Treatment of Limb Spasticity

Abstract

The present invention relates to a modified botulinum neurotoxin A (BoNT/A) for use in treating limb spasticity or paediatric limb spasticity, wherein the modified BoNT/A is administered by intramuscular injection to a plurality of affected muscles of a subject, wherein the modified BoNT/A is administered by way of a unit dose of 53 Units to 948 Units or 26.5 Units to 474 Units of modified BoNT/A at the plurality of affected muscles.

Claims

1-94. (canceled)

95. A method for treating limb spasticity, (BoNT/A) by intramuscular injection to a plurality of affected muscles of a subject, wherein: (i) the subject is an adult subject and the modified BoNT/A is administered by way of a unit dose of 450 pg to 8,000 pg of modified BoNT/A at the plurality of affected muscles, and wherein the total dose administered during the treatment is up to 120,000 pg; or (ii) the subject is a paediatric subject and the modified BoNT/A is administered by way of a unit dose of 225 pg to 4,000 pg of modified BoNT/A at the plurality of affected muscles, and wherein the total dose administered during the treatment is up to 60,000 pg, wherein the plurality of affected muscles are selected from: a first group comprising: the flexor digitorum superficialis, the flexor digitorum profundus, the flexor carpi radialis, the flexor carpi ulnaris, the brachioradialis, the pronator teres, the biceps brachii, the gastrocnemius medial head, the gastrocnemius lateral head, the flexor digitorum longus, the flexor hallucis longus, the gastrocnemius, the deltoid, the levator scapulae, the pronator quadratus, the flexor policis longus, the adductor policis, the flexor policis brevis, the palmaris longus, the lumbricales, the opponens policis, the adductor magnus, the adductor longus, the adductor brevis, the gracilis, the medial hamstrings, the lateral hamstrings, the tensor fascia lata, the rectus femons, the vastus lateralis, the vastus medialis, the vastus intermedius, the gluteus maximus, the tibialis anterior, the flexor digitorum brevis, the extensor hallucis longus, and the flexor hallucis brevis; and a second group comprising: the triceps brachii (long head), the subscapulans, the pectoralis, the latissimus dorsi, the biceps brachii, the brachialis, the soleus, the tibialis posterior, the brachioradialis, the teres major, the iliopsoas, and the gastrocnemius; and wherein a single unit dose is administered at an affected first group muscle and/or multiple unit doses are administered at an affected second group muscle, 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.

96-119. (canceled)

120. A method for treating limb spasticity, the method comprising administering a modified botulinum neurotoxin A (BoNT/A) by intramuscular injection to a plurality of affected muscles of a subject, wherein: (i) the subject is an adult subject and the modified BoNT/A is administered by way of a unit dose of 750 pg to 17,000 pg of modified BoNT/A at the plurality of affected muscles, and wherein the total dose administered during the treatment is up to 255,000 pg; or (ii) the subject is a paediatric subject and the modified BoNT/A is administered by way of a unit dose of 375 pg to 8,500 pg of modified BoNT/A at the plurality of affected muscles, and wherein the total dose administered during the treatment is up to 127,500 pg, wherein the plurality of affected muscles are selected from: a first group comprising: the flexor digitorum superficialis, the flexor digitorum profundus, the flexor carpi radialis, the flexor carpi ulnaris, the brachioradialis, the pronator teres, the biceps brachii, the gastrocnemius medial head, the gastrocnemius lateral head, the flexor digitorum longus, the flexor hallucis longus, the gastrocnemius, the deltoid, the levator scapulae, the pronator quadratus, the flexor policis longus, the adductor policis, the flexor policis brevis, the palmaris longus, the lumbricales, the opponens policis, the adductor magnus, the adductor longus, the adductor brevis, the gracilis, the medial hamstrings, the lateral hamstrings, the tensor fascia lata, the rectus femoris, the vastus lateralis, the vastus medialis, the vastus intermedius, the gluteus maximus, the tibialis anterior, the flexor digitorum brevis, the extensor hallucis longus, and the flexor hallucis brevis; and a second group comprising: the triceps brachii (long head), the subscapularis, the pectoralis, the latissimus dorsi, the biceps brachii, the brachialis, the soleus, the tibialis posterior, the brachioradialis, the teres major, the iliopsoas, and the gastrocnemius; and wherein a single unit dose is administered at an affected first group muscle and/or multiple unit doses are administered at an affected second group muscle, 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).

