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WO2018109447A1 - Neurotoxines - Google Patents

Neurotoxines Download PDF

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Publication number
WO2018109447A1
WO2018109447A1 PCT/GB2017/053705 GB2017053705W WO2018109447A1 WO 2018109447 A1 WO2018109447 A1 WO 2018109447A1 GB 2017053705 W GB2017053705 W GB 2017053705W WO 2018109447 A1 WO2018109447 A1 WO 2018109447A1
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amino acid
acid sequence
seq
polypeptide
neurotoxin
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PCT/GB2017/053705
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English (en)
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Bazbek Davletov
Charlotte LEESE
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The University Of Sheffield
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Publication of WO2018109447A1 publication Critical patent/WO2018109447A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24068—Tentoxilysin (3.4.24.68), i.e. tetanus neurotoxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides neurotoxins comprising a SNARE peptidase domain, a translocation domain, a first Neuronal binding domain (Nbd) and a second Neuronal binding domain (Nbd).
  • the invention also provides compositions (e.g. pharmaceutical compositions) comprising said neurotoxins, and uses thereof.
  • Clostridial neurotoxin family consists of tetanus neurotoxin (TeNT) and seven distinct botulinum neurotoxins (BoNTs; types A to G), which cause the diseases tetanus and botulism respectively.
  • Botulinum neurotoxin types A, B, E and F have been shown to cause disease in humans, with types A, B and E being associated with foodborne illness.
  • Botulinum type C has been shown to cause limberneck in birds, whilst type D causes botulism in other mammals. No disease has currently been attributed to botulinum neurotoxin type G.
  • Each of the eight Clostridial neurotoxins is synthesized as a -150 kDa single chain protein with three structurally independent domains: a SNARE peptidase domain, a translocation domain, and a neuronal binding domain (Rossetto, O. , Pirazzini, M. & Montecucco, C. Nat Rev Microbiol 12, 535-549, (2014)).
  • the single chain protein is subsequently cleaved by Clostridial or host proteases to generate an N-terminal ⁇ 50 kDa enzymatic Light chain (Lc; comprising the SNARE peptidase domain) and a ⁇ 100 kDa Heavy chain (He; comprising the translocation domain and the neuronal binding domain). Both chains remain attached via a single disulphide bond, a peptide loop and further non-covalent interactions.
  • Lc enzymatic Light chain
  • He ⁇ 100 kDa Heavy chain
  • the three protein domains of a Clostridial neurotoxin perform distinct roles in toxin delivery and activity within host cells.
  • the neuronal binding domain is required to specifically bind to target host cells (neurons); the translocation domain facilitates endocytosis of the toxin into the cytosol of the host cell; and the peptidase domain catalyses the proteolysis of one of three soluble /V-ethylmaleimide-sensitive fusion protein attachment receptors (SNAREs) in the cell, namely VAMP/synaptobrevin, a synaptosome-associated protein of 25 kDa (SNAP25), or syntaxin.
  • SNAREs soluble /V-ethylmaleimide-sensitive fusion protein attachment receptors
  • BoNT/A and BoNT/E proteolyse SNAP-25 are essential components of the host vesicular membrane fusion apparatus, cleavage of any one of these proteins blocks neurotransmitter release from the host cell.
  • BoNT/A and BoNT/E proteolyse SNAP-25 while BoNTs B, D, F and G cleave VAMP on the synaptic vesicles.
  • BoNT/A shortened by only nine amino acids by BoNT/A retains its ability to interact with syntaxin and synaptobrevin but cannot mediate the normal vesicle fusion process.
  • BoNT type B binds a simpler ganglioside GD2a and then a synaptic vesicle protein called synaptotagmin (Chai, Q. et al. Nature 444, 1096-1 100, (2006)).
  • botulinum types C and D were shown to rely on gangliosides GD1b and GD1a, respectively, possibly without involving a protein receptor (Strotmeier, J. et al.
