Dkt No. 102320.0037PCT protease activity, etc.) that is statistically distinguishable (e.g., at least 2 standard deviations and / or at least 10%, 25%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, -30-fold, 100-fold, 300- fold, 1,000-fold, 3,000- fold, 10,000-fold, or greater different) between C-BoNT / E and NC- BoNT / E at the same concentration. Such cells can be utilized in cell-based assays that differentiate between C-BoNT / E and NC-BoNT / E. Such differentiation can be useful in qualifying an NC-BoNT / E preparation for pharmaceutical use. Similarly, such differentiation can be useful in determining if a botulinum neurotoxin serotype E present in a sample is natural or man-made.

[0039] Cells suitable for use in these methods can have been genetically modified using a method known in the art to express a reporting construct, where a portion of the reporting construct can act as a substrate for the proteolytic activity of botulinum neurotoxin serotype E.In some embodiments the genetically modified cells are derived from LA-N-2 cells (i.e., LA-N-2 cells are the parental cell line). In some embodiments the genetically modified cells are derived from SH-SY5Y cells (i.e., SH-SY5Y cells are the parental cell line). In some embodiments the genetically modified cells are derived from N2a cells (i.e., N2a cells are the parental cell line).In some embodiments the genetically modified cells are derived from SiMa cells (i.e., SiMa cells are the parental cell line). In some embodiments the genetically modified cells are derived from NS-20Y cells (i.e., NS-20Y cells are the parental cell line). In some embodiments the genetically modified cells are derived from NIE-115 cells (i.e., NIE-1 15 cells are the parental cell line). In some embodiments the genetically modified cells are derived from NG108-C15 cells (i.e., NG1O8-C15 cells are the parental cell line). In some embodiments the genetically modified cells are derived from NSC-34, cells (i.e., NSC-34 cells are the parental cell line). In some embodiments the genetically modified cells are derived from NSC- 19 cells (i.e., NSC- 19 cells are the parental cell line). In some embodiments the genetically modified cells are derived from M4b cells (i.e., M4b cells are the parental cell line). In some embodiments the genetically modified cells are derived from CNh cells (i.e., CNh cells are the parental cell line). In some embodiments the genetically modified cells are derived from G4b cells (i.e., G4b cells are the parental cell line). In some embodiments the genetically modified cells are derived from CNh cells (i.e., CNh cells are the parental cell line). In some embodiments the genetically modified cells are derived from G HT-22 cells (i.e., HT-22 cells are the parental cell line). In someAttny Dkt No. 102320.0037PCT embodiments the genetically modified cells are derived from G PCI 2 cells (i.e., PCI 2 cells are the parental cell line).

[0040] Surprisingly, such genetically modified cells can lack sensitivity to C-BoNT / E but exhibit sensitivity to NC-BoNT / E. In some embodiments one or more cell lines generated by such genetic modification can be screened for differential sensitivity to C-BoNT / E or NC-BoNT / E, for example by identifying reaction products of the proteolytic activity of botulinum neurotoxin serotype E in the cells or in lysates obtained from such cell following exposure to the neurotoxin. Suitable methods for identifying such products of proteolytic activity include SDS-PAGE, Western blot, peptide-specific immunoassay, and / or measurement of fluorescence from a reporting construct as described below.

[0041] Inventors contemplate that such genetically modified cells can be identified that similarly differentiate between complexed and non-complexed forms of botulinum neurotoxin serotypes A, B, C, D, F, and / or G, and that such cells can be utilized in cell-based assays directed to complexed and / or non-complexed forms of these neurotoxins, and can be identified via screening using similar methodologies that identify products of proteolysis by complexed and non-complexed forms of the neurotoxin.

[0042] In some embodiments a reporting construct expressed in such cells can include (a) a reporter-containing portion coupled to (b) a linking portion that includes a cleavage site, which in turn can act as a substrate for the proteolytic activity of botulinum neurotoxin serotype E, and (c) a membrane-binding portion that is coupled to the reporter-containing portion via the linking portion. Interaction with a form of botulinum neurotoxin E to which the cell is susceptible cleaves the reporter-containing portion from a remainder of the reporting construct and releases it into the cytoplasm of the transformed cell. Once released the reporter-containing portion is subject to rapid degradation by proteolytic activity in the cell’s cytoplasm.

