WO2022250405A1 - 렌티바이러스에 의해 특정 유전자가 삽입된 보툴리눔 독소에 민감한 세포 - Google Patents
렌티바이러스에 의해 특정 유전자가 삽입된 보툴리눔 독소에 민감한 세포 Download PDFInfo
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- WO2022250405A1 WO2022250405A1 PCT/KR2022/007323 KR2022007323W WO2022250405A1 WO 2022250405 A1 WO2022250405 A1 WO 2022250405A1 KR 2022007323 W KR2022007323 W KR 2022007323W WO 2022250405 A1 WO2022250405 A1 WO 2022250405A1
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a cell for measuring botulinum toxin activity and a method for measuring botulinum toxin activity using the same.
- Botulinum toxin is a neurotoxin produced by Clostridium botulinum, a gram-positive anaerobic bacterium that grows on rotten canned food and spoiled meat. It is classified into 8 neurotoxins, 7 of which (A, B, C, D, E, F, G) can cause nerve paralysis.
- the size is about 150 kDa, and it is composed of a non-toxin protein complex in addition to the botulinum toxin protein, and the size of each complex is generated up to 900 kDa depending on the type of neurotoxin.
- the type of action, target, and active period are different.
- botulinum toxin type A is known as one of the deadliest biological agents. This botulinum toxin blocks the signal that causes muscle spasm or contraction to cause paralysis. Since being approved by the US FDA in 1989 as a biological agent based on this function, it has been widely used for therapeutic or cosmetic purposes. .
- diseases such as strabismus, torticollis, blepharospasm, inability to relax, dentition, back pain, etc., and for cosmetic purposes, wrinkles, frown lines, square jaw treatment, hyperhidrosis, etc. is used for the treatment of
- mouse LD 50 a lethality test using a mouse.
- the unit on the label of the pharmaceutical preparation is the mouse LD 50 unit.
- it has limitations such as the large number of mice required, the high cost for the test, and the difficulty in seeing differences according to the serotype of botulinum toxin ( Korean Patent Publication No. 10-2012-0134154).
- the present invention was made to solve the above problems in the prior art, and an object of the present invention is to provide a cell for measuring botulinum toxin activity and a method for measuring botulinum toxin activity using the same.
- These cells are cells overexpressing SEPTIN2 or Thioredoxin (TXN) genes, and can evaluate all of botulinum toxin binding, cellular uptake, cytoplasmic translocation and protease activity, as well as significantly improved sensitivity to botulinum toxin, resulting in low
- a dose of botulinum toxin can also be verified with high accuracy.
- the activity of the botulinum toxin pharmaceutical composition in the form of raw materials and finished drugs can also be measured.
- the present invention provides cells for measuring botulinum toxin activity overexpressing SEPTIN2 or Thioredoxin (TXN).
- the cell may overexpress the gene by inserting the SEPTIN2 or TXN gene into the cell, preferably by a method such as transduction or transfection, but generally known It is not limited thereto as long as it is a method of inserting genes into existing cells.
- the SEPTIN2 is preferably a human SEPTIN2 gene, more preferably a gene expressing a 3,726 bp mRNA sequence of NCBI accession number NM_001008491.2, but variants thereof are also included in the scope of the present invention. Specifically, it may include a sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more to the mRNA sequence of NM_001008491.2.
- Percentage of sequence homology is determined by comparing the optimally aligned sequence to the comparison region, wherein a portion of the nucleotide sequence in the comparison region is added or added compared to the reference sequence (not including additions or deletions) to the optimal alignment of the sequences. It may contain deletions (ie gaps).
- the TXN is preferably a human TXN gene, more preferably a gene expressing a 737bp mRNA sequence of NCBI accession number NM_003329.4, but variants thereof are also included in the scope of the present invention.
- sequence homology may include a sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more to the mRNA sequence of NM_003329.4. “Percentage of sequence homology” is determined by comparing the optimally aligned sequence to the comparison region, wherein a portion of the nucleotide sequence in the comparison region is added or added compared to the reference sequence (not including additions or deletions) to the optimal alignment of the sequences. It may contain deletions (ie gaps).
- the cells are characterized in that their susceptibility to botulinum toxin intoxication is increased compared to wild type cells due to overexpression of SEPTIN2 or TXN genes.
- the cells are preferably SiMa cells, LAN-2 cells, PC12 cells, Neuro-2a cells, LA1-55n cells, N18 cells, SH-SY5Y cells, Kelly cells, NB69 cells .
- the botulinum toxin may be selected from the group consisting of botulinum serotypes A, B, C, D, E, F and G.
- the present invention comprises the steps of a) treating and culturing the botulinum toxin to the cells for measuring the activity of the botulinum toxin; b) lysing the cultured cells and collecting the cell lysate; and c) measuring the amount of SNAP-25 cleavage product in the cell lysate.
- the amount of the SNAP-25 cleavage product is characterized by measuring using a sandwich immunoassay (ELISA), Western blot, etc., but is known as a method used to detect proteins If there is a method, it is not limited thereto.
