Treatment of Upper Facial Lines
20230346674 · 2023-11-02
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention relates to a modified BoNT/A for use in treating facial lines, wherein the modified BoNT/A is administered by intramuscular injection at a plurality of sites of the face of the subject, wherein the modified BoNT/A is administered by way of a unit dose of Units to 41 Units of modified BoNT/A per site and wherein 1 Unit is an amount of the modified BoNT/A that corresponds to the calculated median lethal dose (LD.sub.50) in mice, wherein the plurality of sites are selected from: up to two sites of a corrugator muscle and one site of a procerus muscle for treating glabellar lines, up to five sites of a frontalis muscle for treating forehead lines; and up to three sites at the external part of an orbicularis oculi muscle for treating lateral canthal lines, wherein the total dose of modified BoNT/A administered during the treatment is up to 574 Units, and wherein the modified BoNT/A comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 15 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: i. substitution of an acidic surface exposed amino acid residue with a basic amino acid residue; ii. substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; and v. deletion of an acidic surface exposed amino acid residue. Also provided are corresponding methods of treatment and uses, as well as unit dosage forms, and kits.
Claims
1. A modified BoNT/A for use in treating facial lines, wherein the modified BoNT/A is administered by intramuscular injection at a plurality of sites of the face of the subject, wherein the modified BoNT/A is administered by way of a unit dose of 1 Units to 41 Units of modified BoNT/A per site and wherein 1 Unit is an amount of the modified BoNT/A that corresponds to the calculated median lethal dose (LD.sub.50) in mice, wherein the plurality of sites are selected from: up to two sites of a corrugator muscle and one site of a procerus muscle for treating glabellar lines, up to five sites of a frontalis muscle for treating forehead lines; and up to three sites at the external part of an orbicularis oculi muscle for treating lateral canthal lines, wherein the total dose of modified BoNT/A administered during the treatment is up to 574 Units, and wherein the modified BoNT/A comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: i. substitution of an acidic surface exposed amino acid residue with a basic amino acid residue; ii. substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; and v. deletion of an acidic surface exposed amino acid residue.
2. A modified BoNT/A for use in treating facial lines, wherein the modified BoNT/A is administered by intramuscular injection at a plurality of sites of the face of the subject, wherein the modified BoNT/A is administered by way of a unit dose of 8.4 pg to 344.4 pg of modified BoNT/A per site, wherein the plurality of sites are selected from: up to two sites of a corrugator muscle and one site of a procerus muscle for treating glabellar lines, up to five sites of a frontalis muscle for treating forehead lines; and up to three sites at the external part of an orbicularis oculi muscle for treating lateral canthal lines, wherein the total dose of modified BoNT/A administered during the treatment is up to 4821.6 pg, and wherein the modified BoNT/A comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: i. substitution of an acidic surface exposed amino acid residue with a basic amino acid residue; ii. substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; and v. deletion of an acidic surface exposed amino acid residue.
3. The modified BoNT/A for use according to claim 1, wherein 5-10 pg (preferably 8.4 pg) of the modified BoNT/A corresponds to the calculated median lethal dose (LD.sub.50) in mice.
4. The modified BoNT/A for use according to any one of the preceding claims, wherein the unit dose of modified BoNT/A is selected from: 2 Units to 35 Units, 6 to 35 Units, 12 Units to 35 Units, 12 Units to 24 Units, most preferably 12 Units to 18 Units.
5. The modified BoNT/A for use according to claim 4, wherein the total dose of modified BoNT/A administered during the treatment is selected from: 574 Units, 560 Units, 384 Units, most preferably 288 Units; preferably wherein said total dose is based on administration of 16 unit doses across 16 injection sites.
6. The modified BoNT/A for use according to any one of the preceding claims, wherein the unit dose of modified BoNT/A is selected from: 18 pg to 350 pg, 20 pg to 300 pg, most preferably 100 pg to 150 pg.
7. The modified BoNT/A for use according to any one of the preceding claims, wherein the total dose of modified BoNT/A administered during the treatment is selected from up to: 4850 pg, 4800 pg, most preferably 2400 pg.
8. A modified BoNT/A for use in treating facial lines, wherein the modified BoNT/A is administered by intramuscular injection at a plurality of sites of the face of the subject, wherein the modified BoNT/A is administered by way of a unit dose of 0.5 Units to 73 Units of modified BoNT/A per site and wherein 1 Unit is an amount of the modified BoNT/A that corresponds to the calculated median lethal dose (LD.sub.50) in mice, wherein the plurality of sites are selected from: up to two sites of a corrugator muscle and one site of a procerus muscle for treating glabellar lines, up to five sites of a frontalis muscle for treating forehead lines; and up to three sites at the external part of an orbicularis oculi muscle for treating lateral canthal lines, wherein the total dose of modified BoNT/A administered during the treatment is up to 1019 Units, and wherein the modified BoNT/A comprises a BoNT/A light-chain and translocation domain, and a BoNT/B receptor binding domain (H.sub.c domain).
9. A modified BoNT/A for use in treating facial lines, wherein the modified BoNT/A is administered by intramuscular injection at a plurality of sites of the face of the subject, wherein the modified BoNT/A is administered by way of a unit dose of 12 pg to 1754 pg of modified BoNT/A per site, wherein the plurality of sites are selected from: up to two sites of a corrugator muscle and one site of a procerus muscle for treating glabellar lines, up to five sites of a frontalis muscle for treating forehead lines; and up to three sites at the external part of an orbicularis oculi muscle for treating lateral canthal lines, wherein the total dose of modified BoNT/A administered during the treatment is up to 24,500 pg, and wherein the modified BoNT/A comprises a BoNT/A light-chain and translocation domain, and a BoNT/B receptor binding domain (H.sub.c domain).
10. The modified BoNT/A for use according to claim 8, wherein the modified BoNT/A comprises a BoNT/A light-chain and translocation domain, and a BoNT/B H.sub.c domain, and wherein 20-30 pg (preferably 24.04 pg) of the modified BoNT/A corresponds to the calculated median lethal dose (LD.sub.50) in mice.
11. The modified BoNT/A for use according to any one of claims 8-10, wherein the unit dose of modified BoNT/A is selected from: 0.5 Units to 62 Units, 3 Units to 62 Units, 10 to 62 Units, 10 Units to 42 Units, most preferably 10 Units to 21 Units.
12. The modified BoNT/A for use according to claim 11, wherein the total dose of modified BoNT/A administered during the treatment is selected from: 1019 Units, 998 Units, 666 Units, most preferably 333 Units; preferably wherein said total dose is based on administration of 16 unit doses across 16 injection sites.
13. The modified BoNT/A for use according to any one of claims 8-12, wherein the unit dose of modified BoNT/A is selected from: 12 pg to 1750 pg, 20 pg to 1500 pg, most preferably 250 pg to 500 pg.