  • the SNARE peptidase domain also imparts selectivity, in the form of the particular substrate that is cleaved. Substrate cleavage blocks neurotransmitter release, with different clinical symptoms for botulinum neurotoxins and tetanus neurotoxins. Botulinum neurotoxins inhibit the release of neurotransmitters at peripheral nerve terminals, resulting in flaccid paralysis, whereas tetanus neurotoxin causes spastic paralysis by blocking neurotransmitter release at central inhibitory interneurons.
  • Clostridial neurotoxins particularly botulinum neurotoxins (e.g. Botox, or treatment of muscle spasms).
  • botulinum neurotoxins e.g. Botox, or treatment of muscle spasms.
  • their use is typically limited to administration in low doses so as to avoid or minimise a host immune response (Naumann, M., Boo, L. M., Ackerman, A. H. & Gallagher, C. J. J Neural Transm (Vienna) 120, 275-290, (2013)).
  • the invention is based on the surprising finding that neurotoxin efficacy can be improved by modifying the neurotoxin protein to include two neuronal binding domains allowing smaller doses to achieve the same biological effect.
  • novel neurotoxin molecules which include two neuronal (receptor) binding domains. Surprisingly, these novel molecules are more effective in cleaving their SNARE substrate (e.g. SNAP25) in target host cells compared to equivalent neurotoxin molecules with a single neuronal (receptor) binding domain.
  • SNARE substrate e.g. SNAP25
  • the inventors have exemplified the invention using a neurotoxin construct that has been generated using a "stapling method" described in detail below.
  • the method comprises generating different fusion polypeptides (e.g. a fusion protein comprising a synaptobrevin linker and a first neuronal binding domain; a fusion protein comprising a syntaxin linker and a second neuronal binding domain; and a fusion protein comprising a SNARE peptidase domain, a translocation domain and a SNAP linker) and performing the stapling reaction described elsewhere herein to generate a neurotoxin complex comprising the SNARE peptidase domain, translocation domain, first neuronal binding domain and second neuronal binding domain irreversibly linked together by formation of a SNARE complex helical bundle.
  • fusion polypeptides e.g. a fusion protein comprising a synaptobrevin linker and a first neuronal binding domain; a fusion protein compris
  • fusion proteins By inserting a flexible spacer peptide of suitable length into at least one of the fusion proteins (e.g. in between the syntaxin linker and the second neuronal binding domain in the appropriate fusion protein described above), the inventors were able to generate neurotoxins wherein the two binding domains were kept at a favourable distance from each other, with optimal bioactivity.
  • the rational design of fusion proteins (including spacer peptides) used for the construction of the neurotoxins described herein required great consideration.
  • the average length of spacer peptides that connect protein domains in natural multi-domain proteins has previously been calculated to be 6-16 residues, with the spacer peptides being grouped into small, medium, and large spacers with average length of 4.5 ⁇ 0.7, 9.1 ⁇ 2.4, and 21.0 ⁇ 7.6 residues, respectively (see Chen et al 2013, Advanced Drug Delivery Reviews, 65, pp 1357 to 1369).
  • Spacer peptides which connect protein domains to each other can adopt various structures (flexible or rigid) and exert diverse functions to fulfil the optimal joining of fusion proteins (see Table 2, Chen et al, 2013).
  • the flexible spacer peptides used in the invention are unusually long with amino acid lengths being from 31 to 66 (e.g.
  • a fusion protein comprising a SNARE peptidase domain, translation domain, first neuronal binding domain and second neuronal binding domain expressed as a single polypeptide chain, wherein the first neuronal binding domain and second neuronal binding domain may be separated by a spacer peptide as described in more detail elsewhere herein.
  • the first neuronal binding domain and second neuronal binding domain of a neurotoxin of the invention are preferably located C-terminal to the SNARE peptidase domain and translocation domain of the neurotoxin (whether the neurotoxin is generated as a stapled construct or as a single polypeptide chain fusion protein).