[0043] In some embodiments the reporting construct can include a reference reporter that is retained with the membrane-binding portion and is not subject to proteolysis following exposure of the transformed cell to a form of botulinum neurotoxin E to which the cell is susceptible. In such embodiments the reporter-containing portion includes a fluorescent peptide (e.g., a GFP or GFP mutation derived peptide), and the reporting construct can be arranged such that no usefulAttny Dkt No. 102320.0037PCTForster resonance energy transfer (FRET) (e.g., less than 5% energy transfer) occurs between this fluorescent peptide and a fluorophorc (e.g., a second and different fluorescent peptide) of the reference reporter. This advantageously permits the use of measurements from the reference reporter to normalize measurement from the reporter portion across different test wells. For example, emission from a “reporting” fluorophore that is degraded by proteolysis upon release by BoNT activity can be divided by emission from a “reference” fluorophore that is not subjected to such degradation to correct for differences between cell number, expression levels, etc. between different wells of a test plate utilized in such a cell based assay. An example of a suitable reporting construct is found in SEQ ID NO. 1.

[0044] Such transformed cells can be utilized in methods for characterizing a form of botulinum neurotoxin E to which the cell is susceptible, qualitatively and / or quantitatively. An example of such a method can include:(i) providing a composition that includes a cell transformed to express a reporting construct as described above(ii) obtaining a baseline signal from the reporting-containing portion of the reporting construct(iii) exposing the cell to a form of botulinum neurotoxin E to which the cell is susceptible(iv) obtaining a further signal from the reporting-containing portion of the reporting construct after sufficient time has passed for cleavage of the reporting construct by the BoNT and degradation of released reporting portion in the cell’s cytosol.(v) comparing the first emission measurements of step (ii) with the further measurements of step (iv).Typically, these assays are performed at a temperature of 35° C to 39°C (preferably at 37°C), with the testing interval ranging from 24 hours to 72 hours. Temperature, time, and culture media conditions can be optimized to suit specific testing needs.

[0045] The cleaved reporter-containing portion is destroyed or otherwise degraded by the local environment, and presence and / or quantity of the form of botulinum neurotoxin E to which theAttny Dkt No. 102320.0037PCT cell is susceptible is then evidenced by a reduction in signal from the reporter. In the context of this application, it is contemplated that degradation of released reporter will typically proceed intracellularly by at least one of two pathways, by the ubiquitin-dependent process that targets proteins to the proteasome, or by the autophagy-lysosomal pathway. In one pathway, the proteasome is the enzyme. In the lysosome pathways, it is contemplated that the enzymes of interest are hydrolases, including especially a family of proteases called the cathepsins.

[0046] As noted above, a reporter-containing portion of a reporting construct can include a fluorescent protein or peptide, for example a Green Fluorescent Protein (GFP) mutation such as Yellow Fluorescent Protein (YFP), Citrine, Venus, and YPet proteins. The sequence of such fluorescent proteins mentioned herein can be modified to include specific characteristics (e.g., spectral) or be truncated to a desired size.

[0047] As noted above, a portion of the reporting construct that remains associated with the cell membrane following cleavage by the neurotoxin can include a second fluorescent protein, such as Cyan Fluorescent Protein (CFP), mCherry, or mStrawberry. The emission spectra of such a second fluorophore can be selected for minimal overlap with that of a fluorophore of the reporter-containing portion in order to simplify data normalization.

[0048] Any suitable reporting construct can be utilized in cells and methods of the inventive concept. SEQ ID NO. 1 (provided in the accompanying sequence listing) is an example of a suitable reporting construct that provides no useful FRET when intact. This reporting construct includes portion corresponding to SNAP25, flanking spacer peptides, and a YFP portion that is released by the proteolytic activity of the botulinum neurotoxin serotype E light chain, and is rapidly degraded upon release. This reporting construct also includes a CFP portion that is not degraded following proteolysis by a botulinum neurotoxin serotype E light chain, the emission of which can be used to normalize emission measurements made of YFP during the course of a cell based assay.