14. The modified BoNT/A for use according to any one of claims 8-13, wherein the total dose of modified BoNT/A administered during the treatment is selected from up to: 24,500 pg, 24,000 pg, most preferably 8000 pg.
15. The modified BoNT/A for use according to any one of the preceding claims, wherein the modified BoNT/A has a Safety Ratio of greater than 7, wherein the Safety Ratio is calculated as: dose of toxin required for -10% bodyweight change measured as pg/mouse divided by DAS ED.sub.50 measured as pg/mouse, wherein ED.sub.50 = dose required to produce a DAS score of 2.
16. The modified BoNT/A for use according to any one of the preceding claims, wherein the modified BoNT/A is for the use in treating glabellar lines, preferably wherein said modified BoNT/A is administered at two sites of a corrugator muscle and one site of a procerus muscle, more preferably wherein said modified BoNT/A is administered at a total of five sites.
17. The modified BoNT/A for use according to claims 1-15, wherein the modified BoNT/A is for the use in treating forehead lines, preferably wherein said modified BoNT/A is administered at five sites of a frontalis muscle.
18. The modified BoNT/A for use according to claims 1-15, wherein the modified BoNT/A is for the use in treating lateral canthal lines, preferably wherein said modified BoNT/A is administered at three sites at the external part of a orbicularis oculi muscle for treating lateral canthal lines, more preferably wherein said modified BoNT/A is administered at a total of six sites.
19. The modified BoNT/A for use according to claims 16-18, wherein said modified BoNT/A is administered to 8, 10, 12, 14 or 16 sites.
20. The modified BoNT/A for use according to claims 16-18, wherein said modified BoNT/A is administered to 7, 9, 11, 13 or 15 sites.
21. The modified BoNT/A for use according to claims 1-7 or 15-20, wherein said modification comprises (preferably consists of) a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 or THR 1277, and wherein the modified BoNT/A is encoded by a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, and 9, and/or comprises a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 4, 6, 8, and 10.
22. The modified BoNT/A for use according to claims 1-7 or 15-21, wherein said modification comprises (preferably consists of) a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, and GLN 1229, and wherein the modified BoNT/A is encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 3, and/or comprises a polypeptide sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO: 4.
23. The modified BoNT/A for use according to claims 1-7 or 15-22, wherein the modification is a substitution, preferably a substitution with lysine or arginine.
24. The modified BoNT/A for use according to any one of claims 8-20, wherein the modified BoNT/A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14.
25. A unit dosage form of modified botulinum neurotoxin A (BoNT/A), the unit dosage form comprising: a. 1 Units to 41 Units of modified BoNT/A, wherein 1 Unit is an amount of the modified BoNT/A that corresponds to the calculated median lethal dose (LD.sub.50) in mice; or b. 8.4 pg to 350 pg of modified BoNT/A; and c. optionally a pharmaceutically acceptable carrier, excipient, adjuvant, and/or salt, d. wherein the modified BoNT/A comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: i. substitution of an acidic surface exposed amino acid residue with a basic amino acid residue; ii. substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; and v. deletion of an acidic surface exposed amino acid residue.
26. The unit dosage form according to claim 25, comprising: a. 1 Units to 35 Units of modified BoNT/A; or b. 10 pg to 300 pg of modified BoNT/A.
27. A unit dosage form of modified botulinum neurotoxin A (BoNT/A), the unit dosage form comprising: a. 0.5 Units to 73 Units of modified BoNT/A, wherein 1 Unit is an amount of the modified BoNT/A that corresponds to the calculated median lethal dose (LD.sub.50) in mice; or b. 12 pg to 1754 pg of modified BoNT/A; and c. optionally a pharmaceutically acceptable carrier, excipient, adjuvant, and/or salt, d. wherein the modified BoNT/A comprises a BoNT/A light-chain and translocation domain, and a BoNT/B receptor binding domain (H.sub.c domain).
28. The unit dosage form according to claim 27, comprising: a. 0.8 Units to 62 Units of modified BoNT/A; or b. 20 pg to 1500 pg of modified BoNT/A.
29. A kit comprising: a. the unit dosage form according to any one of claims 25-28; and b. instructions for use of the same in treating facial lines; and c. optionally a diluent.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0378] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.
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SEQUENCE LISTING
[0393] Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional.
[0394] SEQ ID NO: 1 (Nucleotide Sequence of Unmodified BoNT/A)
TABLE-US-00004 ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGT CGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTA AGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACC TTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCA AGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAA AAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGC ACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTT CTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACT GCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAAT CTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAG CTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCC AGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTG GAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCC GGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACG GCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATAC TACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGG TGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTC TGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCC AAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTT TAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACC GAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCT GAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACT ACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAAC TTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAA GAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTA TCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTG AATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATC CGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCG ATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAG CAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCAT TGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATA TCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATG TTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGC GCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATA CCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCC GCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGA GACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTA TCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGAC GATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCT ACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCG AAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTG GCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAG AGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAA TACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGA TGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATA TCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATG ATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGA TGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAG TTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCA TTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCAC CGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACG AGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATC GGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTT TAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCT ACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTG TATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCA TTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGT GACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTC GCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGC AACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATAT CTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGA TCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTC TGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTA TGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACA TGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTG AACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAG CGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACG TGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCG GGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCT GAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACA AGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATT GGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTA CAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGT TTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG
[0395] SEQ ID NO: 2 (Polypeptide Sequence of Unmodified BoNT/A)
TABLE-US-00005 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIP KYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHAS NNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYL NSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFI GFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL
[0396] SEQ ID NO: 3 (Nucleotide Sequence of Modified BoNT/A “Cat-A”)
TABLE-US-00006 ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGT CGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTA AGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACC TTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCA AGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAA AAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGC ACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTT CTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACT GCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAAT CTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAG CTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCC AGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTG GAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCC GGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACG GCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATAC TACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGG TGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTC TGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCC AAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTT TAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACC GAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCT GAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACT ACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAAC TTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAA GAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTA TCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTG AATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATC CGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCG ATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAG CAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCAT TGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATA TCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATG TTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGC GCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATA CCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCC GCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGA GACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTA TCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGAC GATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCT ACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCG AAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTG GCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAG AGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAA TACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGA TGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATA TCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATG ATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGA TGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAG TTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCA TTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCAC CGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACG AGAGCAAGCATCTGATTGATCTGAGCCGTTATGCTAGCAAGATCAACATC GGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTT TAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAAGGCCATTGTCT ACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAACAAGATTAGCCTGAACAACGAGTATACTATCATCAACTG TATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCA TTTGGACCTTGCAGGACACCAAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGT GACCATTACGAATAACCGTCTGAATAAGAGCAAGATTTACATCAATGGTC GCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGC AACAAGATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATAT CTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGA TCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTC TGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTA TGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACA TGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTG AACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAG CGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACG TGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCG GGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCT GAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACAAGGGTATCACTAACA AGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATT GGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTA CAATCGTCAGATTGAGCGCAGCAGCcGTACTTTGGGCTGTAGCTGGGAGT TTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG
[0397] SEQ ID NO: 4 (Polypeptide Sequence of Modified BoNT/A “Cat-A”)
TABLE-US-00007 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESKHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIP KYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHAS NKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYL NSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFI GFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL
[0398] SEQ ID NO: 5 (Nucleotide Sequence of Modified BoNT/A “Cat-B″)
TABLE-US-00008 ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGT CGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTA AGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACC TTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCA AGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAA AAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGC ACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTT CTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACT GCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAAT CTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAG CTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCC AGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTG GAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCC GGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACG GCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATAC TACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGG TGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTC TGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCC AAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTT TAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACC GAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCT GAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACT ACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAAC TTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAA GAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTA TCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTG AATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATC CGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCG ATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAG CAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCAT TGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATA TCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATG TTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGC GCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATA CCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCC GCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGA GACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTA TCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGAC GATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCT ACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCG AAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTG GCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAG AGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAA TACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGA TGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATA TCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATG ATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGA TGCGTTGCTGAAATACATTTACGACAaTCGTGGTACGCTGATTGGCCAAG TTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCA TTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCAC CGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACG AGAGCAATCATCTGATTGATCTGAGCCGTTATGCTAGCAAGATCAACATC GGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTT TAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAAGGCCATTGTCT ACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAAGAAGATTAGCCTGAACAACGAGTATACTATCATCAACTG TATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCA TTTGGACCTTGCAGGACACCAAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGT GACCATTACGAATAACCGTCTGAATAAGAGCAAGATTTACATCAATGGTC GCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGC AACAAGATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATAT CTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGA TCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTC TGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTA TGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACA TGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTG AACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAG CGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACG TGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCG GGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCT GAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACAAGGGTATCACTAACA AGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATT GGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTA CAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGT TTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG
[0399] SEQ ID NO: 6 (Polypeptide Sequence of Modified BoNT/A “Cat-B″)
TABLE-US-00009 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIP KYFKKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHAS NKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYL NSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFI GFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL
[0400] SEQ ID NO: 7 (Nucleotide Sequence of Modified BoNT/A “Cat-C″)
TABLE-US-00010 ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGT CGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTA AGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACC TTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCA AGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAA AAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGC ACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTT CTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACT GCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAAT CTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAG CTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCC AGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTG GAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCC GGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACG GCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATAC TACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGG TGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTC TGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCC AAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTT TAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACC GAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCT GAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACT ACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAAC TTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAA GAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTA TCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTG AATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATC CGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCG ATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAG CAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCAT TGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATA TCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATG TTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGC GCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATA CCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCC GCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGA GACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTA TCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGAC GATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCT ACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCG AAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTG GCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAG AGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAA TACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGA TGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATA TCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATG ATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGA TGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAG TTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCA TTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCAC CGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACG AGAGCAATCATCTGATTGATCTGAGCCGTTATGCTAGCAAGATCAACATC GGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTT TAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAAGGCCATTGTCT ACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAACAAGATTAGCCTGAACAACGAGTATACTATCATCAACTG TATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCA TTTGGACCTTGCAGGACACCAAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGT GACCATTACGAATAACCGTCTGAAGAAGAGCAAGATTTACATCAATGGTC GCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGC AACAAGATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATAT CTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGA TCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTC TGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTA TGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACA TGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTG AACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAG CGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACG TGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCG GGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCT GAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACAAGGGTATCACTAACA AGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATT GGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTA CAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGT TTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG
[0401] SEQ ID NO: 8 (Polypeptide Sequence of Modified BoNT/A “Cat-C″)
TABLE-US-00011 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIP KYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRVVFK YSQMINISDYINRWIFVTITNNRLKKSKIYINGRLIDQKPISNLGNIHAS NKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYL NSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFI GFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL
[0402] SEQ ID NO: 9 (Nucleotide Sequence of Modified BoNT/A “Cat-D”)
TABLE-US-00012 ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGT CGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTA AGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACC TTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCA AGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAA AAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGC ACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTT CTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACT GCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAAT CTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAG CTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCC AGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTG GAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCC GGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACG GCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATAC TACGAGATGAGCGGCCTgGAAGTCAGCTTCGAAGAACTGCGCACCTTCGG TGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTC TGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCC AAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTT TAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACC GAGGACAACTTTGTGAAATTCTTCAAaGTGTTGAATCGTAAAACCTATCT GAATTTTGACAAAGCGGTTTTCaAGATTAACATCGTGCCGAAGGTGAACT ACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAAC TTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAA GAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTA TCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTG AATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATC CGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCG ATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAG CAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCAT TGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATA TCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATG TTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGC GCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATA CCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCC GCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGA GACGAGCGAAGTGAGCACTACCGACAAAaTTGCTGATaTTACCATCATTA TCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGAC GATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCT ACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCG AAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTG GCTGGCGAAAGTCaATACCCAGATCGACCTGATCCGTAAGAAAATGAAAG AGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAA TACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGA TGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATA TCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATG ATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGA TGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAG TTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCA TTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCAC CGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACG AGAGCAATCATCTGATtGATCTGAGCCGTTATGCAAGCAAGATCAACATC GGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTT TAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCT ACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTG TATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCA TTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGT GACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTC GCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGC AACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATAT CTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGA TCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTC TGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTA TGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACA TGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTG AACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAG CGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACG TGGTCGTGAAGCGTAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCG GGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTCGTGTCCGTCGTCT GAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACA AGTGCAAGATGAACCTGCAAGACCGTCGTGGTAACGACATCGGCTTTATT GGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTA CAATCGTCAGATTGAGCGCCGTAGCCGTCGTTTGGGCTGTAGCTGGGAGT TTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG
[0403] SEQ ID NO: 10 (Polypeptide Sequence of Modified BoNT/A “Cat-D”)
TABLE-US-00013 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIP KYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHAS NNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYL NSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKRKEYRLATNASQA GVEKILSALEIPRVRRLSQVVVMKSKNDQGITNKCKMNLQDRRGNDIGFI GFHQFNNIAKLVASNWYNRQIERRSRRLGCSWEFIPVDDGWGERPL
[0404] SEQ ID NO: 11 (Polypeptide Sequence of Modified BoNT/A “Chimera 1”)
TABLE-US-00014 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPK YKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVF FEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIA NGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKD FWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINY RDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIG LIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKD EGWTEHHHHHHHHHH
[0405] SEQ ID NO: 12 (Polypeptide Sequence of Modified BoNT/A “Chimera 2”)
TABLE-US-00015 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFL DFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWT LIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLES NTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEER YKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTR SKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLL FKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNL KLGCNWQFIPKDEGWTEHHHHHHHHHH
[0406] SEQ ID NO: 13 (Polypeptide Sequence of Modified BoNT/A “Chimera 3A”)
TABLE-US-00016 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKY KNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFF EYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIAN GEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDF WGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYR DLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGL IGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDE GWTEHHHHHHHHHH
[0407] SEQ ID NO: 14 (Polypeptide Sequence of Modified BoNT/A “Chimera 3B”)
TABLE-US-00017 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKY KNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFF EYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIAN GEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDF WGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYR DLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGL IGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDE GWTE
[0408] SEQ ID NO: 15 (Polypeptide Sequence of Modified BoNT/A “Chimera 3C”)
TABLE-US-00018 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKY KNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFF EYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIAN GEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDF WGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYR DLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGL IGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDE GWTE
[0409] SEQ ID NO: 16 (Polypeptide Sequence of BoNT/B)
TABLE-US-00019 MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERY TFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIK SKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVER KKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYV SVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIV PNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYK SLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNI SDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVD NEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISK IELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDI RDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASI LLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLI VAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNIN IDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDN TLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILI EMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEY TIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYIN RWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDR TQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYM FNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRK SNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVF EEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE
EXAMPLES
Example 1
Cloning, Expression and Purification
[0410] The nucleotide sequence SEQ ID NO: 1, which encodes wild-type BoNT/A (SEQ ID NO: 2) was mutated to introduce the following substitutions to form the four constructs shown in Table 1 below:
TABLE-US-00020 Constructs. Construct Mutations Nucleotide Sequence Polypeptide Sequence Cat-A N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N886K 3 4 Cat-B N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N954K 5 6 Cat-C N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N1025K 7 8 Cat-D* N1188R, D1213R, G1215R, N1216R, N1242R, N1243R, S1274R, T1277R 9 10 *Cat-D had a calculated pl of 7.45, and a molecular weight of 149,859.