121. (canceled)

122. The method of claim 120, 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 is the dose required to produce a DAS score of 2.

123. The method of claim 120, wherein the limb spasticity is upper limb spasticity.

124-129. (canceled)

130. The method of claim 120, wherein the subject is an adult subject and the unit dose is 1,000 pg to 16,000 pg of modified BoNT/A.

131. The method of claim 120, wherein the subject is an adult subject and the total dose administered is 12,750 pg to 255,000 pg.

132. The method of claim 120, wherein the subject is an adult subject and the total dose administered is up to 240,000 pg.

133. The method of claim 120, wherein the subject is an adult subject and the total dose administered is 15,000 pg to 240,000 pg.

134. The method of claim 120, wherein the subject is an adult subject and the total dose administered is 85,333 pg to 240,000 pg.

135. (canceled)

136. The method of claim 120, wherein the modified BoNT/A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14.

137. A unit dosage form of modified botulinum neurotoxin A (BoNT/A), the unit dosage form comprising: (a) 750 pg to 17,000 pg or 375 pg to 8,500 pg of modified BoNT/A comprising a BoNT/A light chain, a BoNT/A translocation domain and a botulinum neurotoxin B (BoNT/B) receptor binding domain (H.sub.c domain): and, optionally a pharmaceutically acceptable carrier, excipient, adjuvant, and/or salt.

138. The unit dosage form of claim 137, comprising 1000 pg to 16,000 pg or 500 pg to 8,000 pg of the modified BoNT/A.

139-177. (canceled)

178. The method of claim 120, wherein the subject is a paediatric subject and the unit dose is 500 pg to 8,000 pg of modified BoNT/A.

179. The method of claim 120, wherein the subject is a paediatric subject and the total dose administered is 6,375 pg to 127,500 pg.

180. The method of claim 120, wherein the subject is a paediatric subject and the total dose administered is up to 120,000 pg.

181. The method of claim 120, wherein the subject is a paediatric subject and the total dose administered is 7,500 pg to 120,000 pg.

182. The method of claim 120, wherein the subject is a paediatric subject and the total dose administered is 42,666.5.00 pg to 120,000 pg.

183-195. (canceled)

196. The method of claim 136, wherein the modified BoNT/A is in a di-chain form.

197. The unit dosage form of claim 137, wherein the modified BoNT/A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14.

198. The unit dosage form of claim 197, wherein the modified BoNT/A is in a di-chain form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0614] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0615] FIG. 1 shows the five most common upper limb spasticity clinical patterns involving upper arm joints.

[0616] FIG. 2 shows the FDA approved dosages of Dysport® for treating spasticity in adults.

[0617] FIG. 3 shows the isoelectric focusing (IEF) gel of cationic constructs.

[0618] FIG. 4 shows the percentage SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) for Cat5v2(K1064H/N954K) (A), Cat5v2(K1064H/N886K) (B) and Cat5v2(K1064H/ N1025K) (C), and summary of pEC50 relative to nBoNT/A1. (A, B, C) Rat embryonic spinal cord neurons were cultured for three weeks and treated with Cat5v4 for 24 h, before Western blotting with SNAP-25 specific antibody. Data is mean ±SEM from three independent experiments in triplicate. (D) Relative potency of Cat5v2(K1064H/N886K), Cat5v2(K1064H/N954K) and Cat5v2(K1064H/ N1025K) to nBoNT/A1 (List Biological Laboratories) in the rat eSCN SNAP-25 cleavage potency assay. Each point corresponds to an individual batch and is a mean of 3 independent pEC50 determinations based on an 8-point concentration response curve (CRC). Each concentration in the CRC was assessed in triplicate. Potency comparisons are made to a mean of List batches, pooled data n=24. Data are mean ±SEM of n=3 batches per Cat5v4.