  • botulinum neurotoxin type A i.e. AA
  • type C i.e. CC
  • type D i.e. DD
  • type E i.e. EE
  • neurotoxins with two botulinum binding domains have improved neurotoxin activity within target cells.
  • neurotoxins with two tetanus neuronal binding domains have improved neurotoxin activity within target cells.
  • the finding that addition of an extra tetanus or botulinum binding domain improves neurotoxin efficacy is unexpected due to the distinct mechanisms and resulting clinical symptoms exhibited by naturally occurring C.tetani and C. botulinum neurotoxins.
  • neurotoxin efficacy in cleaving SNAP25 can be increased massively by the addition of a second binding domain, and this is particularly the case when binding domains are spatially separated using a flexible spacer peptide.
  • selection of specific binding domains (and binding domain combinations) can be used to target the neurotoxin to a specific target neuronal population.
  • the invention provides a new mechanism to improve the efficacy of neurotoxins, and improve the selective targeting of such neurotoxins to target cells.
  • the neurotoxins, compositions and pharmaceutical compositions described herein are particularly useful in the treatment, prevention, regulation and/or reduction of a number of conditions and disorders, as described in more detail below.
  • the invention provides a neurotoxin comprising a SNARE peptidase domain, a translocation domain, a first neuronal binding domain, and a second neuronal binding domain, wherein the first and second neuronal binding domains are each selected from the group consisting of:
  • the neurotoxin may further comprise a spacer peptide to spatially separate the first neuronal binding domain and the second neuronal binding domain in the neurotoxin.
  • the spacer peptide may be a flexible spacer peptide.
  • the first neuronal binding domain and the second neuronal binding domain may be located C-terminal to the SNARE peptidase domain and the translocation domain in the neurotoxin structure.
  • the first and second neuronal binding domains are the same.
  • the polypeptide of (i) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO: 1 , optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide of (ii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO:2, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.
  • the polypeptide of (iii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO:3, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:3.
  • the polypeptide of (iv) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:4.
  • the polypeptide of (v) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:5, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:5.
  • the polypeptide of (vi) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:6, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:6.
  • the polypeptide of (vii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:7, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:7.
  • the polypeptide of (viii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:8.
  • the first neuronal binding domain is attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO:9.
  • the amino acid sequence of SEQ ID NO:9 is N-terminal to the amino acid sequence of the first neuronal binding domain.
  • the second neuronal binding domain is attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO:10 (preferably, wherein the amino acid sequence of SEQ ID NO: 10 is N-terminal to the amino acid sequence of the second neuronal binding domain).
  • the second neuronal binding domain is attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10 via a (flexible) spacer peptide.
  • a (flexible) spacer peptide may be located between the amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of the second neuronal binding domain.
  • the second neuronal binding domain may be attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 17 (preferably wherein the amino acid sequence of SEQ ID NO:17 is N-terminal to the amino acid sequence of the second neuronal binding domain).
  • the SNARE peptidase domain and/or the translocation domain are attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO:1 1.
  • the amino acid sequence of SEQ ID NO:1 1 is C-terminal to the amino acid sequence of SNARE peptidase domain and the translocation domain (i.e. the order of the amino acid sequences in the polypeptide is SNARE peptidase domain, translocation domain, SEQ ID NO:11 (N- terminus to C-terminus)).
  • the SNARE peptidase domain, translocation domain, first neuronal binding domain, and second neuronal binding domain are comprised within a single polypeptide chain.
  • the order of the domains in the single polypeptide domains is SNARE peptidase domain, translocation domain, first neuronal binding domain, [optional (flexible) spacer peptide], second neuronal binding domain (N-terminus to C-terminus)).
  • the SNARE peptidase domain is a botulinum SNARE peptidase domain, optionally wherein the SNARE peptidase domain has at least 80% identity to the amino acid sequence of SEQ ID NO:12, further optionally wherein the SNARE peptidase domain comprises the amino acid sequence of SEQ ID NO: 12.