[0411] DNA constructs encoding the modified BoNT/A molecules above were synthesised, cloned into the pJ401 expression vector and then transformed into BL21 (DE3) E. coli. This allowed for soluble over-expression of the recombinant Cat-A, Cat-B, Cat-C, and Cat-D proteins in BL21(DE3) E. coli.
[0412] The recombinant modified BoNTs were purified using classical chromatography techniques from the E. coli lysates. An initial purification step using a cation-exchange resin was employed, followed by an intermediate purification step using a hydrophobic interaction resin.
[0413] The recombinant modified BoNT single-chain was then cleaved by proteolysis, resulting in the activated di-chain modified BoNT. A final purification step was then employed to remove remaining contaminants. Suitable techniques are taught in WO2015/166242, W02017055274A1, EP2524963B1, EP2677029B1, and US10087432B2.
Example 2
Characterization of Purified Modified BoNT/A
[0414] The modified BoNTs described in Example 1 above were characterised experimentally as follows.
[0415] Measurement of the pl showed that the modified BoNTs had an isoelectric point greater than that of unmodified (native) BoNT/A1 - see
TABLE-US-00021 Modified BoNT/A pl values. BoNT/A1 molecule pl (calculated) pl (observed) Modified, “Cat-A” [Cat5v2(K1064H/N886K] (SEQ ID NO: 4) 6.9 ~8.0 Modified, “Cat-B” [Cat5v2(K1064/N954K)] (SEQ ID NO: 6) 6.9 ~8.0 Modified, “Cat-C” [Cat5v2(K1064H/N1025K)] (SEQ ID NO: 8) 6.9 7.8-8.0 Native BoNT/A1 [rBoNT/A1] (SEQ ID NO: 2) 6.05 ~7.4
[0416] The ability of the modified BoNTs to enter neurons and cleave SNAP-25 (the target of BoNT/A1) was assessed using rat embryonic spinal cord neurons (eSCN).
[0417] Potency of the modified BoNTs was further assessed using the mouse phrenic nerve hemi-diaphragm assay (mPNHD).
[0418] The in vivo mouse Digital Abduction Score (DAS) assay was used to assess potency as well as safety relative to native BoNT/A1. Both molecules (Cat-A [SEQ ID NO: 4] and Cat-B [SEQ ID NO: 6]) displayed a higher safety ratio relative to native BoNT/A1 and were slightly more potent. These data are presented in Table 3 below:
TABLE-US-00022 DAS assay and safety ratio. Molecule DAS ED.sub.50 (pg/mouse) Dose DAS 4 (pg/mouse) Dose for -10% ΔBW (pg/mouse) Safety Ratio Native BoNT/A1 (n=5) (SEQ ID NO: 2) 2 10-20 9.9-14.5 7 Modified, “Cat-A” (SEQ ID NO: 4) 1.16 10-20 27.4 24 Modified, “Cat-B” (SEQ ID NO: 6) 1.79 25 47.6 27 -DAS ED.sub.50: Calculated dose inducing a DAS 2 -Dose DAS 4: Experimental dose inducing a DAS 4 -BW: Body weight -Dose for -10% ΔBW: Calculated dose inducing a decrease of 10% on BW in comparison to BW at D0 -Safety Ratio: Dose for -10% ΔBW / DAS ED.sub.50
[0419] The Safety Ratio is a measure of a negative effect of BoNT treatment (weight loss) with respect to potency (half maximal digital abduction score (DAS)). It is calculated as the ratio between -10% Body Weight (BW) and the DAS ED.sub.50, where -10%BW refers to the amount of BoNT (pg/animal) required for a 10% decrease in body weight, and ED.sub.50 refers to the amount of BoNT (pg/animal) that will produce a DAS of 2.
[0420] The DAS assay is performed by injection of 20.Math.l of modified BoNT/A, formulated in Gelatin Phosphate Buffer, into the mouse gastrocnemius/soleus complex, followed by assessment of Digit Abduction as previously reported by Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001).
Example 3
Cloning, Expression and Purification of BoNT/AB Chimeras
[0421] BoNT/AB chimeric constructs 1, 2, 3A, 3B, and 3C (SEQ ID NO: 11 to 15, respectively) were constructed from DNA encoding the parent serotype molecule and appropriate oligonucleotides using standard molecular biology techniques. These were then cloned into the pJ401 expression vector with or without a C-terminal His.sub.10-tag and transformed into BLR (DE3) E. coli cells for over-expression. These cells were grown at 37° C. and 225 RPM shaking in 2 L baffled conical flasks containing 1 L modified Terrific Broth (mTB) supplemented with the appropriate antibiotic. Once the A.sub.600 reached >0.5, the incubator temperature was decreased to 16° C., and then induced with 1 mM IPTG an hour later for 20 h at 225 RPM shaking, to express the recombinant BoNT/AB construct.