[0619] FIG. 5 shows the potency (t.sub.50) of nBoNT/A1 and Cat5v4 in the mouse phrenic nerve hemi-diaphragm assay (mPNHD). Mouse phrenic nerve hemi-diaphragm tissue was incubated with Cat5v4 or native BoNT/A1 as indicated. Diaphragm contractile force was recorded until the contraction was no longer detectable or after 140 minutes. Each point corresponds to independent determinations. The t.sub.50 value is the time required to inhibit the contractile force of the mouse hemi-diaphragm by 50%.

[0620] FIG. 6 shows SDS-PAGE of purified recombinant BoNT/AB chimera 1, 2 and 3A (SEQ ID NO: 11, 12 and 13 respectively). Lanes are labelled “Marker” (molecular weight marker), “-DTT” (oxidised BoNT/AB chimera sample), and “+DTT” (reduced BoNT/AB chimera sample).

[0621] FIG. 7 shows cleavage of SNAP-25 in rat spinal cord neurones by recombinant BoNT/AB chimera 1, 2 and 3A (SEQ ID NO: 11, 12 and 13 respectively). Cultured rat primary spinal cord neurons (SCN) were exposed to various concentrations of recombinant BoNT/AB chimera 1, 2 or 3A for 24 hours, at 37° C. in a humidified atmosphere with 10% CO.sub.2. Cells were then lysed with 1x NuPAGE buffer supplemented with DTT and Benzonase. The samples were transferred to microcentrifuge tubes, heated for 5 min at 90° C. on heat block and stored at -20° C., before analysis of SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody, that detects both the full length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody.

[0622] FIG. 8 shows mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex muscles of one hind limb, under short general anaesthesia; muscle weakening was measured on a 0-4 scale using the digit abduction score (DAS). DAS max values were determined for each dose and plotted against dose and the data were fitted to a 4-parameter logistic equation, ED50 and dose leading to DAS 4 (DAS 4 dose) values were determined.

[0623] FIG. 9 shows SDS-PAGE of purified recombinant BoNT/AB chimera 3B and 3C (SEQ ID NO: 14 and 15 respectively). Lanes are labelled “Marker” (molecular weight marker), “-DTT” (oxidised BoNT/AB chimera sample), and “+DTT” (reduced BoNT/AB chimera sample).

[0624] FIG. 10 shows cleavage of SNAP-25 by unmodified BoNT/A and BoNT/AB chimera 3B and 3C (SEQ ID NO: 2, 14 and 15 respectively) in human induced pluripotent stem cell derived peripheral neurons (PERL4U - Axiogenesis, Germany). PER1.4U cells were exposed to various concentrations of recombinant BoNT/A, or BoNT/AB chimera 3B or 3C for 24 hours, at 37° C. in a humidified CO.sub.2 atmosphere containing 5% CO.sub.2. Cells were then lysed with 1x NuPAGE buffer supplemented with DTT and Benzonase. The samples were transferred to microcentrifuge tubes, heated for 5 min at 90° C. on heat block and stored at -20° C., before analysis of SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody, that detects both the full length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody.

[0625] FIG. 11 shows duration of muscle weakening over time in the mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex muscles of one hind limb, under short general anaesthesia; muscle weakening was measured on a 0-4 scale using the digit abduction score (DAS). Animals of the group injected with the lowest dose that induced during the first four days of injection a DAS of 4 were monitored until complete recovery of the muscle weakness to a DAS of 0 (no observed muscle weakness).

SEQUENCE LISTING

[0626] 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.