- ELISA sandwich immunoassay
- Western blot etc.
- the present invention provides a kit for measuring botulinum toxin activity, comprising the cell for measuring botulinum toxin activity as an active ingredient.
- the present invention provides a use for measuring botulinum toxin activity of the cell for measuring botulinum toxin activity.
- Cells for measuring botulinum toxin activity according to the present invention have significantly improved sensitivity to botulinum toxin by overexpressing SEPTIN2 or Thioredoxin, so even low doses of botulinum toxin can be verified with high accuracy, as well as botulinum toxin binding and cell absorption , it is expected to be able to replace a number of animal experiments because it can evaluate both translocation into the cytoplasm and protease activity.
- the activity of not only botulinum toxin but also botulinum toxin pharmaceutical compositions in the form of raw materials and finished drugs can be measured, it is expected that the method can be easily applied to industries in various fields using botulinum toxin.
- TXN Thioredoxin
- FIG. 2 is a diagram briefly illustrating a pLenti-C-Myc-DDK-P2A-Puro vector map.
- FIG 3 is a view showing the results of microscopic examination of the cultured 293FT cell line according to an embodiment of the present invention.
- FIG. 4 is a schematic diagram showing a method for preparing a transduced cell line using a lentivirus according to an embodiment of the present invention.
- FIG. 5 is a view showing a method of separating and culturing cell lines into individual colonies using a cloning cylinder according to an embodiment of the present invention.
- FIG. 6 is a view showing the results of Western blotting for proteins expressed in the transduced cell line according to an embodiment of the present invention.
- FIG. 7 is a diagram showing binding sites of primers prepared for PCR according to an embodiment of the present invention.
- FIG. 8 is a view showing the result of confirming the introduced gene by PCR according to an embodiment of the present invention.
- FIG. 9 is a schematic diagram showing the principle of sandwich ELISA.
- FIG. 10 is a diagram showing the result of confirming the sensitivity to botulinum toxin between a control group and a cell line transduced with the SEPTIN2 gene according to an embodiment of the present invention by comparing the amount of SNAP-25 cut.
- 11 is a view showing the result of confirming the sensitivity to botulinum toxin between a control group and a transduced cell line according to an embodiment of the present invention through a raw data ratio between each group.
- FIG. 12 is a view showing the result of confirming the sensitivity to botulinum toxin between a control group and a cell line transduced with a Thioredoxin gene according to an embodiment of the present invention by comparing the amount of SNAP-25 cut.
- FIG. 13 is a diagram showing the results of measuring the activity of a botulinum toxin pharmaceutical composition (raw drug product) according to an embodiment of the present invention using a cell line transduced.
- FIG. 14 is a diagram showing the results of measuring the activity of a botulinum toxin pharmaceutical composition (finished drug product) according to an embodiment of the present invention using a cell line transduced.
- 15 is a diagram briefly showing the entire development process of the present invention.
- the present inventors have prepared a cell line with improved sensitivity for measuring botulinum toxin activity, and confirmed that the activity of botulinum toxin can be stably measured using the cell line of the present invention. has been completed.
- a "botulinum toxin” may be produced bacterially or by recombinant techniques, but includes any known type of botulinum toxin and any subsequently discovered type, including engineered variants or fusion proteins.
- Botulinum toxin is classified into 8 neurotoxins, and 7 types of botulinum toxin serotypes A, B, C, D, E, F and G can cause nerve paralysis. These proteins are divided into proteins that contain complexes and proteins that do not contain complexes.
- the pure toxin protein has a molecular weight of 150 kDa, and various proteins are produced, such as 300 kDa, 500 kDa, and 900 kDa, depending on the presence or absence of complex formation.
- the botulinum toxin of the present invention may alternatively be a botulinum toxin derivative, ie, a compound having botulinum toxin activity but containing one or more chemical or functional modifications relative to the native or recombinant, native botulinum toxin.
- a botulinum toxin can be a modified neurotoxin (e.g., a neurotoxin having one or more amino acid deletions, modifications or substitutions relative to the original or recombinantly produced neurotoxin, derivative or fragment thereof).
- a botulinum toxin can be a botulinum toxin that has been modified in a way that enhances its properties or reduces its undesirable side effects, but still retains the desired botulinum toxin activity.
- the botulinum toxin may be a toxin produced using recombinant or synthetic chemical techniques (e.g., a recombinant peptide, fusion protein, or hybrid neurotoxin prepared from subunits or domains of different botulinum toxin serotypes). (See, eg, US Pat. No. 6,444,209)).
- a botulinum toxin may also be part of an entire molecule that has been shown to have the requisite botulinum toxin activity, and in such cases, is used as such or as part of a combination or conjugate molecule, such as a fusion protein. It can be.
- the botulinum toxin may be in the form of a precursor of botulinum toxin, which may itself be non-toxic, such as a non-toxic zinc protease, which may become toxic upon proteolytic degradation.