  • the translocation domain is a botulinum translocation domain, optionally wherein the translocation domain has at least 80% identity to the amino acid sequence of SEQ ID NO: 13, further optionally wherein the translocation domain comprises the amino acid sequence of SEQ ID NO: 13.
  • the invention provides a composition comprising a neurotoxin according to the invention, and a pharmaceutically acceptable excipient, adjuvant, diluent or carrier.
  • the invention provides the use of a composition according to the invention for (i) preventing, regulating or reducing skin wrinkling (ii) correcting an external appearance distorted due to excessive neuromuscular activity and/or (iii) preventing, regulating or reducing sweating due to excessive neuronal activity, in a subject.
  • the invention provides a method of preventing, regulating or reducing skin wrinkling in a subject, the method comprising administering a composition according to the invention to the subject.
  • the invention provides a composition according to the invention for use in treating or preventing a condition, disorder and/or symptom which is alleviated by the inhibition of neural terminals in a subject.
  • the invention provides a method of treating or preventing a condition, disorder and/or symptom which is alleviated by the inhibition of neural terminals; the method comprising administering a composition according to the invention to a subject.
  • Figure 1 A shows that Botulinum neurotoxin (BoNT) consists of three domains: SNARE peptidase, translocation domain and neuronal binding domain.
  • the peptidase also referred to as Light chain, Lc
  • Td Translocation domain
  • Nbd Neuronal binding domain
  • Figure 1 B shows that the stapling reaction allows non- covalent irreversible linking of large protein domains via formation of an irreversible helical bundle.
  • Crystal PDB structures for BoNT/A and the SNARE complex used are: 3BTA and 1 SFC.
  • Figure 2 shows that duplication of binding domains on the C-terminal end of the
  • translocation domain within a neurotoxin complex of the invention can be achieved by parallel attachment of protein fragments using the SNARE complex (indicated as SC). Note that one Nbd is joined via a flexible spacer peptide to the stapling mechanism.
  • Figure 3 shows that duplication of clostridial binding domains on the C-terminal end of the translocation domain within a neurotoxin complex of the invention and with a flexible spacer peptide separating binding domains allows immense increase in the delivery of botulinum type A enzyme into cultured rat cortical neurons as evidenced by immunoblotting for SNAP25 cleavage (indicated by star * , upper panels).
  • Graphs show 60x and 600x increase in the cleavage of SNAP25 by the double tetanus and double botulinum type D binding domains, respectively, compared to the single liganded constructs.
  • Figure 4A shows that duplication of botulinum binding domains type A and E allows increase in the delivery of botulinum type A enzyme into cultured cortical neurons as evidenced by immunoblotting for SNAP25 cleavage. Note similar potency of SNAP25 cleavage by 80 pM of botulinum-2xNbd/A and 10 nM botulinum-1xNbd/A (125 fold improvement). Similarly, 400 pM botulinum-2xNbd/E leads to the same potency as 10 nM botulinum-1xNbd/E (40 fold improvement).
  • botulinum-2xNbd/A outperforms native BoNT/A as evidenced by cleavage of SNAP25 at 16 pM concentration.
  • Figure 4B shows further immunoblot of cultured cortical neurones treated by native BoNT/A and botulinum-2xNbd/A, and a graph quantifying the efficacy of SNAP25 cleavage by botulinum-2xNbd/A versus native BoNT/A with a 13 fold improvement in the case of botulinum-2xNbd/A.
  • Figure 5 shows that the human SiMa neuroblastoma cells differentiated using retinoic acid are also more susceptible to SNAP25 cleavage when treated with double-liganded botulinum protease type A.
  • Panels A-C show immunoblots with SNAP25 cleavage observed after 65 hour-treatment with botulinum constructs carrying binding domains of botulinum type A, C and tetanus. Note, in the case of type A binding domain (panel A), there is similar cleavage between 2 nM and 16 pM of single- and double-liganded botulinum construct, respectively (125-fold enhancement).
