Botulinum toxin-sensitive cells into which specific genes have been inserted by lentivirus

Abstract

The present invention relates to a cell for measuring botulinum toxin activity that overexpresses SEPTIN2 or Thioredoxin (TXN), and a method for measuring botulinum toxin activity, comprising the steps of treating the cell for measuring botulinum toxin activity with botulinum toxin and culturing it, lysing the cultured cells to collect a cell lysate, and measuring the amount of SNAP-25 cleavage products in the cell lysate.

Description

[Technical Field]

[0001] The present invention relates to a cell for measuring botulinum toxin activity and a method for measuring botulinum toxin activity using the same. [Background technology]

[0002] Botulinum toxin is a neurotoxin produced by Clostridium botulinum, a gram-positive anaerobic bacterium that grows on rotten canned food and rotten meat. It is classified into eight neurotoxins, seven of which (A, B, C, D, E, F, and G) can cause nerve paralysis. Approximately 150 kDa in size, they are composed of a complex of botulinum toxin proteins and non-toxin proteins, with each complex being produced up to 900 kDa in size depending on the neurotoxin type. Different botulinum toxin types have different modes of action, targets, and durations of activity. Botulinum toxin type A is known as one of the deadly biological agents. Botulinum toxins cause paralysis by blocking signals that trigger muscle spasms and contractions. Since being approved by the U.S. FDA in 1989 as a biological agent based on these functions, they have been widely used for therapeutic and cosmetic purposes. It is used therapeutically to treat conditions such as strabismus, torticollis, blepharospasm, impotence, misaligned teeth, and lower back pain, and cosmetically to remove wrinkles, frown lines, square chin, and hyperhidrosis.

[0003] The therapeutic and cosmetic use of botulinum toxin requires biological validation before use, and such validation is generally performed using mouse LD. 50、 In essence, the units on the label of a pharmaceutical preparation are determined to be the mouse LD 50 However, statistically useful mouse LD 50Not only do they require a very large number of mice to provide data, but they also have limitations such as the high cost of testing and the difficulty in detecting differences due to botulinum toxin serotypes (Korean Patent Publication No. 10-2012-0134154).

[0004] Therefore, in order to overcome these drawbacks, a new, simple yet highly sensitive method for measuring botulinum toxin activity is needed that can evaluate all the steps necessary for botulinum toxin absorption without using animals. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention was devised to solve the above-mentioned problems of the prior art, and its object is to provide cells for measuring botulinum toxin activity and a method for measuring botulinum toxin activity using the same. The cells overexpress the SEPTIN2 or Thioredoxin (TXN) gene and can evaluate botulinum toxin binding, cellular absorption, cytoplasmic translocation, and protease activity. Not only can these cells evaluate botulinum toxin binding, cellular absorption, cytoplasmic translocation, and protease activity, but they also have significantly improved sensitivity to botulinum toxin, allowing for highly accurate detection of even low doses of botulinum toxin. Furthermore, the activity of botulinum toxin pharmaceutical compositions in raw drug and finished drug form can also be measured.

[0006] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0007] The present invention provides cells for measuring botulinum toxin activity that overexpress SEPTIN2 or Thioredoxin (TXN).

[0008] In one embodiment of the present invention, the cells may be overexpressed by inserting a SEPTIN2 or TXN gene into the cells, preferably by transduction, transfection, or other methods, but any commonly known method for inserting a gene into cells may be used. The SEPTIN2 gene is preferably the human SEPTIN2 gene, more preferably a gene expressing the 3,726-bp mRNA sequence of NCBI accession number NM_001008491.2. Variants thereof are also within the scope of the present invention. Specifically, the SEPTIN2 gene may have a sequence with 90% or more sequence identity, more preferably 95% or more, and most preferably 98% or more sequence identity with the mRNA sequence of NM_001008491.2. The "percent sequence identity" is determined by comparing the optimally aligned sequence with the comparison region. The comparison region may contain additions or deletions (i.e., gaps) in the nucleotide sequence compared to the reference sequence (which does not contain additions or deletions) for the optimally aligned sequence. Next, the TXN gene is preferably a human TXN gene, more preferably a gene expressing the 737-bp mRNA sequence of NCBI accession number NM_003329.4, although variants thereof are also within the scope of the present invention. Specifically, the TXN gene may include a sequence having 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the mRNA sequence of NM_003329.4. The "percent sequence identity" is determined by comparing the optimally aligned sequence with the comparison region, and a portion of the nucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the sequence.

[0009] In another embodiment of the present invention, the cells are characterized by increased susceptibility to botulinum toxin intoxication compared to wild-type cells due to overexpression of the SEPTIN2 or TXN gene.

[0010] In yet another embodiment of the present invention, the cells may be preferably SiMa cells, LAN-2 cells, PC12 cells, Neuro-2a cells, LA1-55n cells, N18 cells, SH-SY5Y cells, Kelly cells, NB69 cells, N1E-115 cells, BE(2)-M17 cells, SK-N-BE(2) cells, etc., but are not limited thereto as long as they are cell lines generally known to be usable for measuring botulinum toxin activity.

[0011] In yet another embodiment of the invention, the botulinum toxin may be selected from the group consisting of botulinum serotypes A, B, C, D, E, F and G.

[0012] The present invention also provides a method for measuring botulinum toxin activity, comprising the steps of: a) treating the cells for measuring botulinum toxin activity with botulinum toxin and culturing the cells; b) lysing the cultured cells and collecting a cell lysate; and c) measuring the amount of SNAP-25 cleavage products in the cell lysate.