- cell means all eukaryotic cells susceptible to botulinum toxin poisoning by botulinum toxin, or all eukaryotic cells capable of absorbing botulinum toxin.
- Eukaryotic cells refer to cells derived from various mammals, such as, for example, mice, rats, pigs, cattle, sheep, horses, primates and humans.
- manufactured cell line is synonymous with an established cell line, an immortal cell line, or a transformed cell line, and means a cell selected for infinite proliferation.
- the transformed cell line disclosed herein exhibits consistent sensitivity to botulinum toxin activity over multiple cell passages, and "sensitivity to botulinum toxin activity" refers to a signal detected by a non-treated control or background signal. It means the lowest concentration of botulinum toxin that can be measured.
- vector means a DNA fragment, nucleic acid molecule, etc. delivered into a cell, and the vector replicates DNA and can be independently remanufactured in a host cell. May be used interchangeably with the term “delivery vehicle”.
- Expression vector means a recombinant DNA molecule comprising a coding sequence of interest and appropriate nucleic acid sequences necessary to express an operably linked coding sequence in a particular host organism.
- the recombinant vector or plasmid of the present invention includes a gene encoding SEPTIN2 or Thioredoxin, and thus refers to all vectors capable of overexpressing SEPTIN2 or Thioredoxin in cells.
- kit means a device capable of measuring the activity of botulinum toxin by including the cell for measuring botulinum toxin activity of the present invention, and includes botulinum toxin itself, raw material drug of botulinum toxin, and finished drug of botulinum toxin. It can be used to measure the activity of botulinum toxin, such as, etc., and the kit of the present invention may further include a cell lysing agent for lysing cells, antibodies and reagents for ELISA, a manual, etc. in addition to the cells for measuring the botulinum toxin activity of the present invention. However, if it can be used in the botulinum toxin activity measurement method of the present invention, it may be additionally included.
- Example 1 Cultivation of cell line 293FT for lentivirus production
- 293FT a cell line commonly used for lentivirus production, is derived from the 293F cell line, and is a cell line that stably expresses the SV40 large T antigen through pCMVSPORT6TAg.neo plasmid, and is a representative cell line used for lentivirus production. Since the 293FT cell line encodes the neomycin resistance gene in the plasmid, it was cultured using a medium containing geneticin, a neomycin analog, during all cell cultures except for cell thawing.
- 293FT cell stock (Thermo, R700-07) was mixed with DMEM ( Gibco) medium and centrifuged at 200xg for 3 minutes to remove the supernatant and obtain the cells.
- the obtained cells were resuspended using a culture medium, then dispensed into a 100 mm cell culture dish and cultured in a 37°C, 5% CO 2 incubator for 24 hours. Afterwards, the culture medium was removed and 10% FBS (Gibco), 1X NEAA (Gibco), 2 mM L-glutamine (Gibco), 1% penicillin-streptomycin (Gibco) and 500 ⁇ g/mL geneticin selective antibiotic (Gibco) were added.
- DMEM (Gibco) medium After replacement with the DMEM (Gibco) medium, it was cultured. And when the cell confluency increased to 90% or more, subculture was performed again. In the subculture, the existing culture medium was first removed and 5 mL of DPBS (Gibco) was added, which is 50% of the culture volume, and washed with water. Then, DPBS was removed, 2 mL of 20% of the culture volume of TrypLE (Gibco) was added, and reacted for 2 minutes in a 37°C, 5% CO 2 incubator. After the reaction was completed, a culture medium was additionally added, and the supernatant was removed by centrifugation at 800xg and 4° C. for 2 minutes.
- DPBS DPBS
- TrypLE TrypLE
- the number of cells is measured using trypan blue (Gibco) and a hematocytometer, and subcultured by dispensing 2x10 6 cells with 10 mL of culture medium in a 100 mm cell culture dish. And subculture was carried out in the same way as above every 3 to 4 days.
- Example 2 Gene selection and coding plasmid construction for cell line production susceptible to BoNT/A poisoning
- SEPTIN2 (SEPT2) serves to protect BoNT / A from degradation in cells, and Thioredoxin (TXN) acts to separate the disulfide bond between the heavy and light chains of BoNT / A. It is known.
- Origene pLenti-ORF plasmids (SEPTIN2: RC224864L3, TXN: RC208876L3) into which the genes encoding each protein were inserted were inserted into the pLenti-C-Myc-DDK-P2A-Puro vector. It was commissioned by the company.
- SEPTIN2 the amino acid sequence of SEQ ID NO: 9 (DNA sequence: SEQ ID NO: 10) was used, and to prepare Thioredoxin, the amino acid sequence of SEQ ID NO: 11 (DNA sequence: SEQ ID NO: 12) was used.
- the pLenti-C-Myc-DDK-P2A-Puro vector map is shown in FIG. 2 . After the tube containing the lyophilized pLenti-ORF plasmid was centrifuged at 5000xg for 3 minutes, 100 ⁇ L of distilled water was added, pipetted, and stored in a -20°C freezer until use.