[0422] Harvested cells were lysed by ultrasonication and clarified by centrifugation at 4500 RPM for 1 h at 4° C. The recombinant BoNT/AB chimeric molecules were then extracted in ammonium sulphate and purified by standard fast protein liquid chromatography (FPLC) techniques. This involved using a hydrophobic interaction resin for capture and an anion-exchange resin for the intermediate purification step. The partially purified molecules were then proteolytically cleaved with endoproteinase Lys-C to yield the active di-chain. This was further purified with a second hydrophobic interaction resin to obtain the final BoNT/AB chimera.
[0423] For BoNT/AB chimeric molecules with a decahistadine tag (H.sub.10) (chimera 1, 2, 3A), the capture step employed the use of an immobilised nickel resin instead of the hydrophobic interaction resin.
[0424] The sequence of each chimera is presented in Table 4.
TABLE-US-00023 chimeric BoNT/AB constructs Molecule SEQ ID NO Sequence Chimera 1 11 A1:1-871 + B1:858-1291 (E1191M/S1199Y) + His.sub.10-tag Chimera 2 12 A1:1-874 + ELGGGGSEL + B1:858-1291 (E1191M/S1199Y) + His.sub.10-tag Chimera 3A 13 A1:1-872 + B1: 860-1291 (E1191M/S1199Y) + His.sub.10-tag Chimera 3B 14 A1:1-872 + B1: 860-1291 (E1191M/S1199Y) Chimera 3C 15 A1:1-872 + B1: 860-1291
Example 4
Comparison of BoNT/AB Chimera 1, 2 and 3A
[0425] BoNT/AB chimera 1, 2 and 3A which have a C-terminal His.sub.10 tag and E1191M/S1199Y double mutation were purified as described in Example 3 (
Rat Spinal Cord Neurons Snap-25 Cleavage Assay
[0426] Primary cultures of rat spinal cord neurons (SCN) were prepared and grown, for 3 weeks, in 96 well tissue culture plates (as described in: Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B; and in: Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A). Serial dilutions of BoNT/AB were prepared in SCN feeding medium. The growth medium from the wells to be treated was collected and filtered (0.2 .Math.m filter). 125 .Math.L of the filtered medium was added back to each test well. 125 .Math.L of diluted toxin was then added to the plate (triplicate wells).
[0427] The treated cells were incubated at 37° C., 10% CO.sub.2, for 24 ± 1 h).
Analysis of BoNT Activity Using the SNAP-25 Cleavage Assay
[0428] Following treatment, BoNT was removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units/mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684) which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by the BoNT/A endopeptidase. The secondary antibody used was an HRP-conjugated anti-rabbit IgG (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using a pXi6 Access (Synoptics, UK). The intensity of bands was determined using GeneTools software (Syngene, Cambridge, UK) and the percentage of SNAP-25 cleaved at each concentration of BoNT calculated. Data were fitted to a 4-parameter logistic equation and pEC.sub.50 calculated using GraphPad Prism version 6 (GraphPad).
[0429] Table 5 below provides the pEC.sub.50 values determined for Chimera 1, 2 and 3A in the rat SCN SNAP-25 cleavage assay. These results show that the three BoNT/AB chimeras retained the ability to enter rat spinal cord neurons and cleave their target substrate. However, chimera 3A was more potent than chimera 1 and 2 in this assay (see also
TABLE-US-00024 pEC.sub.50 values. pEC.sub.50 ±SEM Chimera 1 12.42 ±0.04 Chimera 2 12.57 ±0.01 Chimera 3A 12.89 ±0.04
Digit Abduction Scoring (DAS) Assay
[0430] The method to measure the activity of BoNT/AB chimera 1, 2 and 3A in the DAS assay is based on the startled response toe spreading reflex of mice, when suspended briefly by the tail. This reflex is scored as Digit Abduction Score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscles of the hind paw. Mice are suspended briefly by the tail to elicit a characteristic startled response in which the animal extends its hind limb and abducts its hind digits. (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).
[0431] On the day of injection, mice were anaesthetized in an induction chamber receiving isoflurane 3% in oxygen. Each mouse received an intramuscular injection of BoNT/AB chimera or vehicle (phosphate buffer containing 0.2 % gelatine) in the gastrocnemius-soleus muscles of the right hind paw.
[0432] Following neurotoxin injection, the varying degrees of digit abduction were scored on a scale from zero to four, where 0= normal and 4= maximal reduction in digit abduction and leg extension. ED50 was determined by nonlinear adjustment analysis using average of maximal effect at each dose. The mathematical model used was the 4 parameters logistic model.
[0433] DAS was performed every 2 hours during the first day after dosing; thereafter it was performed 3 times a day for 4 days.
[0434]
[0435] Table 6 below provides the ED.sub.50 and DAS 4 doses determined for unmodified recombinant BoNT/A1 (rBoNT/A1 — SEQ ID NO: 2) and chimeras 1, 2 and 3A in the mouse DAS assay. These results show that of the three chimeras, chimera 3A has the highest in vivo potency in inducing muscle weakening. Studies shown in
TABLE-US-00025 ED.sub.50 values. ED.sub.50 (pg/mouse) DAS 4 dose (pg/mouse) rBoNT/A1 1 5 Chimera 1 23 200 Chimera 2 89 >300 Chimera 3A 18 133
Example 5
Comparison of BoNT/AB Chimera 3B, 3C and Unmodified BoNT/A1
[0436] Untagged BoNT/AB chimera 3B and 3C, respectively with and without the presence of the E1191M/S1199Y double mutation (SEQ ID NO: 14 and 15) were purified as described in Example 3 (
Human Pluripotent Stem Cells Snap-25 Cleavage Assay
[0437] Cryopreserved PERI.4U-cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of the cells were performed as recommended by the manufacturer. Briefly, cryovials containing the cells were thawed in a water bath at 37° C. for 2 minutes. After gentle resuspension the cells were transferred to a 50 mL tube. The cryovial was washed with 1 mL of Peri.4U® thawing medium supplied by the manufacturer and the medium was transfered drop-wise to the cell suspension to the 50 mL tube, prior to adding a further 2 mL of Peri.4U® thawing medium drop-wise to the 50 mL tube. Cells were then counted using a hemocytometer. After this, a further 6 mL of Peri.4U® thawing medium was added to the cell suspension. A cell pellet was obtained by centrifugation at 260 xg (e.g. 1,100 RPM) for 6 minutes at room temperature. Cells were then resuspended in complete Peri.4U® culture medium supplied by the manufacturer. Cells were plated at a density of 50,000 to 150,000 cells per cm.sup.2 on cell culture plates coated with poly-L-ornithine and laminin. Cells were cultured at 37° C. in a humidified CO.sub.2 atmosphere, and medium was changed completely every 2-3 days during culture.
[0438] For toxin treatment, serial dilutions of BoNTs were prepared in Peri.4U® culture medium. The medium from the wells to be treated was collected and filtered (0.2 .Math.m filter). 125 .Math.L of the filtered medium was added back to each test well. 125 .Math.L of diluted toxin was then added to the plate (triplicate wells). The treated cells were incubated at 37° C., 10% CO.sub.2, for 48 ± 1 h).