[0049] Such a reporting construct can be introduced into a cell by any suitable means, including introduction of a plasmid or viral genome that encodes the reporting construct. Such a plasmid or viral genome can incorporate a transcription promoter positioned to influence transcription of the portion of the plasmid or viral genome encoding the reporting construct. Such a plasmid orAttny Dkt No. 102320.0037PCT viral genome can incorporate additional coding regions that permit screening of transformed cells, such as regions encoding for antibiotic resistance.

[0050] In a preferred cell-based assay for characterizing a form of botulinum neurotoxin E to which the cell is susceptible, a cell containing the reporting construct will exhibit a baseline signal from the reporting portion, and then after exposure exhibits a reduced signal from the reporting portion. In a reporting construct incorporating YFP into the reporting portion and utilizing CFP for normalization separately excited YFP emissions (top, Ex500, Em526) and CFP emissions (middle, Ex434, Em470) are collected. These emissions are then background subtracted and the YFP emission is divided by CFP emission to serve as a reference or control for cell density and reporter expression in the individual cells. That emission ratio (YFP / CFP, bottom) is how the assay is reported.

[0051] Destruction or other degradation of the reporter-containing portion takes place at a much faster rate post-exposure to the form of botulinum neurotoxin E to which the cell is susceptible than pre-exposure. In preferred embodiments, it is contemplated that the destruction or other degradation of the reporter containing portion occurs at least 2x (twice) as fast post-exposure as pre-exposure, but more preferably the rate post-exposure rate is at least 5x, at least lOx, at least lOOx relative to the pre-exposure rate.

[0052] Cells utilized in methods of the inventive concept can be of neuronal or neuronal-derived (e.g., developed from a neuron, neuronal cell line, or neuronal-derived cell line origin. Suitable cell lines include, but are not limited to, EA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC-19, M4b, CNh, G4b, HT-22, and PC12. Surprisingly, Inventors have found unexpected differences in performance characteristics between different transformed cell lines modified to express the same reporting construct. For example, differences in regard to the form of the botulinum neurotoxin (e.g., complexed vs non-complexed forms) sensitivity have been found that have not been recorded previously.

[0053] In some embodiments of the inventive concept, a cell line that susceptible to a specified form of botulinum neurotoxin E (for example, a neuroblastoma cell line) is genetically modified to express a reporting construct (e.g., the reporting construct represented by SEQ ID NO. 1) that is susceptible to cleavage by proteolytic activity of the light chain of a form of botulinumAttny Dkt No. 102320.0037PCT neurotoxin E to which the cell is susceptible. As noted above, such a reporting construct can include a fluorescent moiety that has utility in a cell-based assay (for example, by degrading following scission of the reporting construct or providing a normalizing signal from the reporting construct). Such a reporting construct can be introduced by transformation of the cells with an expression vector, plasmid, or virus that includes genetic information encoding such a reporting construct prepared by and administered by methods as known in the art. Such transformations can be performed by any suitable method, including electroporation, infection via a viral vector (e.g., a lentivirus), direct injection, etc.Examples

[0054] Cells from a human neuroblastoma (human neuroblastoma) cell line were transformed with an expression vector encoding for a reporting construct (SEQ ID NO. I) that included:• a reporter-containing portion that includes a YEP peptide.• a linking portion containing a SNAP-25 derived cleavage site.• a membrane binding portion coupled to the reporter-containing portion via the linking portion.• a control or reference portion containing a CEP peptide and coupled to the membrane binding portion such that the coupling remains following cleavage of the linking portion.The same reporting construct is expressed in murine-derived BoCell® A / E cell lines, data from which is provided for comparative purposes.