- the plasmid was thawed at room temperature, and 100 ⁇ L of competent cells (RBC bioscience) was thawed at 4°C.
- 2 ⁇ L of the thawed plasmid was treated with water-soluble cells, reacted at 4 ° C for 10 minutes, subjected to heat shock at 37 ° C for 1 minute, and then added with 700 ⁇ L of LB broth and stirred in a 37 ° C shaking incubator for 15 minutes. cultured for a while.
- the culture medium was centrifuged at 13,000 rpm for 1 minute to remove the supernatant, and the remaining pellet was used for plasmid purification using the DNA-spin Plasmid DNA Purification Kit (iNtRON) according to the provided protocol.
- the nucleotide sequence of the purified plasmid was analyzed by requesting Cosmogenetech. Primer sequences used for sequencing were shown in Table 1. As a result of sequencing, it was confirmed that SEPTIN2 exactly matches NCBI's accession number NM_001008491.2 and TXN exactly matches NM_003329.4. Through this, it was confirmed that SEPTIN2 or TXN, that is, a plasmid in which the target gene was normally inserted was constructed. did
- Primer sequence (5' -> 3') sequence number SEPTIN2 V2 AGAGCTCGTTTAGTGAA
- the plasmid was inoculated into 100 mL of LB broth supplemented with 34 ⁇ g/mL of chloramphenicol, followed by shaking culture at 37° C. for 16 hours. Then, the culture solution was transferred to a 500 mL Centrifuge bottle (Nalgene), centrifuged at 6000xg, 4° C. for 15 minutes, and the supernatant was removed. Then, the plasmid was purified from the pellet using the 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.
- 293FT cell line which is a cell line with high lentivirus production efficiency, was subcultured in the same manner as in Example 1, and 2.5x10 6 cells were dispensed into a 100 mm cell culture dish. And it was confirmed under a microscope that cell saturation reached 40-50%.
- Figure 3 For transfection, 1.5 mL of opti-MEM (Gibco) and 5 ⁇ g of pLenti-ORF plasmid were added to a 1.5 mL tube and mixed. Then, 6 ⁇ g of 0.5 ⁇ g/ ⁇ L packaging plasmid (Origene) contained in distilled water was added and mixed, and then 33 ⁇ L of TurboFectin (Origene) was additionally added and mixed.
- the mixed solution was reacted at room temperature for 15 minutes, then treated in a cell culture dish and cultured for 2 days.
- the supernatant of the medium containing the virus was collected and stored at 4 ° C., and a new medium was added and cultured for another 1 day.
- the medium supernatant was collected again, mixed with the medium recovered the day before, filtered using a 0.45 ⁇ m pore filter (Sartorius), and stored at 4° C. until use.
- Example 4 Construction of transduced cell lines using lentivirus
- SiMa (DSMZ, ACC164) cell lines subcultured in the same manner as in Example 1 were cultured in RPMI1640 (Gibco) supplemented with 10% FBS, 2 mM L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). It was dispensed into a 60 mm cell culture dish to which 4 mL of medium was added and cultured for 16 hours.
- the trypLE and the cell mixture were mixed by pipetting, and then each was dispensed into a 96-well plate containing 150 ⁇ L of culture medium supplemented with 1 ⁇ g/mL of puromycin.
- the cell lines were named in the order of distribution. Thereafter, the cells were scaled up and cultured according to the growth of the cells.
- the primarily expressed protein was confirmed using Western blotting.
- the medium of the 100 mm cell culture dish cultured by scale-up was removed, and washed with 5 mL of 4° C. DPBS.
- DPBS washed with 5 mL of 4° C.
- cells were treated with 200 ⁇ L of RIPA lysis buffer (iNtRON) supplemented with cOmpleteTM and EDTA-free Protease Inhibitor Cocktail (Roche). After transferring the cell lysate to a 1.5 mL tube using a cell lifter (SPL), it was reacted at 4°C for 20 minutes without movement.
- SPL cell lifter
- the membrane after blocking was treated with a primary antibody (Myc-tag, Cell Signaling Technology, 2278S, 1:1000 v/v in 2% BSA PBST) that specifically binds to the Myc tag, and then subjected to a Digital Orbital Shaker at 4°C. was reacted for 16 hours. After the reaction was completed, the membrane was soaked in PBST and washed 4 times for 5 minutes each using a Digital Orbital Shaker, and then treated with secondary antibodies (Anti-Rabbit HRP, abcam, ab6721, 1:10000 v/v in PBST) at room temperature. reacted for 1 hour.
- a primary antibody Myc-tag, Cell Signaling Technology, 2278S, 1:1000 v/v in 2% BSA PBST
- secondary antibodies Anti-Rabbit HRP, abcam, ab6721, 1:10000 v/v in PBST
- the membrane was immersed in PBST, washed four times for 5 minutes each using a Digital Orbital Shaker, treated with Pierce ECL Western Blotting Substrate (ThermoFisher), and then protein was detected with ImageQuant LAS 500 (Cytiva). The results are shown in FIG. 6 .