[0013] In one embodiment of the present invention, the amount of the SNAP-25 cleavage product is measured using a sandwich immunoassay (ELISA), Western blot, or the like, but is not limited to these methods as long as they are known to be used for detecting proteins.

[0014] The present invention also provides a kit for measuring botulinum toxin activity, which contains the cells for measuring botulinum toxin activity as an active ingredient.

[0015] The present invention also provides use of the cells for measuring botulinum toxin activity for measuring botulinum toxin activity. [Effects of the Invention]

[0016] The cells for measuring botulinum toxin activity according to the present invention have significantly improved sensitivity to botulinum toxin due to overexpression of SEPTIN2 or Thioredoxin, and therefore can not only verify low doses of botulinum toxin with high accuracy, but also evaluate the binding, cellular absorption, intracytoplasmic potential, and protease activity of botulinum toxin, and are therefore expected to replace many animal experiments. Furthermore, because the activity of not only botulinum toxin but also botulinum toxin pharmaceutical compositions in raw drug and finished drug form can be measured, the cells are expected to be easily applicable to various industrial fields that use botulinum toxin. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a simplified diagram showing the functions of SEPTIN2 and Thioredoxin (TXN). [Figure 2] FIG. 2 is a simplified diagram showing the pLenti-C-Myc-DDK-P2A-Puro vector map. [Figure 3] FIG. 3 shows the results of microscopic observation of the 293FT cell line cultured according to one embodiment of the present invention. [Figure 4] FIG. 4 is a simplified diagram illustrating a method for generating lentivirally transduced cell lines according to one embodiment of the present invention. [Figure 5] FIG. 5 is a diagram showing a method for separating and culturing cell lines into colonies using a cloning cylinder according to one embodiment of the present invention. [Figure 6] FIG. 6 shows the results of Western blotting of proteins expressed in a transduced cell line according to one embodiment of the present invention. [Figure 7] FIG. 7 is a diagram showing binding positions of primers prepared for PCR according to one embodiment of the present invention. [Figure 8] FIG. 8 shows the results of PCR confirmation of the introduced gene according to one embodiment of the present invention. [Figure 9] FIG. 9 is a diagram showing the principle of sandwich ELISA in a simplified manner. [Figure 10] FIG. 10 is a diagram showing the results of comparing the amount of SNAP-25 cleaved between a control group and a cell line transfected with the SEPTIN2 gene to determine the sensitivity to botulinum toxin according to an embodiment of the present invention. [Figure 11] FIG. 11 is a diagram showing the results of examining the sensitivity of a control group and transduced cell lines to botulinum toxin based on the raw data ratio between each group according to an embodiment of the present invention. [Figure 12] FIG. 12 is a diagram showing the results of comparing the SNAP-25 cleavage amount to determine the sensitivity to botulinum toxin between a control cell line and a cell line transfected with a thioredoxin gene according to an embodiment of the present invention. [Figure 13] FIG. 13 is a diagram showing the results of measuring the potency of a botulinum toxin pharmaceutical composition (raw drug) according to one embodiment of the present invention using a transduced cell line. [Figure 14] FIG. 14 is a diagram showing the results of measuring the potency of a botulinum toxin pharmaceutical composition (finished drug) according to one embodiment of the present invention using a transduced cell line. [Figure 15] FIG. 15 is a simplified diagram showing the overall development process of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] As a result of extensive research into methods for measuring botulinum toxin activity, the present inventors have produced a cell line with improved sensitivity for measuring botulinum toxin activity, and have confirmed that botulinum toxin activity can be stably measured using this cell line, thereby completing the present invention.

[0019] As used herein, "botulinum toxin" means any known type of botulinum toxin, whether produced by bacteria or by recombinant techniques, and any subsequently discovered type, including engineered variants or fusion proteins. Botulinum toxins are classified into eight neurotoxins, and seven serotypes, A, B, C, D, E, F, and G, can induce nerve paralysis. The protein is divided into complexed and uncomplexed forms. The molecular weight of the pure toxin protein is 150 kDa, with various proteins produced at 300 kDa, 500 kDa, and 900 kDa depending on whether the complex is formed or not. Alternatively, the botulinum toxin of the present invention may be a botulinum toxin derivative, i.e., a compound that has botulinum toxin activity but contains one or more chemical or functional modifications compared to native or recombinant botulinum toxins. For example, the botulinum toxin may be a modified neurotoxin (e.g., a neurotoxin having one or more amino acid deletions, modifications, or substitutions compared to native or recombinantly produced neurotoxins, their derivatives, or fragments). For example, the botulinum toxin may be a botulinum toxin that has been modified in a manner that enhances its properties or reduces its undesirable side effects while still retaining the desired botulinum toxin activity. Alternatively, the botulinum toxin may be a toxin produced using recombinant or synthetic chemical techniques (e.g., a recombinant peptide, fusion protein, or hybrid neurotoxin made from subunits or domains of different botulinum toxin serotypes (see, e.g., U.S. Pat. No. 6,444,209)). The botulinum toxin may also be part of a whole molecule that has been demonstrated to have the required botulinum toxin activity, and in such cases may be used by itself or as part of a combination or conjugate molecule, e.g., a fusion protein. The botulinum toxin may also be in the form of a botulinum toxin precursor, which may be non-toxic itself, e.g., a non-toxic zinc protease that may become toxic upon proteolytic degradation.