[0055] Growth characteristics of several transformed cell lines derived from genetically modified human neuroblastoma (human neuroblastoma) cells and expressing the reporting construct were characterized, as shown in FIGs. 1 to 5. As shown in FIGs. 1 (cell lines A and B) and 3 (cell line C), genetically modified human neuroblastoma cells expressing a reporting construct as described above show vigorous growth in culture. As shown in FIGs. 2 (cell lines A and B) and 4 (cell line C, viability of the genetically modified cell lines remains at 95% or more through at least 168 hours of culture. FIG. 5 provides comparative results for geneticallyAttny Dkt No. 102320.0037PCT modified human neuroblastoma cell lines A, B, and C, showing similar rates of growth over time. These genetically modified cell lines were found to provide EC50S for botulinum neurotoxin serotype A (which can also cleave the linking portion of the reporting construct expressed within these cells) as low as about 0.1 pM to 1.1 pM in optimized cell based assays performed by direct measurement of fluorescence from the cells as described above. It should be appreciated that SNAP25, which forms part of a linker portion of the reporting construct, is a substrate for the proteolytic activity of botulinum neurotoxin serotypes E and A. Accordingly, response to botulinum neurotoxin serotype A can be used to confirm expression of a functional reporting construct within a cell. Within the context of this application, an EC 50 should be understood as a concentration corresponding to the midpoint of a sigmoidal dose response curve. Similarly, within the context of this application a lowest detectable dose (LDD) should be understood as a concentration corresponding to a response along a dose response curve that is distinguishable from a negative control (e.g., a test condition containing no compound to which a response is being tested, such as a botulinum neurotoxin). Such distinction can, for example, be determined as a response that deviates from the negative control condition by at least 2 standard deviations, or that provides clear demonstration of the beginning of a trend in the data.

[0056] Cells from the C genetically modified human neuroblastoma cell line were exposed to various concentrations botulinum neurotoxin serotype E, and emission from both YFP and CFP measured using independent excitation measurements. As noted above, in such a cell-based assay YFP is released by BoNT / A or BoNT / E proteolysis and rapidly degraded in the cytosol, whereas CFP is retained on the cell membrane and is protected from proteolysis.

[0057] Inventors investigated the response to botulinum neurotoxin serotype E by contacting the same cells with NC-BoNT / E, a 150kD protein which does not include the naturally occurring NAPs of the intact C-BoNT / E protein complex. Results are shown in FIG. 6A. Parallel studies performed using C-BoNT / E (panels A and B) and NC-BoNT / E (panel C, performed in triplicate) are shown in FIG. 6B. In these studies, genetically modified C cells were plated on poly-D- lysine assay plates, grown overnight, and then treated with preparations of botulinum neurotoxin type E diluted in half-log dilutions. Emission at 526nm and 470nm were obtained from the cells after a suitable period of time (typically 24 to 72 hours) and the 526nm / 479nm ratios were plotted as a function of the log of the botulinum neurotoxin type E concentration. MolarAttny Dkt No. 102320.0037PCT concentrations are reported using a molecular weight of 150 kD for the botulinum neurotoxin serotype E I component.

[0058] Surprisingly, genetically modified cells of the inventive concept that showed little to no intoxication with C-BoNT / E at concentrations as high as 0.5 nM (500 pM) showed sensitivity to as little as about 1 ng / mL (about 7 pM) of NC-BoNT / E.

[0059] BoCell® cells are a murine cell line expressing the same reporting construct that is expressed in the C cell line. FIG. 7 shows comparative C-BoNT / E dose response curves for BoCell® and C cell lines in cell based assays performed under the same testing conditions (time, temperature, etc.). As shown, the cells of the BoCell® cell line show a high degree of sensitivity to C-BoNT / E holotoxin when compared to C cells. Accordingly, the reporting construct utilized in these cells does not appear to be selective for either C-BoNT / E or NC-BoNT / E.

[0060] Discrimination between different forms of botulinum neurotoxin serotype E by such genetically modified cells can be sufficient to render the genetically modified cells essentially nonresponsive to the complexed C-BoNT / E form of the neurotoxin. For example, in the studies shown in FIGs. 6 A and 8 the C cell line was unresponsive to C-BoNT / E to the point where an EC50 could not be calculated, despite the use of high nM concentrations of the neurotoxin. Within the context of this application, genetically modified cells that are selectively responsive to a target form of a botulinum neurotoxin arc essentially nonresponsive to a non-target form of a botulinum neurotoxin when a 1,000-fold, 2,00-fold, 3,000-fold, 5,000-fold, 8,000-fold, 10,000- fold or greater excess of the non-target BoNT is required to generate a response (e.g., an LDD, EC50, etc.) from the cell equivalent to that produced by the target BoNT. For example, as shown in FIG. 7, when C cells are exposed to BoNT / A or BoNT / E and approximately 0.5 nM of BoNT / E is required to produce a response that corresponds that provided by 0.05 pM of BoNT / A (i.e., an approximately 10,000-fold difference) under the same testing conditions.