- genomic DNA was isolated from the cell line and then the transgene portion was amplified using PCR.
- primers for PCR were designed using the 3'-terminal sequence of the transduced gene only, and then commissioned to CosmoGenetech to manufacture. did Primer sequences used for PCR are shown in Table 3.
- forward primers for amplification of the TXN or SEPTIN2 genes were designed to bind to the 5'-end sequence of each gene, and the reverse primer common to both genes was DDK- It was designed to bind to the tag sequence.
- primers GH20 and GH21 capable of amplifying the ⁇ -globin gene were used as positive controls.
- the transduced SiMa-TXN-3, SiMa-SEPTIN2-1, SiMa-SEPTIN2-2, and SiMa-SEPTIN2-3 cell lines were each 1 ⁇ g/mL of puromycin added to the culture medium containing 100 ml. Divided into mm cell culture dishes, and when the cell confluency reached about 80%, the genomic DNA of each cell line was extracted using the Wizard® Genomic DNA Purification Kit (Promega) according to the protocol provided, and the extracted genomic DNA was It was quantified using Life Science UV/Vis Spectrophotometer DU 730 (Beckman Coulter). The results are shown in Table 4.
- Electrophoresis was performed using a 1.5% (w/v) DNA agarose gel mixed with 0.01% (v/v) EcoDyeTM Nucleic Acid Staining Solution (Biofact) and the electrophoresis kit Mupid-One (Advance). moved Electrophoresis was performed at 100 V for 35 minutes, and imaging was performed using a Gel Documentation System LSG 1000 (iNtRON). The results are shown in FIG. 8 .
- the beta globin gene corresponding to the positive control and the TXN gene including the transduced DDK-tag were normally amplified in the SiMa-TXN-3 cell line.
- all of the SEPTIN2 genes including the transduced DDK-tag were amplified in the cell lines of SiMa-SEPTIN2-1, SiMa-SEPTIN2-2, and SiMa-SEPTIN2-3.
- a cell lysate of cells treated with the botulinum toxin was first prepared.
- the SiMa cell line was cultured in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin at 1.2 X 10 5 cells/100 ⁇ L/
- the experimental group SiMa-SEPTIN2-3 cell line was placed in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 ⁇ g/mL puromycin.
- the medium was removed, and the BoNT/A complex was serially diluted 1.35 times in RPMI1640 supplemented with 1X B-27TM Plus Supplement, 1X N-2 Supplement, and 0.25% Human Serum Albumin (Green Cross). and finally added to each well to make 100 ⁇ L.
- the medium was removed and lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton X-100) supplemented with protease inhibitor cocktail 110 ⁇ L was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.
- lysis buffer 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton
- Botulinum toxin serotype A acts on the presynapse of the neuromuscular junction and causes the cleavage of Synaptosomal-Associated Protein, 25kDa (SNAP-25) molecule bound to the presynaptic cell membrane. Since it is known that this principle is applied, a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) test method using each antibody capable of recognizing cleaved SNAP-25 and intact SNAP-25 is designed did The principle is briefly shown in FIG. 9 .
- cleaved Monoclonal Antibody Mybiosource, MBS312597
- a capture antibody that specifically binds to cleaved SNAP-25
- 0.1 M sodium carbonate coating buffer coating After diluting to a concentration of 0.2% (v/v) using buffer, pH 9.6), 100 ⁇ L was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and moved at 4°C for 16 hours.
- Anti-SNAP-25 antibody (Sigma-Aldrich, S9684) to a concentration of 0.1% (v/v) using a blocking buffer, 100 ⁇ L was treated in each well, It was reacted for 1 hour in a shaker at 75 rpm at room temperature. Thereafter, all unbound antibodies were removed by washing three times with 200 ⁇ L of PBST, and the secondary antibody, Anti-Rabbit HRP (abcam, ab6721) was diluted to 0.01% (v/v) using a blocking buffer, , 100 ⁇ L of the diluted antibody solution was treated in each well and reacted for 1 hour on a shaker at 75 rpm at room temperature.
- the TMB solution of the TMB Peroxidase EIA Substrate Kit (Bio-Rad) and the hydrogen peroxide solution were mixed at a ratio of 9: 1, and 50 ⁇ L was applied to each well, and then incubated at 37 ° C in an incubator. Reacted for 30 minutes. Then, after treating each well with 50 ⁇ L of 2 N sulfuric acid, the absorbance (OD450) in the 450 nm wavelength band 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.), and the EC 50 value was calculated based on the result of the company's mouse titer test (unit). Calculated. Afterwards, all experiments were repeated at least three times, and the results were expressed as mean values ⁇ standard deviations. The results are shown in FIG. 10 .
