SG11201903523YA - In vitro and cell based assays for measuring the activity of botulinum neurotoxins - Google Patents

In vitro and cell based assays for measuring the activity of botulinum neurotoxins

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Publication number
SG11201903523YA
SG11201903523YA SG11201903523YA SG11201903523YA SG11201903523YA SG 11201903523Y A SG11201903523Y A SG 11201903523YA SG 11201903523Y A SG11201903523Y A SG 11201903523YA SG 11201903523Y A SG11201903523Y A SG 11201903523YA SG 11201903523Y A SG11201903523Y A SG 11201903523YA
Authority
SG
Singapore
Prior art keywords
international
activity
neurotoxin
polypeptides
rule
Prior art date
Application number
SG11201903523YA
Inventor
Min Dong
Feifan Yu
Original Assignee
Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harvard College filed Critical Harvard College
Publication of SG11201903523YA publication Critical patent/SG11201903523YA/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Abstract

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property 111111101111 0 1110101011111 011101 0 01111H111001H1111111011111111111110111111 Organization International Bureau (10) International Publication Number (43) International Publication Date .....0\"\" WO 2018/075783 A2 26 April 2018 (26.04.2018) WIP0 I PCT (51) International Patent Classification: Declarations under Rule 4.17: G01N 33/542 (2006.01) Cl 2N 9/02 (2006.01) C12Q 1/66 (2006.01) — as to applicant's entitlement to apply for and be granted a patent (Rule 4.17(H)) (21) International Application Number: PCT/US2017/057411 Published: — without international search report and to be republished (22) International Filing Date: upon receipt of that report (Rule 48.2(g)) 19 October 2017 (19.10.2017) — with sequence listing part of description (Rule 5.2(a)) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/410,558 20 October 2016 (20.10.2016) US (71) Applicant: PRESIDENT AND FELLOWS OF HAR- VARD COLLEGE [US/US]; 17 Quincy Street, Cam- bridge, Massachusetts 02138 (US). (72) Inventors: DONG, Min; 12 Croft Lane, Weatogue, Con- necticut 06089 (US). YU, Feifan; 98 Dundee Court, Ab- erdeen, New Jersey 07747 (US). Agent: JARRELL, Brenda Herschbach et al.; Choate, Hall & Stewart LLP, Two International Place, Boston, (74) _ _ Massachusetts 02110 (US). = Designated States (unless otherwise indicated, for every (81) = kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, _ DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, = KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, _ MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, = = = TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. _ Designated States (unless otherwise indicated, for every — (84) _ _ kind of regional protection available): ARIPO (BW, GH, = GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, = UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, = TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, = EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, — MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). = ei M QC (54) Title: IN VITRO AND CELL BASED ASSAYS FOR MEASURING THE ACTIVITY OF BOTULINUM NEUROTOXINS IN Ir) (57) : Disclosed herein are means for the detection and characterization of neurotoxins such as botulinum neurotoxin (BoNT) I r ---- or tetanus neurotoxin. The present disclosure provides methods for determining potency and activity of neurotoxins in vitro and in vivo. 0 —. Also disclosed are polypeptides comprising N- and C- terminal fragments of a reporter protein that are split by a linker comprising GO a 1-1 neurotoxin cleavage site. Cleavage of the linker by a neurotoxin decreases reporter protein activity, thereby indicating activity of © the neurotoxin. Compositions and kits comprising the disclosed polypeptides, nucleic acids comprising nucleotide sequences encoding f'1 such polypeptides, and cells expressing such polypeptides are also disclosed. 0
SG11201903523YA 2016-10-20 2017-10-19 In vitro and cell based assays for measuring the activity of botulinum neurotoxins SG11201903523YA (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662410558P 2016-10-20 2016-10-20
PCT/US2017/057411 WO2018075783A2 (en) 2016-10-20 2017-10-19 In vitro and cell based assays for measuring the activity of botulinum neurotoxins

Publications (1)

Publication Number Publication Date
SG11201903523YA true SG11201903523YA (en) 2019-05-30