[0061] As noted above, cell-based assays utilizing genetically modified cells of the inventive concept can provide botulinum neurotoxin assays with excellent sensitivity and neurotoxin form specificity. Such cell-based assays can be applied to any type of sample in which the presence of a botulinum neurotoxin of a serotype appropriate for the expressed reporting construct (i.e., capable of cleaving the reporting construct) is suspected or in which botulinum neurotoxinAttny Dkt No. 102320.0037PCT content needs to be characterized. Suitable sample types include environmental samples (e.g., soil, foliage, samples obtained from surfaces, clothing, and / or protective gear, etc.), foods, beverages, human or animal samples (e.g., tissue samples, blood, plasma, serum, lymphatic fluid, tears, cerebrospinal fluid, saliva, urine, feces, etc.), and botulinum neurotoxin-containing pharmaceutical products. Such human or animal samples can be freshly obtained or preserved (e.g., by freezing or storage at reduced temperature). In some embodiments such human or animal samples can be treated to reduce degradation of BoNT content prior to testing, for example by the addition of a protease inhibitor.

[0062] Genetically modified cells derived from the cell line can be identified by any suitable means. In some embodiments such cells can be identified by selection for selection markers incorporated into the expression vector, plasmid, or viral genome encoding the construct (for example, by encoding for resistance to an antibiotic). Alternatively, or in addition, genetically modified cells expressing the reporting construct can be identified and isolated by fluorescence activated cell sorting (FACS) utilizing emission from a fluorescent moiety of the expressed reporting construct. For example, droplets containing cells showing fluorescence associated with the reporting construct can be separated and expanded in culture. As noted above, further selection steps can be utilized to identify cell lines having the desired selectively (e.g., selectivity for NC-BoNT / E over C-BoNT / E)

[0063] As noted above, the Inventors have surprisingly identified cells that can exhibit a high degree of selectivity (e.g., showing a 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900- fold, 1,000 fold or greater difference if ECso) between different forms of a botulinum neurotoxin of a given serotype, for example between NC-BoNT / E and C- BoNT / E. Such cells can be identified by comparison of the results of exposure to a range of concentrations of different forms of the neurotoxin and any resulting cleavage of the reporting construct (e.g., by monitoring loss of fluorescence in the fluorescent moiety so released, as described above). In some embodiments this can be performed by culture of the genetically modified cells in a series of wells in a microwell plate in the presence of the BoNT, followed by fluorescence measurements of the wells.Attny Dkt No. 102320.0037PCT

[0064] Alternatively, such cells can be isolated using FACS to segregate cells responsive to a desired form of the neurotoxin, for example by contacting the genetically modified cells with a high concentration of s desired form of the neurotoxin and measuring a low ratio (e.g., 0.1, 0.05, 0.05, 0.01 or less) of fluorescence between the fluorescent moiety released into the cytoplasm by the neurotoxin and the fluorescent moiety that is not released by the neurotoxin. Cells within this population that are relatively non-responsive to the non-desired form of the neurotoxin can be identified from this population, for example by contacting then with a high concentration of the non-desired form of the neurotoxin and identifying cells that maintain a high ratio (e.g., greater than 50% of that observed for untreated cells) of fluorescence between the respective fluorophores.

[0065] Exemplary methods for generating or identifying neuronal-derived cell lines that are selectively responsive to a specific form of botulinum neurotoxin serotype E are depicted in FIGs. 8 to 11. As shown in FIG. 8, a population of neuronal-derived cells that express a reporting construct that is responsive to the protease activity of botulinum neurotoxin serotype E can be generated (for example, by transfection with an expression vector encoding a protein comprising SEQ ID NO. 1). Expression of such a reporting construct can, for example, be verified by measurement of fluorescence emission from fluorescent peptide sequences that form part of the reporting construct. Such cells can be isolated by any suitable means, including (but not limited to) incorporation of a selection factor (e.g. drug resistance) into the expression vector, selective culture, and / or fluorescence activated cell-sorting (e.g., collection of cells exhibiting fluorescence corresponding to the reporting construct). In some embodiments such cells can be contacted with a different neurotoxin that can also cleave the reporting construct in order to verify appropriate expression and localization. For example, if a reporting construct that utilizes a portion of SNAP-25 that includes both botulinum neurotoxin serotype E and serotype A cleavage and recognition sites is utilized, botulinum neurotoxin serotype A can be used to verify proper function and localization of the reporting construct.