[0627] SEQ ID NO: 1 (Nucleotide Sequence of Unmodified BoNT/A)

TABLE-US-00009 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

[0628] SEQ ID NO: 2 (Polypeptide Sequence of Unmodified BoNT/A)

TABLE-US-00010 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIP KYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRWFKY SQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASN NIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFW GDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLN SSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIG FHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0629] SEQ ID NO: 3 (Nucleotide Sequence of Modified BoNT/A “Cat-A”)

TABLE-US-00011 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

[0630] SEQ ID NO: 4 (Polypeptide Sequence of Modified BoNT/A “Cat-A”)

TABLE-US-00012 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESKHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIP KYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRWFKY SQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHASN KIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFW GDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLN SSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIG FHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0631] SEQ ID NO: 5 (Nucleotide Sequence of Modified BoNT/A “Cat-B″)

TABLE-US-00013 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

[0632] SEQ ID NO: 6 (Polypeptide Sequence of Modified BoNT/A “Cat-B″)

TABLE-US-00014 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIP KYFKKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRWFKY SQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHASN KIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFW GDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLN SSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIG FHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0633] SEQ ID NO: 7 (Nucleotide Sequence of Modified BoNT/A “Cat-C″)

TABLE-US-00015 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

[0634] SEQ ID NO: 8 (Polypeptide Sequence of Modified BoNT/A “Cat-C″)

TABLE-US-00016 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

[0635] SEQ ID NO: 9 (Nucleotide Sequence of Modified BoNT/A “Cat-D”)

TABLE-US-00017 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

[0636] SEQ ID NO: 10 (Polypeptide Sequence of Modified BoNT/A “Cat-D”)

TABLE-US-00018 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIP KYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRWFKY SQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASN NIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFW GDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLN SSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKRKEYRLATNASQAG VEKILSALEIPRVRRLSQVVVMKSKNDQGITNKCKMNLQDRRGNDIGFIG FHQFNNIAKLVASNWYNRQIERRSRRLGCSWEFIPVDDGWGERPL

[0637] SEQ ID NO: 11 (Polypeptide Sequence of Modified BoNT/A “Chimera 1”)

TABLE-US-00019 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

[0638] SEQ ID NO: 12 (Polypeptide Sequence of Modified BoNT/A “Chimera 2”)

TABLE-US-00020 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

[0639] SEQ ID NO: 13 (Polypeptide Sequence of Modified BoNT/A “Chimera 3A”)

TABLE-US-00021 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

[0640] SEQ ID NO: 14 (Polypeptide Sequence of Modified BoNT/A “Chimera 3B”)

TABLE-US-00022 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

[0641] SEQ ID NO: 15 (Polypeptide Sequence of Modified BoNT/A “Chimera 3C”)

TABLE-US-00023 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

[0642] SEQ ID NO: 16 (Polypeptide Sequence of BoNT/B)

TABLE-US-00024 MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERY TFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIK SKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVER KKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYV SVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIV PNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYK SLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNI SDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVD NEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISK IELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDI RDISLTSSFDDALLFSNKVYSFFSMDYIKTANKWEAGLFAGWVKQIVNDF VIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASIL LEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIV AQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINI DFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNT LKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIE MFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKL TSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYT IINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINR WFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRT QFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMF NAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKS NSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDS DEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFE EYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

EXAMPLES

Example 1

Cloning, Expression and Purification

[0643] 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-00025 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.

[0644] 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.

[0645] 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. 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, WO2017055274A1, EP2524963B1, EP2677029B1, and US10087432B2.

Example 2

Characterization of Purified Modified BoNT/A

[0646] The modified BoNTs described in Example 1 above were characterised experimentally as follows.

[0647] Measurement of the pl showed that the modified BoNTs had an isoelectric point greater than that of unmodified (native) BoNT/A1 - see FIG. 3 and Table 2 below.

TABLE-US-00026 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

[0648] 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). FIG. 4 shows that the modified BoNTs retained the same ability to enter the neuron and cleave SNAP-25 as native BoNT/A1.

[0649] Potency of the modified BoNTs was further assessed using the mouse phrenic nerve hemi-diaphragm assay (mPNHD). FIG. 5 shows that the modified BoNTs retained the same ability to inhibit the contractile abilities of the mouse hemi-diaphragm as native BoNT/A1.

[0650] 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-00027 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

[0651] 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.

[0652] 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

[0653] 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.

[0654] 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.

[0655] 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.

[0656] The sequence of each chimera is presented in Table 4.

TABLE-US-00028 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

[0657] 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 (FIG. 6) and tested for functional activity.