  • Figure 6 shows increased binding to mouse motor neurons by duplicated tetanus neuronal binding domain (Nbd/T).
  • Figure 6A is a schematic of formation of single- and double- liganded fluorescent constructs with SDS-PAGE gels showing the assembled fluorescent constructs (A, upper panel). Note that one Nbd is joined via a flexible spacer peptide to the stapling mechanism. Injection of fluorescent constructs (2 microgram) into the mouse hind paw leads to apparently stronger accumulation of fluorescent Nbd/T in motor neurons located in the ventral horn of the spinal cord (A, lower panel).
  • Figure 6B shows quantification of bound fluorescence to cultured mouse motor neurons confirming increased binding (by 150%) of the double-liganded construct.
  • Figure 7 shows that duplication of tetanus binding domain linked to the botulinum enzyme type A (right panel) leads to increased cleavage of SNAP25 in rat spinal cord following injection into the hind paw compared to single-liganded molecule (left panel).
  • Spinal cord sections were immunostained using an antibody which recognises strictly the botulinum- cleaved end of neuronal SNAP25.
  • Intraplantar injection of botulinum-2xNbd/T resulted in an approximately three times larger area of SNAP-25 cleavage in comparison to botulinum- 1xNbd/T.
  • Figure 8 shows that native botulinum neurotoxin type A (PDB 3BTA, 4JRA and 2VU9) binds SV2 protein (SV2) and ganglioside GT1b in the neuronal membrane via its Neuronal binding domain (Nbd) (Benoit, R. M., Frey, D., Hilbert, M., Kevenaar, J. T., Wieser, M.
  • the translocation domain and the botulinum peptidase are labelled as Td and Lc, respectively.
  • Joining of the C-terminal leucine 1295 (left star) to a 39 aa flexible spacer peptide sequence attached to the Isoleucine 872 of a second binding domain (right star) allows both binding domains to be located on the C-terminal end of the translocation domain within the neurotoxin complex to bind SV2 and GT1b ganglioside in parallel and in optimal orientation in the plane of the neuronal membrane.
  • Figure 9 shows a Botulinum A (874-1296) Neuronal binding domain amino acid sequence (SEQ ID NO: 1 ).
  • Figure 10 shows a Botulinum B Neuronal binding domain amino acid sequence (SEQ ID NO:2)
  • Figure 11 shows a Botulinum C (872-1291) Neuronal binding domain amino acid sequence (SEQ ID NO:3).
  • Figure 12 shows a Botulinum D (865 - 1275) Neuronal binding domain amino acid sequence (SEQ ID NO: 4).
  • Figure 13 shows a Botulinum E (853 - 1251) Neuronal binding domain amino acid sequence (SEQ ID NO:5).
  • Figure 14 shows a Botulinum F Neuronal binding domain amino acid sequence (SEQ ID NO:6).
  • Figure 15 shows a Botulinum G Neuronal binding domain amino acid sequence (SEQ ID NO:7).
  • Figure 16 shows a Tetanus (856 - 1315) Neuronal binding domain amino acid sequence (SEQ ID NO: 8).
  • Figure 17 shows a Minimal Synaptobrevin SNARE linkerl (25-84) amino acid sequence (SEQ ID NO: 9).
  • Figure 18 shows a Syntaxin 3 SNARE amino acid sequence (Iinker2) for recombinant expression (195-253) (SEQ ID NO: 10) which is expressed better in E.coli compared to that of Syntaxin 1.
  • Figure 19 shows a SNAP25 linker amino acid sequence (SEQ ID NO: 11).
  • Figure 20 shows a Botulinum neurotoxin type A SNARE peptidase domain amino acid sequence (SEQ ID NO:12).
  • Figure 21 shows a Botulinum neurotoxin type A translocation domain amino acid sequence (SEQ ID NO: 13). The sequence includes a Thrombin cleavage site for functional activation of the disulphide bond between the peptidase and translocation domain.