[0066] As shown in FIG. 8. such a population of genetically modified neuronal-derived cells can be treated with NC-BoNT / E. Following such treatment, a first subset of cells that are responsive to NC-BoNT / E can be identified, based on a change in the state of the reporting construct. For example, if a fluorescent reporting construct (e.g., SEQ ID NO. 1) is utilized, a reduction inAttny Dkt No. 102320.0037PCT fluorescence from a fluorescent amino acid sequence of the reporting construct (e.g. relative to fluorescence from a control or reference fluorescent amino acid sequence of the reporting construct) can indicate that the cell is responsive to NC-BoNT / E. In a preferred embodiment a concentration of NC-BoNT / E is utilized in such an initial screening step that is appropriate for a cell-based assay for NC-BoNT / E (e.g., from 1 pM to 1 nM, or any concentration between these values). Such a first population, however, can include cells that are not selectively responsive to the non-complexed form of the neurotoxin.

[0067] As shown in FIG. 8, cells from this first subset can be treated with C-BoNT / E (for example, at a concentration equal to or greater than that utilized in screening with NC-BoNT / E) and a second subset of cells identified that is not responsive to C-BoNT / E. This second subset of cells comprises cells that are selectively responsive to the non-complexed form of botulinum neurotoxin serotype E.

[0068] As shown in FIG. 9, a related method can be used to generate neuronal derived cels that are selectively responsive to C-BoNT / E. As shown, a population of genetically modified neuronal-derived cells can be treated with C-BoNT / E. Following such treatment, a first subset of cells that are responsive to C-BoNT / E can be identified, based on a change in the state of the reporting construct. For example, if a fluorescent reporting construct (e.g., SEQ ID NO. 1) is utilized, a reduction in fluorescence from a fluorescent amino acid sequence of the reporting construct (e.g. relative to fluorescence from a control or reference fluorescent amino acid sequence of the reporting construct) can indicate that the cell is responsive to C-BoNT / E. In a preferred embodiment a concentration of C-BoNT / E is utilized in such an initial screening step that is appropriate for a cell-based assay for C-BoNT / E (e.g., from 1 pM to 1 nM, or any concentration between these values). Such a first population, however, can include cells that are not selectively responsive to the complexed form of the neurotoxin.

[0069] As shown in FIG. 9, cells from this first subset can be treated with NC-BoNT / E (for example, at a concentration equal to or greater than that utilized in screening with C-BoNT / E) and a second subset of cells identified that is not responsive to NC-BoNT / E. This second subset of cells comprises cells that are selectively responsive to the complexed form of botulinum neurotoxin serotype E.Attny Dkt No. 102320.0037PCT

[0070] FIG. 10 schematically depicts an alternative method that can be used to generate neuronal derived cells that arc selectively responsive to NC-BoNT / E. As shown, a population of genetically modified neuronal-derived cells can be treated with C-BoNT / E. In such embodiments such genetically modified neuronal-derived cells can be initially screened using an alternative neurotoxin to which the reporting construct is also responsive (e.g., botulinum neurotoxin serotype A) in order to verify useful expression and localization of the reporting construct). Following such treatment, a first subset of cells that are not responsive to C-BoNT / E can be identified, based on a lack of change in the state of the reporting construct. For example, if a fluorescent reporting construct (e.g., SEQ ID NO. 1) is utilized, a lack of decrease in fluorescence from a fluorescent amino acid sequence of the reporting construct (e.g. relative to fluorescence from a control or reference fluorescent amino acid sequence of the reporting construct) can indicate that the cell is not responsive to C-BoNT / E. In a preferred embodiment a concentration of C-BoNT / E is utilized in such an initial screening step that is greater than would be appropriate for a cell-based assay for NC-BoNT / E (e.g., from 1 nM to 500 nM, or any concentration between these values). Such a first population, however, can include cells that are not responsive to the non-complexed form of the neurotoxin.