Rat Spinal Cord Neurons Snap-25 Cleavage Assay

[0658] 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). 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

[0659] 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).

[0660] 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 FIG. 7).

TABLE-US-00029 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

[0661] 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).

[0662] 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.

[0663] 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.

[0664] DAS was performed every 2 hours during the first day after dosing; thereafter it was performed 3 times a day for 4 days.

[0665] FIG. 8 shows the fitted curves for chimera 1, 2 and 3A (SEQ ID NO: 11, 12 and 13 respectively). The chimera 3A curve is shifted to the left, meaning lower doses of chimera 3A achieved a similar DAS response compared to chimera 1 and 2, therefore showing that chimera 3A is more potent than the others in the mouse DAS assay; see also the table below (Table 6) that provides the values for the calculated ED50 and the dose leading to DAS 4 (highest score) for each chimera.

[0666] 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 FIG. 8 and Table 6 were performed in mice obtained from Charles River laboratories.

TABLE-US-00030 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

[0667] 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 (FIG. 9), and tested for functional activity using unmodified BoNT/A (SEQ ID NO: 2) as a reference.

Human Pluripotent Stem Cells Snap-25 Cleavage Assay

[0668] 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.

[0669] 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

[0670] 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).

[0671] FIG. 10 shows that chimera 3B and 3C displayed greater potency than rBoNT/A1 in cleaving SNAP-25 in induced human pluripotent stem cells but the former significantly more so. This can be explained by the double mutation which increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells (FIG. 10, Table 7).

TABLE-US-00031 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

[0672] 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).

[0673] 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.

[0674] 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.

[0675] 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).

[0676] 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.

[0677] FIG. 11 shows the duration of muscle weakening over time in the mouse digit abduction scoring assay for unmodified BoNT/A, chimera 3B and chimera 3C (SEQ ID NO: 2, 14 and 15), showing that the chimera has longer duration of action.

[0678] 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 FIG. 11 and Table 8 were performed in mice obtained from Janvier laboratories.

TABLE-US-00032 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)

[0679] 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

[0680] 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. 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.

[0681] 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

[0682] 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

[0683] 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

[0684] 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

[0685] 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

[0686] 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-00033 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 Monkey Single Dose Estimated NOAEL 0.125 ng/kg Lethal 0.375 ng/kg Rat (Pregnant pEFD Maternal NOAEL and fetal NOEL 0.1 ng/kg/day Female) Rabbit (Pregnant Female) pEFD Maternal NOAEL 0.005 ng/kg/day Fetal NOEL 0.01 ng/kg/day

[0687] 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-00034 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

[0688] 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 Limb Spasticity

[0689] 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.

[0690] As modified BoNT/A shares the same mechanism of action as Dysport® (albeit with an increased Safety Ratio due to its modified properties), the lowest dose of modified BoNT/A for treating subjects with spasticity has been positioned for context relative to the labelled doses of Dysport® in that same muscle group: [0691] In the Digit Abduction Score rat model, the ED.sub.50 of modified BoNT/A 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. In the same rat model, the ED.sub.50 of Dysport® is . Based on these animal data, a dose of 2.6 ng of modified BoNT/A would estimate to a dose of 100 U Dysport® which is the lowest limit of the labelled range for treating a single spastic finger flexor muscle in an adult subject with upper limb spasticity. [0692] The intraperitoneal mouse LD.sub.50 was established at 8.44 pg. Under these conditions, a dose of 0.84 ng of modified BoNT/A corresponds to a dose of 100 U Dysport®.

[0693] The calculated lowest dose is thus 500 pg (0.5 ng). To provide some context and using the intraperitoneal mouse LD.sub.50 data above, 0.5 ng modified BoNT/A equates to approximately 60 U Dysport®, and would thus be active when administered intramuscularly for treatment of limb spasticity.

[0694] The estimated NOAEL of 1.5 ng/kg of modified BoNT/A in rats corresponds to a 90 ng dose for a human of 60 kg body weight. In monkeys, the more sensitive of the two nonclinical species tested, the estimated NOAEL of 0.125 ng/kg of modified BoNT/A corresponds to a 7.5 ng dose for a human of 60 kg body weight.