Family

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Application Number Title Priority Date Filing Date
SG11201903523YA SG11201903523YA (en) 2016-10-20 2017-10-19 In vitro and cell based assays for measuring the activity of botulinum neurotoxins

Country Status (19)

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US (2) US11306347B2 (en)
EP (1) EP3529616B1 (en)
JP (2) JP7227901B2 (en)
KR (1) KR102556363B1 (en)
CN (1) CN110088624B (en)
AU (1) AU2017345560A1 (en)
BR (1) BR112019007831A2 (en)
CA (1) CA3040507A1 (en)
DK (1) DK3529616T3 (en)
EA (1) EA039761B1 (en)
ES (1) ES2963830T3 (en)
FI (1) FI3529616T3 (en)
HU (1) HUE064332T2 (en)
MX (2) MX2019004431A (en)
PL (1) PL3529616T3 (en)
PT (1) PT3529616T (en)
SA (1) SA519401607B1 (en)
SG (1) SG11201903523YA (en)
WO (1) WO2018075783A2 (en)

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EP2655614B1 (en) 2010-12-22 2017-03-15 President and Fellows of Harvard College Continuous directed evolution
US10920208B2 (en) 2014-10-22 2021-02-16 President And Fellows Of Harvard College Evolution of proteases
WO2016168631A1 (en) 2015-04-17 2016-10-20 President And Fellows Of Harvard College Vector-based mutagenesis system
US10392674B2 (en) 2015-07-22 2019-08-27 President And Fellows Of Harvard College Evolution of site-specific recombinases
WO2017015559A2 (en) 2015-07-23 2017-01-26 President And Fellows Of Harvard College Evolution of bt toxins
US10612011B2 (en) 2015-07-30 2020-04-07 President And Fellows Of Harvard College Evolution of TALENs
MX2018014066A (en) * 2016-05-16 2019-04-04 Harvard College Method for purification and activation of botulinum neurotoxin.
JP7227901B2 (en) 2016-10-20 2023-02-22 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ In vitro and cell-based assays for measuring activity of botulinum neurotoxin
WO2019010164A1 (en) 2017-07-06 2019-01-10 President And Fellows Of Harvard College Evolution of trna synthetases
US11624130B2 (en) 2017-09-18 2023-04-11 President And Fellows Of Harvard College Continuous evolution for stabilized proteins
US11913044B2 (en) 2018-06-14 2024-02-27 President And Fellows Of Harvard College Evolution of cytidine deaminases
US20220259269A1 (en) * 2019-07-15 2022-08-18 President And Fellows Of Harvard College Evolved botulinum neurotoxins and uses thereof
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Publication number Publication date
CN110088624B (en) 2024-01-16
KR20190068582A (en) 2019-06-18
CA3040507A1 (en) 2018-04-26
BR112019007831A2 (en) 2019-10-01
US11306347B2 (en) 2022-04-19
CN110088624A (en) 2019-08-02
US11788069B2 (en) 2023-10-17
EP3529616B1 (en) 2023-09-06
HUE064332T2 (en) 2024-03-28
EA039761B1 (en) 2022-03-10
PT3529616T (en) 2023-12-06
WO2018075783A2 (en) 2018-04-26
MX2019004431A (en) 2019-08-14
WO2018075783A3 (en) 2018-06-21
US20220356508A1 (en) 2022-11-10
PL3529616T3 (en) 2024-03-18
US20190276873A1 (en) 2019-09-12
JP2019532651A (en) 2019-11-14
EP3529616A2 (en) 2019-08-28
EA201990929A1 (en) 2019-10-31
SA519401607B1 (en) 2022-08-04
AU2017345560A1 (en) 2019-05-02
ES2963830T3 (en) 2024-04-02
DK3529616T3 (en) 2023-12-11
MX2023004733A (en) 2023-05-10
JP2023011700A (en) 2023-01-24
JP7227901B2 (en) 2023-02-22
FI3529616T3 (en) 2023-12-04
KR102556363B1 (en) 2023-07-18

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