[0071] As shown in FIG. 10, cells from this first subset can be treated with NC-BoNT / E (for example, at a concentration appropriate for a cell-based assay directed to NC-BoNT / E as described above) and a second subset of cells identified that is responsive to NC-BoNT / E. This second subset of cells comprises cells that are selectively responsive to the non-complexed form of botulinum neurotoxin serotype E.

[0072] FIG. 11 schematically depicts an alternative method that can be used to generate neuronal derived cells that are selectively responsive to C-BoNT / E. As shown, a population of genetically modified neuronal-derived cells can be treated with NC-BoNT / E. In such embodiments such genetically modified neuronal-derived cells can be initially screened using an alternative neurotoxin to which the reporting construct is also responsive (e.g., botulinum neurotoxin serotype A) in order to verify useful expression and localization of the reporting construct). Following such treatment, a first subset of cells that are not responsive to NC-BoNT / E can be identified, based on a lack of change in the state of the reporting construct. For example, if a fluorescent reporting construct (e.g., SEQ ID NO. 1) is utilized, a lack of decrease inAttny Dkt No. 102320.0037PCT fluorescence from a fluorescent amino acid sequence of the reporting construct (e.g. relative to fluorescence from a control or reference fluorescent amino acid sequence of the reporting construct) can indicate that the cell is not responsive to NC-BoNT / E. In a preferred embodiment a concentration of NC-BoNT / E is utilized in such an initial screening step that is greater than would be appropriate for a cell-based assay for C-BoNT / E (e.g., from 1 nM to 500 nM, or any concentration between these values). Such a first population, however, can include cells that are not responsive to the complexed form of the neurotoxin.

[0073] As shown in FIG. 11, cells from this first subset can be treated with C-BoNT / E (for example, at a concentration appropriate for a cell-based assay directed to C-BoNT / E, such as from 1 pM to 1 nM) and a second subset of cells identified that is responsive to C-BoNT / E. This second subset of cells comprises cells that are selectively responsive to the complexed form of botulinum neurotoxin serotype E.

[0074] Notably, the capability to distinguish between a form of a botulinum neurotoxin that is complexed with NAPs and a non-complexed form in which such NAPs are not present can provide insight into the origin of the neurotoxin. For example, selective detection of a noncomplexed form can indicate manmade origin, as such forms are likely the result of extensive processing of the natural product or synthesis in a genetically modified organism. Conversely, detection of a form of the neurotoxin that is complexed with NAPs can indicate a potential natural origin for the neurotoxin. In some embodiments, a sample can be split and characterized using a cell-based assay that is selective for a form of a botulinum neurotoxin from which NAPs are present and also characterized using a cell-based assay that is sensitive to a complexed form of the botulinum neurotoxin in which NAPs are associated with the neurotoxin. A positive result from the selective cell-based assay would indicate a man-made origin for the botulinum neurotoxin, whereas a lack of response from the selective cell based assay and a response from the cell based assay that is sensitive to a complexed form of the botulinum neurotoxin in which NAPs are associated with the neurotoxin can indicate a potential natural origin.

[0075] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appendedAttny Dkt No. 102320.0037PCT claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMSWhat is claimed is:

1. A cell produced by genetic modification of a parental neuronal cell line and comprising a reporting construct, wherein the cell is greater than 100-fold more selective for intoxication by a non-complexed botulinum neurotoxin serotype E (NC-BoNT / E) comprising botulinum neurotoxin serotype E without a neurotoxin associated protein (NAP) than by complexed botulinum neurotoxin serotype E (C-BoNT / E) comprising botulinum neurotoxin serotype E complexed with a NAP.

2. The cell of claim 1, wherein the parental neuronal cell line is a human neuronal, human neuronal-derived, or human neuroblastoma cell line.

3. The cell of claim 1, wherein the parental neuronal cell line is selected from the group consisting of LA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC- 19, M4b, CNh, G4b, HT-22, and PC12.

4. The cell of one of claims 1 to 3, wherein the reporting construct comprises: a membrane binding portion; a linking portion comprising a cleavage site, wherein the cleavage site is a substrate for the BoNT protease activity; and a reporting portion comprising a labile reporter, wherein the reporting portion is coupled to the membrane binding portion via the linking portion, such that cleavage of the cleavage sites results in release of the labile reporter into the genetically modified cell’s cytoplasm, and wherein the labile reporter is subject to proteolysis in the genetically modified cell’s cytoplasm within a time course of the cell-based assay.