  • Figure 22 shows LcTd/A fused to SNAP25 (SEQ ID NO: 14). The sequences that are shown correspond to BoNT/A Lc (1-449, bold), a Thrombin cleavage site (Underlined), an optimized BoNT/A Td (449-872, italics) and Mouse SNAP25 (1-206, bold, underlined and italics) with four cysteines mutated to alanines. Cleaved protein size: 1095aa, 124.84 kDa.
  • Figure 22b shows LcTd/B fused to SNAP25 (SEQ ID NO: 14).
  • Figure 23 shows a Synaptobrevin SNARE linker! (2-84) amino acid sequence (SEQ ID NO: 15).
  • Figure 24 shows a Syntaxin 1 Iinker2 (Synthesised Peptide also known as Staple) amino acid sequence (SEQ ID NO: 16).
  • Figure 25 shows an extended Syntaxin 3 Iinker2 amino acid sequence with a flexible spacer peptide (SEQ ID NO: 17).
  • Syntaxin 3 (195-253, Bold), Amino acids from restriction sites (Italics) and 66 aa Flexible spacer peptide (Underlined).
  • FIG 26 illustrates that for making single liganded-botulinum constructs, the Iinker2 (SEQ ID NO: 16) was utilised as a staple to link the enzymatic portion of the botulinum type A1 neurotoxin with any clostridial binding domain (Darios, F. et al. Proc Natl Acad Sci U S A 107, 18197-18201).
  • the Iinker2 amino acid sequence is based on the Syntaxin 1 SNARE helix prepared as a chemically synthetic peptide (SEQ ID NO: 16).
  • Figure 27 shows the cleaved end of SNAP25 (SEQ ID NO: 18).
  • Figure 28 shows that duplication of Neuronal binding domains on the C-terminal end of the translocation domain within a neurotoxin complex of the invention using linkers with a 66 aa flexible spacer peptide increases efficacy and rate of SNAP25 cleavage.
  • Figure 29 shows that duplication of Neuronal binding domain type B on the C-terminal end of the translocation domain using stapling mechanism with a flexible 66 aa spacer peptide increases efficacy and rate of SNAP25 cleavage.
  • Figure 30 shows that BoNT/C, comprising a SNARE peptidase domain, a translocation domain, a first neuronal binding domain, a 39 aa flexible spacer peptide (FE), and a second neuronal binding domain, within a single polypeptide chain (BoNT-2xNbd/C) allows more efficient and faster SNAP25 cleavage compared to native BoNT/C.
  • A Schematic and SDS- PAGE gel showing the molecular weight difference between the native BoNT C and BoNT- 2xNbd/C.
  • B Western blot showing an increased efficacy of SNAP25 cleavage in
  • Figure 31 shows the amino acid sequence for BoNT-2xNbd/C (SEQ ID NO: 19).
  • the sequences that are shown correspond to BoNT/C Lc (1-439, bold), a Thrombin cleavage site (underlined), BoNT/C Td (440-866, italics), a first BoNT/C Nbd (867-1291 , bold, underlined and italics), a spacer peptide sequence (underlined, italics) and a second BoNT/C Nbd (867- 1291 , bold, underlined and italics) Cleaved protein size: 1760aa, 201.39 kDa.
  • the invention is based on the surprising finding that neurotoxin efficacy can be improved by modifying the neurotoxin protein to include two neuronal binding domains (e.g. on the C- terminal end of the translocation domain and with these domains being separated by a flexible spacer peptide).
  • the inventors have constructed novel neurotoxin molecules which include two neuronal (receptor) binding domains in such configuration. Surprisingly, these novel molecules are more effective in cleaving their SNARE substrate (e.g. SNAP25) in target cells compared to equivalent neurotoxin molecules with a single neuronal (receptor) binding domain.
  • the inventors have exemplified the invention using novel neurotoxin molecules comprising the SNARE peptidase domain and translocation domain of botulinum neurotoxin type A, together with two neuronal binding domains of botulinum neurotoxin (botulinum neurotoxin type A (i.e. AA), type C (i.e. CC), type D (i.e. DD) and type E (i.e. EE)) or two neuronal binding domains of tetanus neurotoxin.