[0020] As used herein, the term "cell" refers to any eukaryotic cell that is susceptible to botulinum toxin intoxication or that can absorb botulinum toxin. Eukaryotic cells refer to cells derived from various mammals, such as mice, rats, pigs, cattle, sheep, horses, primates, and humans. As used herein, the term "engineered cell line" is synonymous with established cell line, immortal cell line, or transformed cell line, and refers to cells selected for indefinite proliferation. The transformed cell lines disclosed herein exhibit consistent sensitivity to botulinum toxin activity over multiple cell passages, and "sensitivity to botulinum toxin activity" refers to the lowest botulinum toxin concentration at which a signal detected by the cell line can be consistently measured relative to the untreated control or background signal.

[0021] As used herein, the terms "vector" and "plasmid" refer to DNA fragments, nucleic acid molecules, etc., that are transferred into cells, and the vector can replicate DNA and reproduce independently of the host cell. They may be used interchangeably with the term "transmitter." The term "expression vector" refers to a recombinant DNA molecule that contains a coding sequence of interest and a titered nucleic acid sequence necessary for expressing the operably linked coding sequence in a specific host organism. Furthermore, the recombinant vector or plasmid of the present invention collectively refers to all vectors that contain a gene encoding SEPTIN2 or thioredoxin and can overexpress SEPTIN2 or thioredoxin in cells.

[0022] As used herein, the term "kit" refers to an instrument capable of measuring the activity of a botulinum toxin by including the cells for measuring botulinum toxin activity of the present invention, and may be used to measure the activity of a botulinum toxin itself, a raw material pharmaceutical product for a botulinum toxin, or a finished pharmaceutical product for a botulinum toxin. The kit of the present invention may further include, in addition to the cells for measuring botulinum toxin activity of the present invention, a cell lysing agent for lysing cells, antibodies and reagents for ELISA, a manual, and the like, provided that such further items can be used in the method for measuring botulinum toxin activity of the present invention.

[0023] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention.

[0024] [Example] Example 1: Cultivation of the lentivirus-producing cell line 293FT 293FT, a cell line commonly used for lentivirus production, is derived from the 293F cell line and stably expresses the SV40 large T antigen via the pCMVSPORT6TAg.neo plasmid. It is a representative cell line used for lentivirus production. Because the 293FT cell line encodes a neomycin resistance gene within the plasmid, it was cultured in medium containing geneticin, a neomycin analog, during all cell culture steps except for thawing. Specifically, 293FT cell stock (Thermo, R700-07) was suspended in DMEM (Gibco) medium supplemented with 10% FBS (Gibco), 1X NEAA (Gibco), 2 mM L-glutamine (Gibco), and 1% penicillin-streptomycin (Gibco), followed by centrifugation at 200 x g for 3 minutes, and the supernatant was removed to obtain cells. The obtained cells were resuspended in culture medium and then distributed into 100 mm cell culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was then removed and replaced with DMEM (Gibco) medium supplemented with 10% FBS (Gibco), 1X NEAA (Gibco), 2 mM L-glutamine (Gibco), 1% penicillin-streptomycin (Gibco), and 500 μg / mL geneticin selective antibiotic (Gibco). Once cell confluency reached 90% or greater, the cells were subcultured again. To subculture, the culture medium was removed and 5 mL of DPBS (Gibco) (50% of the culture volume) was added to wash the cells. Next, DPBS was removed and 2 mL of TrypLE (Gibco) (20% of the culture volume) was added. The cells were then incubated at 37°C in a 5% CO2 incubator for 2 minutes. After the reaction was completed, additional culture medium was added, and the mixture was centrifuged at 800 x g for 2 minutes at 4°C to remove the supernatant. The cells were then resuspended in culture medium, and the cell number was measured using Trypan blue (Gibco) and a hemocytometer. 2 x 10 cells were then plated onto a 100 mm cell culture dish. 6The cells were dispensed together with 10 mL of culture medium and subcultured, and the subculture was carried out every 3 to 4 days in the same manner as above.

[0025] Example 2: Gene selection and construction of encoding plasmids for the generation of cell lines vulnerable to BoNT / A intoxication As shown in Figure 1, SEPTIN2 (SEPT2) protects BoNT / A from intracellular degradation, while thioredoxin (TXN) acts to separate the disulfide bond between the heavy and light chains of BoNT / A. To overexpress these two proteins in cells, we commissioned Origene to construct the pLenti-ORF plasmids (SEPTIN2: RC224864L3, TXN: RC208876L3) in which the genes encoding each protein were inserted into the pLenti-C-Myc-DDK-P2A-Puro vector. The amino acid sequence of SEQ ID NO: 9 (DNA sequence: SEQ ID NO: 10) was used to construct SEPTIN2, and the amino acid sequence of SEQ ID NO: 11 (DNA sequence: SEQ ID NO: 12) was used to construct thioredoxin. The pLenti-C-Myc-DDK-P2A-Puro vector map is shown in Figure 2. The tube containing the freeze-dried pLenti-ORF plasmid was centrifuged at 5000×g for 3 minutes, after which 100 μL of distilled water was added, pipetted, and then stored in a freezer at −20°C until use.