Analysis of BoNT Activity Using the SNAP-25 Cleavage Assay
[0439] Following treatment, BoNT was removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units/mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684) which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by the BoNT/A endopeptidase. The secondary antibody used was an HRP-conjugated anti-rabbit IgG (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using a pXi6 Access (Synoptics, UK). The intensity of bands was determined using GeneTools software (Syngene, Cambridge, UK) and the percentage of SNAP-25 cleaved at each concentration of BoNT calculated. Data were fitted to a 4-parameter logistic equation and pEC.sub.50 calculated using GraphPad Prism version 6 (GraphPad).
[0440]
TABLE-US-00026 pEC.sub.50 values. pEC.sub.50 ±SEM rBoNT/A1 10.21 ±0.05 Chimera 3B 12.38 ±0.06 Chimera 3C 10.72 ±0.08
Digit Abduction Scoring (DAS) Assay — Safety Ratio
[0441] The method to measure the activity of BoNTs in the DAS assay is based on the startled response toe spreading reflex of mice, when suspended briefly by the tail. This reflex is scored as Digit Abduction Score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscles of the hind paw. Mice are suspended briefly by the tail to elicit a characteristic startled response in which the animal extends its hind limb and abducts its hind digits. (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).
[0442] On the day of injection, mice were anaesthetized in an induction chamber receiving isoflurane 3% in oxygen. Each mouse received an intramuscular injection of BoNT or vehicle (phosphate buffer containing 0.2 % gelatine) in the gastrocnemius-soleus muscles of the right hind paw.
[0443] Following neurotoxin injection, the varying degrees of digit abduction were scored on a scale from zero to four, where 0= normal and 4= maximal reduction in digit abduction and leg extension. ED50 was determined by nonlinear adjustment analysis using average of maximal effect at each dose. The mathematical model used was the 4 parameters logistic model.
[0444] DAS was performed every 2 hours during the first day after dosing; thereafter it was performed 3 times a day for 4 days for all doses. Animals of the groups injected with vehicle and the lowest dose that induced during the first four days of injection a DAS of 4 were thereafter monitored until complete recovery of the muscle weakness to a DAS of 0 (no observed muscle weakness).
[0445] For calculation of the safety ratio all animals were weighed the day before toxin injection (D0) and thereafter once daily throughout the duration of the study. The average body weight, its standard deviation, and the standard error mean were calculated daily for each dose-group. To obtain the safety ratio for a BoNT (-10%ΔBW/ED.sub.50), the dose at which at any time during the study the average weight of a dose-group was lower than 10% of the average weight at D0 of that same dose-group was divided by the ED.sub.50 for the BoNT studied. The lethal dose was defined as the dose at which one or more of the animals within that dose-group died.
[0446]
[0447] Table 8 below provides the ED.sub.50 and DAS 4 doses determined for rBoNT/A1 and chimeras 3B and 3C in the mouse DAS assay. The table also provide the total duration of action for the DAS 4 dose until complete recovery of the muscle weakness to a DAS of 0 (no observed muscle weakness). In addition, the table shows the mouse lethal dose and the safety ratio (-10%ΔBW/ED.sub.50), as defined in the text above. In comparison to rBoNT/A1, chimeras 3B and 3C have longer duration of action, a better safety ratio, and a higher lethal dose. Studies shown in
TABLE-US-00027 DAS and Safety Ratios of the BoNT/AB chimeras. ED.sub.50 (DAS 2) Dose (pg/mouse) DAS 4 dose (pg/mouse) Total duration of action (day) with lowest DAS 4 dose Mouse lethal dose (pg) Safety ratio (-10%ΔBW/ED.sub.50) rBoNT/A1 0.9 2.3 29 18 4.5 Chimera 3B 8.0 89 42 200 14.1 Chimera 3C 5.0 26 42 8.9 7.4
Example 6
Pre-Clinical Testing of Modified BoNT/A (SEQ ID NO: 4)
[0448] The modified BoNT/A “Cat-A” (SEQ ID NO: 4) was subjected to additional pre-clinical testing.
Materials & Methods
Rat Digit Abduction Score (DAS) Assay
[0449] To assess the effects of modified BoNT/A (SEQ ID NO: 4) on in vivo muscular activity, dose-response studies were conducted using the rat DAS assay. The rat DAS assay is based on the toe spreading reflex, a characteristic startle response, when the animal is briefly grasped.
[0450] Following a single neurotoxin injection into the left peroneus muscle complex, the muscular weakness results in a reduction in digit abduction. The varying degrees of digit abduction are scored on a 5-point scale: 0=normal to 4=maximal reduction in digit abduction and leg extension (Broide RS, Rubino J, Nicholson GS, et al. The rat Digit Abduction Score (DAS) assay: A physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis. Toxicon 2013;71:18-24). DAS values were measured for the first five consecutive days after toxin injection and after this at intervals of two to three days until complete disappearance of the effect of modified BoNT/A (SEQ ID NO: 4) on the toe spreading reflex for lower doses and until recovery to DAS2 for doses resulting in DAS4. Transient BoNT-induced dose-dependent effects on body weight gain are considered evidence of a generalised toxin effect (Torii Y, Goto Y, Nakahira S, et al. Comparison of Systemic Toxicity between Botulinum Toxin Subtypes A1 and A2 in Mice and Rats. Basic Clin. Pharmacol. Toxicol. 2015;116:524-528.). At each evaluation time point rats were consequently weighed and side effects were noted. Dosing solutions of BoNT were masked (assigned random letters) before injection and until the end of the study. Potency was determined as the dose required to induce 50% of the effect (ED.sub.50: dose leading to a DAS value of 2). To determine ED.sub.50 and the 95% confidence intervals (Cls), doses ranging between 2.5 and 750 pg/kg were tested. Higher doses of 1, 1.5, 2, 2.4, 3, 4 and 5 ng/kg were also administered to assess possible side effects.
[0451] To evaluate the duration of action of modified BoNT/A (SEQ ID NO: 4) and compare it to the duration of action of unmodified BoNT/A (SEQ ID NO: 2), the median time necessary to return to a DAS2 reading of 2 was evaluated for the highest tolerated dose (no impact on body weight evolution compared to untreated rats) for both toxins in two independent, direct head-to-head studies.
Rat Single Dose Studies
[0452] Rats received a single intramuscular (i.m.) injection of modified BoNT/A (SEQ ID NO: 4) at doses of 0, 0.1, 1 and 3 ng/kg administered into the right gastrocnemius muscle. Control animals received SEQ ID NO: 4 diluent in the right gastrocnemius. Animals were euthanised 7 days after treatment (ten males and ten females per group) or after a 13 or 26-week observation period (five males and five females per dose). Irwin test observations, for assessment of central nervous system function, were performed pretest (Day -1), on Day 8 and during Weeks 13 and 27. Other clinical (adverse) signs assessed for were limping, small toxin injected muscle size, and soft distended abdomen.