- the OD 450 nm value of each group of SiMa cell line was divided by the OD 450 nm value of each group of SEPTIN2-3 cell line, and the degree of response of the cell line to the same botulinum toxin treatment was compared. The results are shown in FIG. 11 .
- the OD450 SEPTIN2 /OD450 WT value without toxin treatment was 1, whereas when toxin was treated, the value rose to about 1.7, confirming the difference in sensitivity to toxin between cell lines. .
- the transduced cell line overexpressing the SEPTIN2 gene had increased sensitivity to botulinum toxin compared to the control group (wild type).
- a cell lysate of cells treated with the botulinum toxin was first prepared.
- the SiMa cell line was cultured in 8 X 10 4 cells/100 ⁇ L/well in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin.
- SiMa-TXN3 cell line as an experimental group was plated 8 X in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 ⁇ g/mL puromycin. Each was dispensed at a concentration of 10 4 cells/100 ⁇ L/well.
- lysis buffer 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton X-100 supplemented with protease inhibitor cocktail 110 ⁇ L was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.
- lysis buffer 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton X-100
- Example 6.2 In the same manner as in Example 6.2, the sensitivity of botulinum toxin to the transduced cell line overexpressing the TXN gene was confirmed using the cell lysate of Example 6.3. The results are shown in FIG. 12 .
- the transduced cell line overexpressing the Thioredoxin gene had increased sensitivity to botulinum toxin compared to the control group (wild type).
- Example 7 Verification of Biological Activity of BoNT/A Pharmaceutical Composition (Raw Material) by Sandwich ELISA
- sandwich ELISA was performed using the SiMa-SEPTIN2-3 cell line. More specifically, the SiMa-SEPTIN2-3 cell line was cultured in 1.2 X 10 5 in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 ⁇ g/mL puromycin. It was dispensed at a concentration of cells/100 ⁇ L/well.
- the medium was removed and treated with 100 ⁇ L of differentiation medium supplemented with 1X B-27TM Plus Supplement, 1X N-2 Supplement, 2 mM L-glutamine and 25 ⁇ g/mL Trisialoganglioside GT 1B . and cultured for 2 days to induce differentiation.
- the medium was removed, and the BoNT/A complex (botulinum, a raw material from which other substances such as preservation solutions were removed from ATGC-100 was removed) was added to RPMI1640 to which 1X B-27TM Plus Supplement and 1X N-2 Supplement were added. toxin) was serially diluted 1.55 times (serial dilution), and the cells were treated by concentration.
- lysis buffer 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton X-100 supplemented with protease inhibitor cocktail 110 ⁇ L was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.
- lysis buffer 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton X-100
- cleaved Monoclonal Antibody (Mybiosource), a capture antibody that specifically binds to truncated SNAP-25, was added to 0.1 M sodium carbonate coating buffer , pH 9.6) after dilution to a concentration of 0.8% (v/v), 100 ⁇ L was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and left at 4°C for 16 hours without movement. reacted Thereafter, antibodies that did not bind to the wells were removed, and washed three times using 200 ⁇ L of 0.1% polysorbate 20 in PBS (PBST).
- PBST PBS
- Anti-SNAP-25 antibody (Sigma-Aldrich, S9684) to a concentration of 0.1% (v/v) using a blocking buffer, 100 ⁇ L was treated in each well, It was reacted for 1 hour in a shaker at 75 rpm at room temperature. Thereafter, all unbound antibodies were removed by washing three times with 200 ⁇ L of PBST, and the secondary antibody, Anti-Rabbit HRP (abcam, ab6721) was diluted to 0.01% (v/v) using a blocking buffer, , 100 ⁇ L of the diluted antibody solution was treated in each well and treated for 1 hour on a shaker at 75 rpm at room temperature.
- Example 8 Verification of biological activity of BoNT/A pharmaceutical composition (drug product) through sandwich ELISA
- sandwich ELISA was performed using the SiMa-SEPTIN2-3 cell line. More specifically, the SiMa-SEPTIN2-3 cell line was cultured in 1.2 X 10 5 in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 ⁇ g/mL puromycin. It was dispensed at a concentration of cells/100 ⁇ L/well.
- the medium was removed and treated with 100 ⁇ L of differentiation medium supplemented with 1X B-27TM Plus Supplement, 1X N-2 Supplement, 2 mM L-glutamine and 25 ⁇ g/mL Trisialoganglioside GT 1B . and cultured for 2 days to induce differentiation.
- the medium was removed, and the BoNT/A finished product (ATGC-100) was serially diluted 1.4 times in RPMI1640 to which 1X B-27TM Plus Supplement and 1X N-2 Supplement had been added. It was treated by concentration.
- lysis buffer 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton X-100 supplemented with protease inhibitor cocktail 110 ⁇ L was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.