[0026] To prepare strains transformed with the pLenti-ORF plasmid, the plasmid was thawed at room temperature, and 100 μL of competent cells (RBC Bioscience) was thawed at 4°C. Next, 2 μL of the thawed plasmid was added to the soluble cells, which were then incubated at 4°C for 10 minutes. After 1 minute of heat shock at 37°C, 700 μL of LB broth was added and incubated in a shaking incubator at 37°C for 15 minutes. After incubation was complete, 20 μL of the culture medium was added to an LB agar plate supplemented with 34 μg / mL chloramphenicol and spread evenly with a spreader. The treated agar plate was incubated for 16 hours in a 37°C incubator. Next, each colony formed on the agar plate was inoculated into 1.5 mL of LB broth supplemented with chloramphenicol and incubated in a shaking incubator at 37°C for 16 hours. After incubation, the culture medium was centrifuged at 13,000 rpm for 1 minute to remove the supernatant, and the remaining pellet was used to purify the plasmid using the DNA-spin Plasmid DNA Purification Kit (iNtRON) according to the provided protocol. The purified plasmids were then sequenced by Cosmogenetek. The primer sequences used for sequence analysis are listed in Table 1. Sequence analysis confirmed that SEPTIN2 and TXN matched NCBI accession numbers NM_001008491.2 and NM_003329.4, respectively, confirming that the plasmids containing the target genes, i.e., SEPTIN2 and TXN, had been successfully inserted.

[0027] [Table 1]

[0028] To mass-produce the pLenti-ORF plasmid, 500 μL of the transformed strain culture was inoculated into 100 mL of LB broth supplemented with 34 μg / mL chloramphenicol and incubated at 37°C for 16 hours with shaking. The culture was then transferred to a 500 mL centrifuge bottle (Nalgene) and centrifuged at 6000 xg for 15 minutes at 4°C. The supernatant was removed. The plasmid was purified from the pellet using a HiSpeed ​​Plasmid Midi Kit (Qiagen). The purified plasmid was quantified using a Life Science UV / vis Spectrophotometer DU 730 (Beckman Coulter). The results are shown in Table 2.

[0029] [Table 2]

[0030] Example 3: Production of lentivirus by transfection in the 293FT cell line The 293FT cell line, which is a cell line with high lentivirus production efficiency, was subcultured in the same manner as in Example 1 to obtain 2.5 x 10 6Cells were dispensed into 100 mm cell culture dishes. Cell saturation was confirmed to be 40-50% under a microscope. The results are shown in Figure 3. Next, for transfection, 1.5 mL of opti-MEM (Gibco) and 5 μg of pLenti-ORF plasmid were mixed in a 1.5 mL tube. Next, 6 μg of 0.5 μg / μL packaging plasmid (Origene) in distilled water was added and mixed, followed by 33 μL of TurboFectin (Origene). The mixture was incubated at room temperature for 15 minutes, then transferred to a cell culture dish and cultured for two days. The virus-containing culture supernatant was then collected and stored at 4°C. Fresh medium was added and cultured again for one day. The culture supernatant was then collected again, mixed with the medium collected the previous day, filtered through a 0.45 μm syringe filter (Sartorius), and stored at 4°C until use.

[0031] Example 4: Generation of transduced cell lines using lentivirus 4x10 subcultured in the same manner as in Example 1 6 SiMa (DSMZ, ACC164) cell lines were dispensed into 60 mm cell culture dishes containing 4 mL of RPMI 1640 (Gibco) culture medium supplemented with 10% FBS, 2 mM L-glutamine (Gibco), and 1% penicillin-streptomycin (Gibco) and cultured for 16 hours. After removing the culture medium, 1 mL of the lentivirus solution obtained using the same method as in Example 3 was mixed with 3 mL of fresh medium, and polybrene (Sigma-Aldrich) was added to a final concentration of 8 μg / mL. The following day, the culture medium was removed, fresh medium was added, and the cells were cultured for another day. The medium was then replaced with fresh culture medium supplemented with 1 μg / mL puromycin (Sigma-Aldrich). The medium was then replaced with puromycin-containing medium every 3–4 days, and transduced puromycin-resistant cells were selected. A simplified diagram of the method for generating lentivirus-transduced cell lines is shown in Figure 4.

[0032] Next, to separate the cultured cell lines into individual colonies, a cloning cylinder (Sigma-Aldrich) was used to physically separate the colonies within the culture dish, as shown in Figure 5. Specifically, the culture medium was removed, and the cells were washed with 3 mL of DPBS. After that, a cloning cylinder was attached to each colony, and the DPBS inside the cylinder was removed separately. Next, 50 μL of trypLE was added to each cloning cylinder and incubated in a 37°C, 5% CO2 incubator for 3 minutes. The trypLE and cell mixture was then mixed by pipetting and dispensed into a 96-well plate containing 150 μL of culture medium supplemented with 1 μg / mL puromycin. The cell lines were then named in the order in which they were dispensed. Subsequently, the cultures were scaled up and cultured according to cell growth.