[0695] The upper limit of the unit dose is thus calculated to be 7,500 pg (7.5 ng) as this remains below the rat NOAEL translated in human dose.

[0696] Thus, a suitable unit dose for treatment of limb spasticity using modified BoNT/A has been calculated at 500-7500 pg. Based on the pre-clinical data obtained, this is 59-889 Units of modified BoNT/A (and also corresponds to 59-889 Units of Dysport®) based on the calculated median lethal intraperitoneal dose (LD.sub.50) in mice as determined using the mouse intraperitoneal Lethal Dose Assay.

[0697] In view of the improved safety profile when compared to Dysport® as determined by the pre-clinical data of Example 6, total dosages (in units) administered in treating limb spasticity are expected to be almost 10x greater than that for Dysport®. The maximum total dose of Dysport® for treatment of upper and lower limb spasticity in adults is 1500 Units (see FIG. 2).

[0698] Advantageously, modified BoNT/A can be injected to a greater number of muscles in the treatment of limb spasticity before reaching the maximum dose. This is a significant and advantageous finding leading to improved treatment of limb spasticity while providing clinicians with a greater range of treatment options. For the first time, it also provides the option of being able to treat additional large muscles such as those of the shoulder, while also treating the elbow, forearm, and/or wrist well within the maximum dose.

Example 8

Dosage Regimen for Treating Adult Upper Limb Spasticity

[0699] Modified BoNT/A (e.g. SEQ ID NO:4) 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.

[0700] The unit dose (UD) is 500-7,500 pg (59-889 Units).

[0701] Adult upper limb spasticity is treated by intramuscular injection according to the following dosage regimen (Table 11):

TABLE-US-00035 Dosage regimen Clinical Patterns Muscles Injected Dosage Total Volume Clenched fist Flexor Digitorum Superficialis (FDS) 1x UD 1 mL Flexor Digitorum Profundus (FDP) 1x UD 1 mL Flexed wrist Flexor Carpi Radialis (FCR) 1 x UD 1 mL Flexor Carpi Ulnaris (FCU) 1 x UD 1 mL Flexed elbow Brachioradialis 1 x UD 1 mL Brachialis 2 x UD 2 mL Pronator Teres 1 x UD 1 mL Biceps Brachii 2 x UD 1 x or 2 x UD 2 mL 1-2 mL Adducted/rotated shoulder Triceps Brachii (long head) 2 x UD 2 mL Pectoralis Major 2 x UD 2 mL Subscapularis 2 x UD 2 mL Latissimus Dorsi 2 x UD 2 mL

[0702] A maximum total dosage administered is 15x UD. This corresponds to 112,500 pg/13,335 Units. This is more than 10x greater than the maximum total dosage of Dysport® that can be administered during treatment of adult upper limb spasticity without approaching toxic limits (a concern with conventional treatment regimens). Thus, the clinician is able to tailor treatment to the patient with the knowledge that 15x UD can be administered without any concern of toxicity, thereby allowing the treatment of additional muscles of the subject, including the shoulder, and/or ensuring each muscle receives a pharmaceutically effective dose.

Example 9

Dosage Regimen for Treating Adult Lower Limb Spasticity

[0703] Modified BoNT/A (e.g. SEQ ID NO:4) 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.

[0704] The unit dose (UD) is 500-7,500 pg (59-889 Units).

[0705] Adult lower limb spasticity is treated by intramuscular injection according to the following dosage regimen (Table 12):

TABLE-US-00036 Dosage regimen Muscles Injected Dosage Gastrocnemius (Medial head) 1x UD Gastrocnemius (Lateral head) 1x UD Soleus 3x UD Tibialis posterior 2x UD Flexor digitorum longus 1x UD Flexor hallucis longus 1x or 2x UD

[0706] A maximum total dosage administered is 15x UD. This corresponds to 112,500 pg/13,335 Units. This is almost 10x greater than the maximum total dosage of Dysport® that can be administered during treatment of adult upper limb spasticity without approaching toxic limits (a concern with conventional treatment regimens). Thus, the clinician is able to tailor treatment to the patient with the knowledge that 15x UD can be administered without any concern of toxicity, thereby allowing the treatment of additional muscles of the subject and/or ensuring each muscle receives a pharmaceutically effective dose.