- lysis buffer 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 and 1% Triton X-100
- cleaved Monoclonal Antibody (Mybiosource), a capture antibody that specifically binds to truncated SNAP-25, was added to 0.1 M sodium carbonate coating buffer , pH 9.6) after dilution to a concentration of 0.8% (v/v), 100 ⁇ L was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and left at 4°C for 16 hours without movement. reacted Thereafter, antibodies that did not bind to the wells were removed, and washed three times using 200 ⁇ L of 0.1% polysorbate 20 in PBS (PBST).
- PBST PBS
- Anti-SNAP-25 antibody (Sigma-Aldrich, S9684) to a concentration of 0.1% (v/v) using a blocking buffer, 100 ⁇ L was treated in each well, It was reacted for 1 hour in a shaker at 75 rpm at room temperature. Thereafter, all unbound antibodies were removed by washing three times with 200 ⁇ L of PBST, and the secondary antibody, Anti-Rabbit HRP (abcam, ab6721) was diluted to 0.01% (v/v) using a blocking buffer, , 100 ⁇ L of the diluted antibody solution was treated in each well and treated for 1 hour on a shaker at 75 rpm at room temperature.
- the potency of not only botulinum toxin but also botulinum toxin pharmaceutical compositions, that is, raw materials and finished drugs It was confirmed that 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 using botulinum toxin.
- the cell for measuring botulinum toxin activity of the present invention has significantly improved sensitivity to botulinum toxin, it replaces a number of animal experiments and measures the activity of not only botulinum toxin but also botulinum toxin pharmaceutical compositions in the form of raw materials or finished drugs. It can be widely used in various industrial fields.
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Abstract
Description
Name | Primer sequence (5' -> 3') | 서열번호 |
SEPTIN2 V2 | AGAGCTCGTTTAGTGAA | 1 |
SEPTIN2-600F | GGATGAAATTGAAGAACATA | 2 |
TXN V2 | AGAGCTCGTTTAGTGAA | 3 |
Gene | A260 | A280 | 260/280 ratio | DNA concentration (ng/μL) |
TXN | 1.119 | 0.595 | 1.880 | 559 |
SEPTIN2 | 1.059 | 0.562 | 1.884 | 529 |
Purpose | Name | Primer sequence (5' -> 3') | 서열번호 |
PCR | TXN forward | CTTTTCAGGAAGCCTTGG | 4 |
SEPTIN2 forward | GTCTAAGCAACAGCCAAC | 5 | |
DDK reverse (common) | CTTATCGTCGTCATCCTTG | 6 | |
Positive control | GH20 | GAAGAGCCAAGGACAGGTAC | 7 |
GH21 | GGAAAATAGACCAATAGGCAG | 8 |
Cell line | 260/280 ratio | DNA concentration (ng/uL) |
SiMa-TXN-3 | 1.937 | 1072.0 |
SiMa-SEPTIN2-1 | 1.830 | 887.5 |
SiMa-SEPTIN2-2 | 1.939 | 947.0 |
SiMa-SEPTIN2-3 | 1.916 | 1053.0 |
Cycle | Procedure | Temperature (℃) | Time |
1 | Denaturation | 95 | 30 sec |
30 | Denaturation | 95 | 30 sec |
Annealing | 50 | 30 sec | |
Elongation | 72 | 1 min 30 sec | |
1 | Elongation | 72 | 5 min |
1 | Storage | 4 | Overnight |
Claims (8)
- SEPTIN2 또는 Thioredoxin(TXN)을 과발현하는 보툴리눔 독소 활성 측정용 세포.
- 제 1 항에 있어서,상기 세포는 SEPTIN2 또는 TXN 유전자가 형질도입(transduction)되어 과발현되는 것을 특징으로 하는, 보툴리눔 독소 활성 측정용 세포.
- 제 1 항에 있어서,상기 세포는 보툴리눔 독소 중독(intoxication)에 대한 감수성이 증가된 것을 특징으로 하는, 보툴리눔 독소 활성 측정용 세포.
- 제 1 항에 있어서,상기 세포는 SiMa 세포, LAN-2 세포, PC12 세포, Neuro-2a 세포, LA1-55n 세포, N18 세포, SH-SY5Y 세포, Kelly 세포, NB69 세포, N1E-115 세포, BE(2)-M17 세포, 및 SK-N-BE(2) 세포로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는, 보툴리눔 독소 활성 측정용 세포.
- 제 1 항에 있어서,상기 보툴리눔 독소는 보툴리눔 세로타입(serotype) A, B, C, D, E, F 및 G로 이루어진 군으로부터 선택되는 어느 하나인 것을 특징으로 하는, 보툴리눔 독소 활성 측정용 세포.
- a) 제 1 항 내지 제 5 항 중 어느 한 항의 보툴리눔 독소 활성 측정용 세포에 보툴리눔 독소를 처리하고 배양하는 단계;b) 상기 배양된 세포를 용해시키고 세포 용해액을 수집하는 단계; 및c) 상기 세포 용해액 내의 SNAP-25 절단 생성물의 양을 측정하는 단계를 포함하는, 보툴리눔 독소 활성 측정 방법.