[0033] Example 5: Verification of transduced cell lines 5.1. Verification of expression of introduced proteins To confirm the transduced cell line prepared in the same manner as in Example 4, the primarily expressed proteins were examined by Western blotting. More specifically, the medium was removed from the 100 mm cell culture dish where the cell line had been cultured at a scaled-up level, and the dish was washed with 5 mL of DPBS at 4°C. Next, the DPBS was removed, and the cells were washed with 5 mL of Complete PBS. TM The cells were treated with 200 μL of RIPA lysis buffer (iNtRON) containing EDTA-free Protease Inhibitor Cocktail (Roche). The cell lysate was transferred to a 1.5 mL tube using a cell lifter (SPL) and incubated at 4°C for 20 minutes without moving. The cells were then centrifuged at 17,000 rpm and 4°C for 30 minutes, and the supernatant was transferred to a new 1.5 mL tube. TMProtein content in the supernatant was quantified using a BCA Protein Assay Kit (ThermoFisher). Samples were then prepared for Western blotting by adding 4X Laemmli Sample Buffer (Bio-Rad) and heating at 100°C for 10 minutes. Western blotting was then performed using a 15% polyacrylamide gel. Proteins in the sample were separated by size using electrophoresis and transferred to an Immobilon-P PVDF membrane (Merck) at 100V for 1 hour. After protein transfer, the membrane was stained with Ponceau S (Sigma-Aldrich) to confirm the protein expression pattern. The membrane was immersed in 0.1% polysorbate 20 in PBS (PBST) and washed four times for 5 minutes each using a Digital Orbital Shaker (DAIHAN Scientific) to remove the Ponceau S. Blocking buffer (5% BSA in PBST) was then added and the membrane was blocked at room temperature for 1 hour using a Digital Orbital Shaker. When the target gene in the pLenti-C-Myc-DDK-P2A-Puro vector, i.e., the inserted gene, is expressed, Myc and DDK-TAG are co-expressed at the C-terminus of the expressed protein. Therefore, after blocking, the membrane was treated with a primary antibody that specifically binds to the Myc tag (Myc-tag, Cell Signaling Technology, 2278S, 1:1000 v / v in 2% BSA PBST) and incubated for 16 hours at 4°C using a digital orbital shaker. After the incubation, the membrane was immersed in PBST and washed four times for 5 minutes each using a digital orbital shaker. Then, it was treated with a secondary antibody (Anti-Rabbit HRP, Abcam, ab6721, 1:10000 v / v in PBST) and incubated for 1 hour at room temperature.The membrane was then immersed in PBST and washed four times for 5 minutes each using a Digital Orbital Shaker. After treatment with Pierce ECL Western Blotting Substrate (ThermoFisher), proteins were detected using ImageQuant LAS 500 (Cytiva). The results are shown in Figure 6.

[0034] As shown in Figure 6, expression of Thioredoxin or Septin-2 protein was confirmed in the transduced SiMa cell line. However, in the SEPTIN2-1 cell line, an unidentified band was detected in addition to Septin-2.

[0035] 5.2. Verification of gene transfer feasibility To confirm the introduction of the target gene into the transduced cell line prepared in the same manner as in Example 4, DNA was isolated from the cell line and the transgene region was amplified by PCR. PCR primers were designed using the 3'-terminal sequence of the transduced gene only to distinguish between the transduced gene and the endogenous gene. These primers were then prepared by Cosmogene Tech. The primer sequences used for PCR are shown in Table 3. As shown in Figure 7, the forward primers for amplifying the TXN or SEPTIN2 gene were designed to bind to the 5'-terminal sequence of the respective gene, and the reverse primer common to both genes was designed to bind to the DDK-tag sequence. Primers (GH20 and GH21) capable of amplifying the beta-globin gene were used as a positive control.

[0036] [Table 3]

[0037] For PCR, the transduced SiMa-TXN-3, SiMa-SEPTIN2-1, SiMa-SEPTIN2-2, and SiMa-SEPTIN2-3 cell lines were each plated into a 100 mm cell culture dish containing culture medium supplemented with 1 μg / mL puromycin. When cell saturation reached approximately 80%, DNA was extracted from each cell line using the Wizard® Genomic DNA Purification Kit (Promega) according to the provided protocol. The extracted DNA was quantified using a Life Science UV / Vis Spectrophotometer DU 730 (Beckman Coulter). The results are shown in Table 4.

[0038] [Table 4]

[0039] Next, 50 ng of DNA was mixed with 25 μL of 2X Platinum SuperFi PCR Master Mix, 2.5 μL each of 10 μM forward and reverse primers, and distilled water was added to a final volume of 50 μL to prepare the PCR sample. PCR conditions are shown in Table 5.

[0040] [Table 5]

[0041] After PCR was completed, 0.01% (v / v) EcoDye TM Electrophoresis was performed using a 1.5% (w / v) DNA agarose gel containing Nucleic Acid Staining Solution (Biofact) and the Mupid-One (Advance) electrophoresis kit. Electrophoresis was performed at 100 V for 35 minutes, and imaging was performed using the Gel Documentation System LSG1000 (iNtRON). The results are shown in Figure 8.

[0042] As shown in Figure 8, the beta globin gene (positive control) and the transduced TXN gene containing the DDK tag were all successfully amplified in the SiMa-TXN-3 cell line. Furthermore, the transduced SEPTIN2 gene containing the DDK tag was also successfully amplified in the SiMa-SEPTIN2-1, SiMa-SEPTIN2-2, and SiMa-SEPTIN2-3 cell lines. These results confirmed that transduced cell lines overexpressing the transduced genes were successfully constructed.