Monkey Studies
[0453] Monkeys received single i.m. doses of 0, 0.1, 0.25 and 0.75 ng/kg modified BoNT/A (SEQ ID NO: 4) administered into the right gastrocnemius muscle. Animals were euthanised 7 days after treatment (three males and three females per group) or after a 13 or 26-week observation period (two males and two females per dose). Cardiovascular examinations, including haemodynamic, electrocardiogram and respiratory parameters, were performed by external telemetry pretest, on Days 8 and 15.
Preliminary Enhanced EFD in Pregnant Rat
[0454] The objective of the study was to provide initial information on the effects of modified BoNT/A (SEQ ID NO: 4) on embryonic and foetal development of the rat when administered by the i.m. route throughout the period of organogenesis. Modified BoNT/A (SEQ ID NO: 4) was administered by daily i.m. injection (gastrocnemius) at dose levels of 0.02, 0.05 and 0.1 ng/kg/day to groups of nine mated female Sprague-Dawley rats from days 6 (G6) to 17 (G17) of gestation, inclusive. Clinical condition, body weight and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on G21 and litter parameters were recorded. At necropsy, the females were examined macroscopically, the gravid uteri were weighed and for those who presented a small injected gastrocnemius muscle, this muscle and the contralateral muscle were weighed. All foetuses were weighed. The foetuses were then examined for external and visceral abnormalities and sexed. The heads of approximately half of the foetuses were fixed for internal examination by serial sectioning. The eviscerated carcasses of all fetuses were processed for skeletal examination.
Preliminary Extended EFD in Pregnant Rabbit
[0455] The objective of the study was to provide initial information on the effects of modified BoNT/A (SEQ ID NO: 4) on embryonic and foetal development of the rabbit when administered by the i.m. route throughout the period of organogenesis. Modified BoNT/A (SEQ ID NO: 4) was administered by daily i.m. injection (gastrocnemius) at dose levels of 0.002, 0.005 and 0.01 ng/kg/day to groups of nine mated female New Zealand White rabbits from days 6 (G6) to 19 (G19) of gestation, inclusive. Clinical condition, body weight and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on G29 and litter parameters were recorded. At necropsy, the females were examined macroscopically, the gravid uteri were weighed and for those who presented a small injected gastrocnemius muscle, this muscle and the contralateral muscle were weighed. All foetuses were weighed. The foetuses were then examined for external and visceral abnormalities and sexed. The heads of approximately half of the foetuses were fixed for internal examination by serial sectioning.
Results
[0456] By carrying out the studies as indicated above, the following pharmacological data (indicated in Table 9 below) were obtained for a number of different species administered the modified BoNT/A.
TABLE-US-00028 Pre-clinical results. Animal Study Type Results Mouse LD50 IP 0.422 ng/kg Rat DAS ED50 0.013 ng/kg DAS4 0.125 pg/kg CMAP Single Dose Distant Spread 0.002 ng/kg: No spread 0.3 ng/kg: -25% 0.8 ng/kg: -56% Single Dose Estimated NOAEL 1.5 ng/kg Estimated Lethal 3 ng/kg
[0457] Additionally, modified BoNT/A (SEQ ID NO: 4) was tested in a rat DAS assay to determine the duration of action when compared to Dysport®. Results are presented in Table 10 below:
TABLE-US-00029 Duration of action. Dysport® 3 U/rat 15 U/kg Modified BoNT/A 150 pg/rat 0.750 ng/kg Duration of Action (median days) 21.9 46.4
[0458] These data show that the modified BoNT/A has a duration of action that is more than double that of Dysport®.
Example 7
Calculation of a Unit Dose of Modified BoNT/A (SEQ ID NO: 4) for Treating Facial Lines
[0459] In view of the pre-clinical pharmacology data obtained in Example 6 above, a suitable unit dose range (UD) for administration of modified BoNT/A in humans has been calculated. The studies show that modified BoNT/A provides a longer duration of action than unmodified BoNT/A while at the same time exhibiting an improved safety profile. This improved safety profile may be expressed by the high Safety Ratio described herein for the modified BoNT/A.
[0460] As modified BoNT/A shares the same mechanism of action as Dysport® (albeit with an increased therapeutic index (i.e. an increased Safety Ratio) due to its modified properties), the lowest dose of modified BoNT/A for treating subjects with facial lines has been positioned for context relative to the labelled doses of Dysport® in the same muscle groups: [0461] In the Digit Abduction Score rat model, the ED.sub.50 of modified BoNT/A generated with the batch intended for clinical use is 13 pg/kg, and is more than 100-fold lower than the estimated no-observed-adverse-effect-level (NOAEL) of 1500 pg/kg in the same animal species. Following application of the corresponding allometric factor, this ED50 can be translated into a human equivalent dose (HED) of 130 pg. Based on DAS rat findings, 100 pg of modified BoNT/AB would eastimate to a dose of approximately 4 U of marketed Azzalure/Dysport. If the same approach is applied for calculation of a starting dose using the DAS rat ED50 established for Azzalure/Dysport (i.e., an ED50 of 0.5 U/kg), this would equate to a total starting dose of 5 U which is ⅒ of the total registered dose of 50 U for the temporary improvement of moderate to severe GL. [0462] The intraperitoneal mouse LD50 was established at 0.422 ng/kg (8.44 pg/animal). Under these conditions, a dose of 0.84 ng of modified BoNT/A corresponds to a dose of 100 U Dysport®.
[0463] The calculated lowest starting total dose is thus 100 pg. To provide some context and using the intraperitoneal mouse LD.sub.50 data above, 100 pg modified BoNT/A equates to approximately 12 U Dysport® and would thus be active when administered intramuscularly for treatment of facial lines.
[0464] The upper limit of the unit dose is thus calculated to be 300 pg (0.3 ng), with a total dose administered to the subject of 4800 pg as this remains below the rat NOAEL translated in human dose.
[0465] Thus, a suitable unit dose for treatment of facial lines using modified BoNT/A has been calculated at 20-300 pg. Based on the pre-clinical data obtained, this is 2-35 Units of modified BoNT/A based on the calculated median lethal intraperitoneal dose (LD.sub.50) in mice as determined using the mouse intraperitoneal Lethal Dose Assay.
[0466] Advantageously, modified BoNT/A can be injected to a greater number of muscles in the treatment of facial lines before reaching the maximum dose. This is a significant and advantageous finding leading to improved treatment of upper facial lines while providing clinicians with a greater range of treatment options.
Example 8
Dosage Regimen for Treating Glabellar Lines
[0467] Modified BoNT/A is provided as a lyophilised powder in 2 mL clear glass vials containing 15 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted with a mixture of sterile sodium chloride 0.9% v/w preservative free solution and diluent (formulation buffer containing only the excipients of modified BoNT/A). After reconstitution, the solution is further diluted as necessary.