- 제 6 항에 있어서,상기 SNAP-25 절단 생성물의 양은 샌드위치 면역검정법(ELISA) 또는 웨스턴블롯(Western blot)을 이용하여 측정하는 것을 특징으로 하는, 보툴리눔 독소 활성 측정 방법.
- 제 1 항 내지 제 5 항 중 어느 한 항의 보툴리눔 독소 활성 측정용 세포를 유효성분으로 포함하는, 보툴리눔 독소 활성 측정용 키트.
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CA3219818A CA3219818A1 (en) | 2021-05-24 | 2022-05-24 | Cells sensitive to botulinum toxin into which specific gene has been inserted by lentivirus |
EP22811598.6A EP4349992A1 (en) | 2021-05-24 | 2022-05-24 | Cells sensitive to botulinum toxin into which specific gene has been inserted by lentivirus |
BR112023024599A BR112023024599A2 (pt) | 2021-05-24 | 2022-05-24 | Células sensíveis à toxina botulínica dentro das quais um gene específico foi inserido por um lentivírus |
CN202280037765.5A CN117529557A (zh) | 2021-05-24 | 2022-05-24 | 通过慢病毒插入有特定基因的对肉毒杆菌毒素敏感的细胞 |
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KR1020220063008A KR20220159283A (ko) | 2021-05-24 | 2022-05-23 | 렌티바이러스에 의해 특정 유전자가 삽입된 보툴리눔 독소에 민감한 세포 |
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Citations (5)
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US6444209B1 (en) | 1996-10-28 | 2002-09-03 | Wisconsin Alumni Research Foundation | Hybrid botulinal neurotoxins |
US20120208271A1 (en) * | 2009-03-13 | 2012-08-16 | Allergan, Inc. | Cells Useful for Immuno-Based Botulinum Toxin Serotype A Activity Assays |
KR20120134154A (ko) | 2008-03-14 | 2012-12-11 | 알러간, 인코포레이티드 | 면역-기반 보툴리눔 독소 세로타입 a 활성 검정 |
KR101609894B1 (ko) * | 2008-03-14 | 2016-04-08 | 알러간, 인코포레이티드 | 면역-기반 보툴리눔 독소 세로타입 a 활성 검정 |
KR20200072008A (ko) * | 2018-12-12 | 2020-06-22 | 주식회사 에이비바이오 | 보툴리눔 독소 활성을 결정하는 세포 기반 방법 |
-
2022
- 2022-05-24 EP EP22811598.6A patent/EP4349992A1/en active Pending
- 2022-05-24 CA CA3219818A patent/CA3219818A1/en active Pending
- 2022-05-24 BR BR112023024599A patent/BR112023024599A2/pt unknown
- 2022-05-24 WO PCT/KR2022/007323 patent/WO2022250405A1/ko active Application Filing
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US6444209B1 (en) | 1996-10-28 | 2002-09-03 | Wisconsin Alumni Research Foundation | Hybrid botulinal neurotoxins |
KR20120134154A (ko) | 2008-03-14 | 2012-12-11 | 알러간, 인코포레이티드 | 면역-기반 보툴리눔 독소 세로타입 a 활성 검정 |
KR101609894B1 (ko) * | 2008-03-14 | 2016-04-08 | 알러간, 인코포레이티드 | 면역-기반 보툴리눔 독소 세로타입 a 활성 검정 |
US20120208271A1 (en) * | 2009-03-13 | 2012-08-16 | Allergan, Inc. | Cells Useful for Immuno-Based Botulinum Toxin Serotype A Activity Assays |
KR20200072008A (ko) * | 2018-12-12 | 2020-06-22 | 주식회사 에이비바이오 | 보툴리눔 독소 활성을 결정하는 세포 기반 방법 |
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PIRAZZINI MARCO, AZARNIA TEHRAN DOMENICO, ZANETTI GIULIA, ROSSETTO ORNELLA, MONTECUCCO CESARE: "Hsp90 and Thioredoxin-Thioredoxin Reductase enable the catalytic activity of Clostridial neurotoxins inside nerve terminals", TOXICON, ELMSFORD, NY, US, vol. 147, 1 June 2018 (2018-06-01), US , pages 32 - 37, XP093008794, ISSN: 0041-0101, DOI: 10.1016/j.toxicon.2017.10.028 * |
VAGIN OLGA, TOKHTAEVA ELMIRA, GARAY PATTON E., SOUDA PUNEET, BASSILIAN SARA, WHITELEGGE JULIAN P., LEWIS RAMILLA, SACHS GEORGE, WH: "Recruitment of septin cytoskeletal proteins by Botulinum toxin A protease determines its remarkable stability", JOURNAL OF CELL SCIENCE, COMPANY OF BIOLOGISTS LIMITED, CAMBRIDGE, Cambridge , XP093008795, ISSN: 0021-9533, DOI: 10.1242/jcs.146324 * |
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