[0043] Example 6: Verification of BoNT / A biological activity by sandwich ELISA 6.1. Preparation of cell lysates from BoNT / A-treated SiMa-SEPTIN2-3 cells To confirm the cell-based biological activity verification system for botulinum toxin, we first prepared cell lysates from cells treated with botulinum toxin. As a control, SiMa cells were cultured at 1.2 x 10 cells per well in a 96-well plate containing RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin. 5 For the experimental group, the SiMa-SEPTIN2-3 cell line was plated at a concentration of 1.2 × 10 cells / 100 μL / well in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 μg / mL puromycin. 5 After culturing each cell line for 2 days, the medium was removed and 1X B-27 TM Plus Supplement (Gibco), 1X N-2 Supplement (Gibco), 2mM L-glutamine and 25μg / mL Trisialoganglioside GT 1B The cells were treated with 100 μL of differentiation medium containing Matreya (SEQ ID NO: 1) and cultured for 2 days to induce differentiation. After 2 days of differentiation, the medium was removed and 1X B-27 TMThe BoNT / A complex was serially diluted 1.35-fold in RPMI 1640 supplemented with 1X N-2 Supplement and 0.25% Human Serum Albumin (Green Cross), and the final volume was 100 μL. After 4 days of botulinum toxin treatment and incubation, the medium was removed, and each well was treated with 110 μL of lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, and 1% Triton X-100) supplemented with a protease inhibitor cocktail to lyse the cells. The cell lysate was then transferred to a 1.5 mL tube and centrifuged at 17,000 rpm at 4°C for 5 minutes to obtain the supernatant. This cell lysate was then used for sandwich ELISA.

[0044] 6.2. Confirmation of EC50 using sandwich ELISA Botulinum toxin serotype A (BoNT / A) is known to act at the presynapse of the neuromuscular junction, causing cleavage of the Synaptosomal-Associated Protein, 25 kDa (SNAP-25) molecule, which is bound to the presynaptic plasma membrane. Based on this principle, we designed a sandwich enzyme-linked immunosorbent assay (ELISA) using antibodies that recognize cleaved SNAP-25 and intact SNAP-25, respectively. The principle is shown in Figure 9.

[0045] Mouse Synaptosomal Protein 25kDa (SNAP-25, aa183-197) cleaved monoclonal antibody (Mybiosource, MBS312597), which specifically binds to cleaved SNAP-25, was diluted to a concentration of 0.2% (v / v) in 0.1M sodium carbonate coating buffer (pH 9.6) and 100μL was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and incubated at 4℃ for 16 hours without moving. Unbound antibody was then removed from the wells, and the plates were washed three times with 200μL of 0.1% polysorbate 20 in PBS (PBST). Next, 200μL of blocking buffer (5% BSA in PBST) was applied to each well and placed on a shaker at 75 rpm for 1 hour at room temperature. After 1 hour, the blocking buffer was removed, and the wells were washed three times with 200 μL of PBST. 90 μL of cell lysate obtained by the same method as in Example 6.1 was added, and the wells were incubated at room temperature for 2 hours on a shaker at 75 rpm. The cell lysate was then removed, and the wells were washed three times with 200 μL of PBST. The detection antibody, anti-SNAP-25 antibody (Sigma-Aldrich, S9684), was diluted to a concentration of 0.1% (v / v) in the blocking buffer, and 100 μL of the diluted solution was added to each well. The wells were incubated at room temperature for 1 hour on a shaker at 75 rpm. The plate was then washed three times with 200 μL of PBST to remove all unbound antibody. The secondary antibody, Anti-Rabbit HRP (Abcam, ab6721), was diluted to 0.01% (v / v) using blocking buffer, and 100 μL of the diluted antibody solution was applied to each well. The plate was then incubated at room temperature for 1 hour on a shaker at 75 rpm.After washing three times with 200 μL of PBST, the TMB solution and hydrogen peroxide solution from the TMB Peroxidase EIA Substrate Kit (Bio-Rad) were mixed at a 9:1 ratio, and 50 μL was added to each well and incubated in a 37°C incubator for 30 minutes. Then, 50 μL of 2N sulfuric acid was added to each well, and the absorbance (OD450) at 450 nm was measured using a SpectraMax Plus 384 Microplate Reader (Molecular Devices). A dose-response curve (four parameters) was created using GraphPad Prism Version 7.00 (GraphPad Software, Inc.) from the obtained raw data, and the EC was calculated based on the results of our mouse titer test (units). 50 The values ​​were calculated. All experiments were then repeated at least three times, and the results were expressed as the mean ± standard deviation. The results are shown in Figure 10.

[0046] As shown in Figure 10, the EC of the control SiMa cell line 50 EC50 of SEPTIN2-3 transduced cell lines was 7.35 U / mL (0.49 pM). 50 The value of 5.38 U / mL (0.36 pM) was confirmed, confirming that sensitivity can be improved by overexpressing SEPTIN2.

[0047] In addition, to compare the differences in sensitivity between cell lines in more detail, the OD of each group of SiMa cell lines was 450nm The OD values ​​for each group of SEPTIN2-3 cell lines were calculated. 450nm The results are shown in Figure 11.

[0048] As shown in Figure 11, the OD450 without toxin treatment SEPTIN2 / OD450 WTThe value of β-glucan was 1, but when treated with toxin, the value rose to approximately 1.7, indicating a difference in sensitivity to the toxin between the cell lines. This confirmed that the transfected cell lines overexpressing the SEPTIN2 gene had increased sensitivity to botulinum toxin compared to the control group (wild type).