Example 10

Further Characterisation of a BoNT/AB Chimera (SEQ ID NO: 14)

[0707] 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.

[0708] 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-00037 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

[0709] 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 limb spasticity.

Example 11

Calculation of a Unit Dose of Modified BoNT/A (SEQ ID NO: 14) for Treating Limb Spasticity

[0710] In view of pre-clinical pharmacology data, a suitable unit dose range (UD) for administration of modified BoNT/A in humans has been calculated.

[0711] 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.

[0712] Thus, the lower limit of a unit dose of 1000 pg was selected. An upper limit of the unit dose of 16,000 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.

[0713] In view of the improved safety profile the maximum total dose for the treatment of limb spasticity was set at 240,000 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.

[0714] Advantageously, modified BoNT/A (SEQ ID NO: 14) can be injected to a greater number of muscles in the treatment of limb spasticity before reaching the maximum dose. This is a significant and advantageous finding leading to improved treatment of limb spasticity while providing clinicians with a greater range of treatment options. For the first time, it also provides the option of being able to treat additional large muscles such as those of the shoulder, while also treating the elbow, forearm, and/or wrist well within the maximum dose.

Example 12

Dosage Regimen for Treating Adult Upper Limb Spasticity Using a Modified BoNT/A (SEQ ID NO: 14)

[0715] 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.

[0716] The unit dose (UD) is 1000-16,000 pg (42-666 Units [measured by mouse LD.sub.50]).

[0717] Adult upper limb spasticity is treated by intramuscular injection according to the following dosage regimen (Table 14):

TABLE-US-00038 Dosage regimen Clinical Patterns Muscles Injected Dosage Total Volume Clenched fist Flexor Digitorum Superficialis (FDS) 1x UD 1 mL Flexor Digitorum Profundus (FDP) 1x UD 1 mL Flexed wrist Flexor Carpi Radialis (FCR) 1 x UD 1 mL Flexor Carpi Ulnaris (FCU) 1 x UD 1 mL Flexed elbow Brachioradialis 1 x UD 1 mL Brachialis 2 x UD 2 mL Pronator Teres 1 x UD 1 mL Biceps Brachii 2 x UD 1 x or 2 x UD 2 mL 1-2 mL Adducted/rotated shoulder Triceps Brachii (long head) 2 x UD 2 mL Pectoralis Major 2 x UD 2 mL Subscapularis 2 x UD 2 mL Latissimus Dorsi 2 x UD 2 mL

[0718] A maximum total dosage administered is 15x UD. This corresponds to 240,000 pg/9,990 Units. Thus, the clinician is able to tailor treatment to the patient with the knowledge that 15x UD can be administered without any concern of toxicity, thereby allowing the treatment of additional muscles of the subject, including the shoulder, and/or ensuring each muscle receives a pharmaceutically effective dose.

Example 13

Dosage Regimen for Treating Adult Lower Limb Spasticity Using a Modified BoNT/A (SEQ ID NO: 14)

[0719] 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.

[0720] The unit dose (UD) is 1000-16,000 pg (42-666 Units).

[0721] Adult lower limb spasticity is treated by intramuscular injection according to the following dosage regimen (Table 15):

TABLE-US-00039 Dosage regimen Muscles Injected Dosage Gastrocnemius (Medial head) 1x UD Gastrocnemius (Lateral head) 1x UD Soleus 3x UD Tibialis posterior 2x UD Flexor digitorum longus 1x UD Flexor hallucis longus 1x or 2x UD

[0722] A maximum total dosage administered is 15x UD. This corresponds to 240,000 pg/9,990 Units. Thus, the clinician is able to tailor treatment to the patient with the knowledge that 15x UD can be administered without any concern of toxicity, thereby allowing the treatment of additional muscles of the subject and/or ensuring each muscle receives a pharmaceutically effective dose.

[0723] 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.