[0468] Moderate to severe glabellar lines are treated according to the injection regimen shown in
[0469] The unit dose is 20-300 pg (2-35 Units).
[0470] Intramuscular injections are administered at up to five sites according to the unit dose. A maximum total dosage administered is 1500 pg (177 Units).
Example 9
Dosage Regimen for Treating Glabellar Lines and Forehead Lines
[0471] Modified BoNT/A is provided as a lyophilised powder in 2 mL clear glass vials containing 15 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted with a mixture of sterile sodium chloride 0.9% v/w preservative free solution and diluent (formulation buffer containing only the excipients of modified BoNT/A). After reconstitution, the solution is further diluted as necessary.
[0472] Moderate to severe glabellar and forehead lines are treated according to the injection regimen shown in
[0473] The unit dose is 20-300 pg (2-35 Units).
[0474] Intramuscular injections are administered at up to ten sites according to the unit dose. A maximum total dosage administered is 3000 pg (355 Units).
Example 10
Dosage Regimen for Treating Lateral Canthal Lines
[0475] Modified BoNT/A is provided as a lyophilised powder in 2 mL clear glass vials containing 15 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted with a mixture of sterile sodium chloride 0.9% v/w preservative free solution and diluent (formulation buffer containing only the excipients of modified BoNT/A). After reconstitution, the solution is further diluted as necessary.
[0476] Moderate to severe lateral canthal lines are treated according to the injection regimen shown in
[0477] The unit dose is 20-300 pg (2-35 Units).
[0478] Intramuscular injections are administered at up to six sites according to the unit dose. A maximum total dosage administered is 1800 pg (213 Units).
Example 11
Dosage Regimen for Treating Glabellar, Forehead and Lateral Canthal Lines
[0479] Modified BoNT/A is provided as a lyophilised powder in 2 mL clear glass vials containing 15 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted with a mixture of sterile sodium chloride 0.9% v/w preservative free solution and diluent (formulation buffer containing only the excipients of modified BoNT/A). After reconstitution, the solution is further diluted as necessary.
[0480] Moderate to severe glabellar, forehead and lateral canthal lines are treated according to the injection regimen shown in
[0481] The unit dose is 20-300 pg (2-35 Units).
[0482] Intramuscular injections are administered at up to sixteen sites according to the unit dose. A maximum total dosage administered is 4800 pg (569 Units).
Example 12
Further Characterisation of a Modified BoNT/A (SEQ ID NO: 14)
[0483] BoNT/AB chimera SEQ ID NO: 14 was tested in a mouse LD.sub.50 assay yielding a result of 1.202 ng/kg. 1 Unit of SEQ ID NO: 14 therefore corresponds to 24.04 pg in this assay.
[0484] Additionally, said BoNT/AB chimera was tested in a rat DAS assay to determine the duration of action (as per Example 6) when compared to Dysport®. Results are presented in Table 13 below:
TABLE-US-00030 Duration of action. Dysport® 3 U/rat 15 U/kg BoNT/AB 300 pg/rat 1.5 ng/kg Duration of Action (median days) 21.9 47.7
[0485] In conclusion, the duration of action of BoNT/AB was much higher than Dysport® and similar to that of SEQ ID NO: 4. Thus, it is expected that the unit doses and dosage regimen for SEQ ID NO: 4 could similarly be applied to BoNT/AB to provide an improved treatment of facial lines.
Example 13
Calculation of a Unit Dose of Modified BoNT/A (SEQ ID NO: 14) for Upper Facial Lines
[0486] In view of pre-clinical pharmacology data, a suitable unit dose range (UD) for administration of modified BoNT/A in humans has been calculated.
[0487] A DAS ED.sub.50 of 13 pg/kg was calculated for SEQ ID NO: 14. ED.sub.50 is considered as a minimal pharmacologically active dose, which is approximately 300-fold lower than the no observed adverse effect level (NOAEL) of 4 ng/kg in the same animal species. An ED.sub.50 of 13 pg/kg of SEQ ID NO: 14 in rats corresponds to a 0.8 ng dose for a human of 60 kg body weight.
[0488] Thus, the lower limit of a unit dose of 20 pg was selected. An upper limit of the unit dose of 1500 pg was selected, which is lower than the NOAEL of 4 ng/kg from both nonclinical safety species (rat and monkey) converted into human dose for 60 kg body weight.
[0489] In view of the improved safety profile the maximum total dose for the treatment of upper facial lines was set at 24,000 pg, which is derived from the NOAEL of 4 ng/kg from both nonclinical safety species (rat and monkey) converted into human dose for 60 kg body weight.
Example 14
Dosage Regimen for Treating Glabellar Lines Using a Modified BoNT/A (SEQ ID NO: 14)
[0490] Modified BoNT/A is provided as a lyophilised powder in a vial containing 36 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted.
[0491] Moderate to severe glabellar lines are treated according to the injection regimen shown in
[0492] The unit dose is 20-1500 pg (0.8-62 Units).
[0493] Intramuscular injections are administered at up to five sites according to the unit dose. A maximum total dosage administered is 7500 pg (312 Units).
Example 15
Dosage Regimen for Treating Glabellar Lines and Forehead Lines Using a modified BoNT/A (SEQ ID NO: 14)
[0494] Modified BoNT/A is provided as a lyophilised powder in a vial containing 36 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted.
[0495] Moderate to severe glabellar and forehead lines are treated according to the injection regimen shown in
[0496] The unit dose is 20-1500 pg (0.8-62 Units).
[0497] Intramuscular injections are administered at up to ten sites according to the unit dose. A maximum total dosage administered is 15,000 pg (624 Units).
Example 16
Dosage Regimen for Treating Lateral Canthal Lines Using a Modified BoNT/A (SEQ ID NO: 14)
[0498] Modified BoNT/A is provided as a lyophilised powder in a vial containing 36 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted.
[0499] Moderate to severe lateral canthal lines are treated according to the injection regimen shown in
[0500] The unit dose is 20-1500 pg (0.8-62 Units).
[0501] Intramuscular injections are administered at up to six sites according to the unit dose. A maximum total dosage administered is 9000 pg (374 Units).
Example 17
Dosage Regimen for Treating Glabellar, Forehead and Lateral Canthal Lines Using A Modified BoNT/A (SEQ ID NO: 14)
[0502] Modified BoNT/A is provided as a lyophilised powder in a vial containing 36 ng of modified BoNT/A per vial. The lyophilised powder is reconstituted.
[0503] Moderate to severe glabellar, forehead and lateral canthal lines are treated according to the injection regimen shown in
[0504] The unit dose is 20-1500 pg (0.8-62 Units).
[0505] Intramuscular injections are administered at up to sixteen sites according to the unit dose. A maximum total dosage administered is 24,000 pg (998 Units).
[0506] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.