[0049] 6.3. Preparation of cell lysates from BoNT / A-treated SiMa-Thioredoxin cells To confirm the cell-based biological activity verification system for botulinum toxin, we first prepared cell lysates from cells treated with botulinum toxin. As a control, SiMa cells were cultured at 8 × 10 cells per well in a 96-well plate containing RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin. 4 The experimental group was cultured in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 μg / mL puromycin at a concentration of 8 × 10 cells / 100 μL / well. 4 After culturing each cell line for 2 days, the medium was removed and 1X B-27 TM Plus Supplement (Gibco), 1X N-2 Supplement (Gibco), 2mM L-glutamine and 25μg / mL Trisialoganglioside GT 1BCells were treated with 100 μL of differentiation medium supplemented with Matreya (Matreya) and cultured for 2 days to induce differentiation. After 2 days of differentiation, the medium was removed, and 100 μL of BoNT / A complexes were prepared in RPMI 1640 at concentrations of 0, 1, 5, 10, 50, 100, 500, 1000, and 2000 pM, respectively, and added to each well. After 4 days of culture, the medium was removed, and cells were lysed by adding 110 μL of lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, and 1% Triton X-100) supplemented with a protease inhibitor cocktail to each well. The cell lysates were then transferred to 1.5 mL tubes and centrifuged at 17,000 rpm at 4°C for 5 minutes to obtain the supernatant, which was then used to prepare the cell lysates for sandwich ELISA.

[0050] 6.4. Confirmation of EC50 using sandwich ELISA The sensitivity of the transfected cell line overexpressing the TXN gene to botulinum toxin was confirmed using the cell lysate of Example 6.3 in the same manner as in Example 6.2. The results are shown in Figure 12.

[0051] As shown in FIG. 12, it was confirmed that the transfected cell line overexpressing the thioredoxin gene had increased sensitivity to botulinum toxin compared to the control group (wild type).

[0052] Example 7: Verification of the biological activity of BoNT / A pharmaceutical compositions (raw drug products) by sandwich ELISA To confirm whether the cell-based botulinum toxin biological activity verification system can also be applied to botulinum toxin pharmaceutical compositions (raw drug substances), sandwich ELISA was performed using the SiMa-SEPTIN2-3 cell line. More specifically, the SiMa-SEPTIN2-3 cell line was cultured at 1.2 × 10 cells per well in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 μg / mL puromycin. 5After culturing for 2 days, the medium was removed and 1X B-27 TM Plus Supplement, 1X N-2 Supplement, 2mM L-glutamine and 25μg / ml Trisialoganglioside GT 1B After 2 days of differentiation, the medium was removed and the cells were treated with 100 μL of 1X B-27. TM BoNT / A complex (botulinum toxin, a raw drug derived from ATGC-100, free of other substances such as stock solutions) was serially diluted 1.55-fold in RPMI 1640 supplemented with 1X Plus Supplement and 1X N-2 Supplement and treated with different concentrations. After 4 days of botulinum toxin treatment and incubation, the medium was removed, and 110 μL of lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, and 1% Triton X-100) supplemented with a protease inhibitor cocktail was added to each well to lyse the cells. The cell lysate was then transferred to a 1.5 mL tube and centrifuged at 17,000 rpm at 4°C for 5 minutes to obtain the supernatant, which was then used to prepare the cell lysate for sandwich ELISA.

[0053] Next, a capture antibody specifically binding to cleaved SNAP-25, Mouse Synaptosomal Protein 25kDa (SNAP-25, aa183-197) Cleaved Monoclonal Antibody (Mybiosource), was diluted to a concentration of 0.8% (v / v) in 0.1M sodium carbonate coating buffer (pH 9.6). 100 μL was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and incubated at 4°C for 16 hours without moving. Unbound antibody was then removed from the wells, and the plates were washed three times with 200 μL of 0.1% polysorbate 20 in PBS (PBST). Next, 200 μL of blocking buffer (5% BSA in PBST) was applied to each well and placed on a shaker at 75 rpm for 2 hours at room temperature. After 2 hours, the blocking buffer was removed, and 90 μL of the resulting cell lysate was added. The wells were shaken at 75 rpm at room temperature for 2 hours. The cell lysate was then removed, and the wells were washed three times with 200 μL of PBST. The detection antibody, anti-SNAP-25 antibody (Sigma-Aldrich, S9684), was diluted to 0.1% (v / v) in blocking buffer, and 100 μL of the solution was added to each well. The wells were shaken at 75 rpm at room temperature for 1 hour. The wells were then washed three times with 200 μL of PBST to remove any unbound antibody. The secondary antibody, anti-rabbit HRP (Abcam, ab6721), was diluted to 0.01% (v / v) in blocking buffer, and 100 μL of the diluted antibody solution was added to each well. The wells were shaken at 75 rpm at room temperature for 1 hour. Next, after washing three times with 200 μL of PBST, 100 μL of TMB (ThermoFisher, 34028) solution was added to each well, and the reaction was carried out at room temperature for 15 minutes.After that, 100 μL of 2N sulfuric acid was added to each well, and the absorbance at 450 nm (OD450) was measured using a SpectraMax Plus 384 Microplate Reader (Molecular Devices). A dose-response curve (four parameters) was created using GraphPad Prism Version 7.00 (GraphPad Software, Inc.) from the obtained raw data, and the EC200 was calculated based on the results of our mouse titer test (units). 50 The results are shown in Figure 13.

[0054] As shown in Figure 13, it was confirmed that the amount of SNAP-25 cleaved increased in a botulinum toxin dose-dependent manner. 50 The value was confirmed to be 16.98 U / mL.