5. The cell of claim 4, wherein the labile reporter is a first fluorophorc.

6. The cell of claim 5, wherein the first fluorophore is a fluorescent peptide.

7. The cell of one of claims 4 to 6, wherein the reporting construct further comprises a reference reporter coupled to the membrane binding portion, wherein the reference reporter remains coupled to the membrane binding portion of cleavage of the cleavage site.

8. The cell of claim 7, wherein the reference reporter is a second fluorophore.

9. The cell of claim 7 or 8, wherein the reference reporter is not subject to degradation upon cleavage of the cleavage site.

10. A method of selectively quantifying NC-BoNT / E in a sample, comprising: contacting a cell of one of claims 1 to 3 with the sample; obtaining a first emission measurement from the cell at a first wavelength; obtaining a second emission measurement from the cell at a second wavelength; dividing the first measurement by the second measurement to obtain a first emission ratio; contacting the cell with a solution comprising a botulinum neurotoxin serotype E; after a period of time, obtaining a third measurement from the cell at the first wavelength and a fourth measurement from the cell at the second wavelength; and dividing the third measurement by the fourth measurement to obtain a second emission ratio, wherein reduction of the second emission ratio relative to the first emission ratio is indicative of the presence of NC-BoNT / E.11 . The method of claim 10, wherein the first emission ratio is not distinguishable from the second emission ratio in the absence of NC-BoNT / E and when C-BoNT / E is present in the sample at a concentration of 0.5 nM.

12. The method of claim 10 or 11, wherein the period of time is 72 hours.

13. The method of one of claims 10 to 12, wherein the sample is obtained from a human tissue, animal tissue, food, beverage, or pharmaceutical preparation.

14. The method of claim 13, wherein the human tissue or animal tissue is selected from the group consisting of blood, serum, and plasma.

15. A method of improving form specificity of a cell-based assay for botulinum neurotoxin serotype E, comprising:transfecting cells of a neuronal-derived cell line to generate a plurality of genetically modified cell lines expressing a reporting construct that is a substrate of for botulinum neurotoxin serotype E; contacting the plurality of genetically modified cells with NC-BoNT / E at a first concentration; selecting, from among the plurality of genetically modified cells contacted with NC- BoNT / E, a first subset of genetically modified cells comprising cells responsive to NC-BoNT / E; contacting the first subset of genetically modified cells with C-BoNT / E at a second concentration that exceeds the first concentration; and selecting, from among the first subset of genetically modified cells contacted with C- BoNT / E, a second subset of genetically modified cells comprising cells that are not responsive to C-BoNT / E, wherein cells of the second subset of genetically modified cells comprise cells with improved selectivity for NC-BoNT / E.

16. The method of claim 15, wherein the neuronal -derived cell line is a human neuronal or human neuroblastoma cell line.

17. The method of claim 1 , wherein neuronal-derived cell line is selected from the group consisting of LA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC- 19, M4b, CNh, G4b, HT-22, and PC1217.

18. The method of one of claims 10 to 17, wherein the reporting construct comprises” a membrane binding portion; a linking portion comprising a cleavage site, wherein the cleavage site is a substrate for the BoNT protease activity; and a reporting portion comprising a labile reporter, wherein the reporting portion is coupled to the membrane binding portion via the linking portion, such that cleavage of the cleavage sites results in release of the labile reporter into the genetically modified cell’s cytoplasm, and wherein the labile reporter is subject to proteolysis in the genetically modified cell’s cytoplasm within a time course of the cell-based assay.

19. The method of claim 18, wherein the labile reporter is a first fluorophore.

20. The method of claim 19, wherein the first fluorophore is a fluorescent peptide.

21. The method of one of claims 18 to 21, wherein the reporting construct further comprises a reference reporter coupled to the membrane binding portion, wherein the reference reporter remains coupled to the membrane binding portion of cleavage of the cleavage site.

22. The method of claim 22, wherein the reference reporter is a second fluorophore.

23. The method of claim 21 or 22, wherein the reference reporter is not subject to degradation upon cleavage of the cleavage site.

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