[0055] Example 8: Verification of the biological activity of BoNT / A pharmaceutical compositions (finished drugs) by sandwich ELISA To confirm whether the cell-based botulinum toxin biological activity verification system can be applied to other botulinum toxin pharmaceutical compositions (finished drugs), sandwich ELISA was performed using the SiMa-SEPTIN2-3 cell line. More specifically, the SiMa-SEPTIN2-3 cell line was cultured at 1.2 × 10 cells per well in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 μg / mL puromycin. 5 After culturing for 2 days, the medium was removed and 1X B-27 TM Plus Supplement, 1X N-2 Supplement, 2mM L-glutamine and 25μg / ml Trisialoganglioside GT 1B After 2 days of differentiation, the medium was removed and the cells were treated with 100 μL of 1X B-27. TMThe BoNT / A complete drug product (ATGC-100) was serially diluted 1.4 times in RPMI 1640 supplemented with 1X Plus Supplement and 1X N-2 Supplement, and the cells were treated with different concentrations. After 4 days of botulinum toxin treatment and incubation, the medium was removed, and 110 μL of lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, and 1% Triton X-100) supplemented with a protease inhibitor cocktail was added to each well to lyse the cells. The cell lysates were then transferred to 1.5 mL tubes and centrifuged at 17,000 rpm at 4°C for 5 minutes to obtain the supernatant, which was then used to prepare the cell lysates for sandwich ELISA.

[0056] Next, a capture antibody specifically binding to cleaved SNAP-25, Mouse Synaptosomal Protein 25kDa (SNAP-25, aa183-197) Cleaved Monoclonal Antibody (Mybiosource), was diluted to a concentration of 0.8% (v / v) in 0.1M sodium carbonate coating buffer (pH 9.6). 100 μL was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and incubated at 4°C for 16 hours without moving. Unbound antibody was then removed from the wells, and the plates were washed three times with 200 μL of 0.1% polysorbate 20 in PBS (PBST). Next, 200 μL of blocking buffer (5% BSA in PBST) was applied to each well and placed on a shaker at 75 rpm for 2 hours at room temperature. After 2 hours, the blocking buffer was removed, and 90 μL of the resulting cell lysate was added. The wells were shaken at 75 rpm at room temperature for 2 hours. The cell lysate was then removed, and the wells were washed three times with 200 μL of PBST. The detection antibody, anti-SNAP-25 antibody (Sigma-Aldrich, S9684), was diluted to 0.1% (v / v) in blocking buffer, and 100 μL of this solution was added to each well. The wells were shaken at 75 rpm at room temperature for 1 hour. The wells were then washed three times with 200 μL of PBST to remove any unbound antibody. The secondary antibody, anti-rabbit HRP (Abcam, ab6721), was diluted to 0.01% (v / v) in blocking buffer, and 100 μL of the diluted antibody solution was added to each well. The wells were shaken at 75 rpm at room temperature for 1 hour. Next, after washing three times with 200 μL of PBST, 100 μL of TMB (ThermoFisher, 34028) solution was added to each well, and the plate was allowed to react at room temperature for 15 minutes.After that, 100 μL of 2N sulfuric acid was added to each well, and the absorbance at 450 nm (OD450) was measured using a SpectraMax Plus 384 Microplate Reader (Molecular Devices). A dose-response curve (four parameters) was created using GraphPad Prism Version 7.00 (GraphPad Software, Inc.) from the obtained raw data, and the EC200 was calculated based on the results of our mouse titer test (units). 50 The results are shown in Figure 14.

[0057] As shown in Figure 14, it was confirmed that the amount of SNAP-25 cleaved increased in a botulinum toxin dose-dependent manner. 50 The value was confirmed to be 5.42 U / mL.

[0058] From the above results, as shown in Figure 15, it was confirmed that by using a cell line overexpressing the SEPTIN2 or TXN gene using the lentivirus of the present invention, the potency of not only botulinum toxin but also botulinum toxin pharmaceutical compositions, i.e., raw drug and finished drug, can be easily measured on a cell basis, and the sensitivity can be significantly improved. Therefore, it is expected that the cell line of the present invention can be applied to various industrial fields that use botulinum toxin.

[0059] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. [Industrial Applicability]

[0060] The cells for measuring botulinum toxin activity of the present invention have significantly improved sensitivity to botulinum toxin, and can therefore be widely used in various industrial fields to measure the activity of not only botulinum toxin but also botulinum toxin pharmaceutical compositions in the form of raw pharmaceuticals or finished pharmaceuticals, replacing the numerous animal experiments.

Claims

1. A cell line for measuring botulinum toxin activity that overexpresses SEPTIN2 or thioredoxin (TXN), the cell is any one selected from the group consisting of SiMa cells, LAN-2 cells, PC12 cells, Neuro-2a cells, LA1-55n cells, N18 cells, SH-SY5Y cells, Kelly cells, NB69 cells, N1E-115 cells, BE(2)-M17 cells, and SK-N-BE(2) cells; the cells are characterized by increased susceptibility to botulinum toxin intoxication; The botulinum toxin is any one selected from botulinum serotypes A, C, and E.

2. The cell for measuring botulinum toxin activity according to claim 1, wherein the cell is transduced with a SEPTIN2 or TXN gene and overexpressed.

3. a) treating the cells for measuring botulinum toxin activity according to claim 1 with botulinum toxin and culturing the cells; b) lysing the cultured cells and collecting a cell lysate; c) measuring the amount of SNAP-25 cleavage products in the cell lysate.

4. The method for measuring botulinum toxin activity according to claim 3, wherein the amount of the SNAP-25 cleavage product is measured using a sandwich immunoassay (ELISA) or Western blot.

5. A kit for measuring botulinum toxin activity, comprising the cells for measuring botulinum toxin activity according to claim 1 as an active ingredient.

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