  • botulinum neurotoxin type A i.e. AA
  • type C i.e. CC
  • type D i.e. DD
  • type E i.e. EE
  • neurotoxin efficacy can be increased by the addition of a second neuronal binding domain (e.g. on the C-terminal end of the translocation domain with a flexible spacer peptide separating the two binding domains).
  • a second neuronal binding domain e.g. on the C-terminal end of the translocation domain with a flexible spacer peptide separating the two binding domains.
  • selection of specific binding domains (and binding domain combinations) can be used to target the neurotoxin to a specific target neuronal population.
  • the invention provides a new
  • neurotoxins, compositions and pharmaceutical compositions described herein are particularly useful in the treatment, prevention, regulation and/or reduction of a number of conditions and disorders, as described in more detail below.
  • Neurotoxins that comprise a SNARE peptidase domain, a translocation domain, a first neuronal binding domain and a second neuronal binding domain are provided herein.
  • the neurotoxin may therefore further comprise a spacer peptide to spatially separate the first neuronal binding domain and the second neuronal binding domain in the neurotoxin.
  • the spacer peptide may be a flexible spacer peptide.
  • the first neuronal binding domain and the second neuronal binding domain may be located C-terminal to the SNARE peptidase domain and the translocation domain in the neurotoxin structure.
  • the term "Neuronal binding domain” (abbreviated to "Nbd") refers to a protein domain within a polypeptide, wherein the protein domain facilitates binding of the polypeptide to neuronal cells.
  • the neuronal binding domain interacts with a receptor on the surface of the target neuronal cell.
  • the neuronal binding domain may also be considered as a ligand for the corresponding receptor.
  • Neuronal binding domain and “ligand” are used herein as alternative terminology to describe a neuronal binding domain (unless the context specifies otherwise).
  • a neurotoxin that has two neuronal binding domains may also be described herein as a “double liganded" neurotoxin (or as "2xNbd", for example 2xNbd/T refers to a neurotoxin with two tetanus neurotoxin neuronal binding domains).
  • the neurotoxins provided herein comprise at least two (a first and a second) neuronal binding domains.
  • the neurotoxins may further comprise additional neuronal binding domains.
  • a neurotoxin may comprise two, three, four, or more neuronal binding domains.
  • Each of the neuronal binding domains (e.g. the first and the second neuronal binding domains) of the neurotoxin may be the same, or they may be different.
  • Neurotoxins with more than two neuronal binding domains may have two, three, four or more neuronal binding domains that are the same (or each neuronal binding domain may be different).
  • the first and second neuronal binding domains are the same.
  • the first and second binding domains are selected from polypeptides having botulinum neurotoxin binding domain activity, and/or from polypeptides having tetanus neurotoxin binding domain activity.
  • the term "activity" is used to refer to the physiological function of the binding domain i.e. its binding capacity for its target (e.g. receptor).
  • first and second neuronal binding domains of the neurotoxins provided herein are each selected from the group consisting of:
  • the first neuronal binding domain may therefore be selected from (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii), and may be combined with any second neuronal binding domain also selected from (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii).
  • the first and second bind domains may be any one of the following combinations: (i) and (i), (i) and (ii), (i) and (iii), (i) and (iv), (i) and (iv),
  • Particularly suitable combinations include the first and second neuronal binding domain both being selected as (i); the first and second neuronal binding domain both being selected as (ii); the first and second neuronal binding domain both being selected as (iii); the first and second neuronal binding domain both being selected as (iv); the first and second neuronal binding domain both being selected as (v); or the first and second neuronal binding domain both being selected as (viii).
  • Any additional neuronal binding domains present in the neurotoxin may also be selected from the group consisting of (i) to (viii) listed above.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type A activity” refers to a polypeptide that retains the functional binding capacity of a BoNT type A binding domain i.e. it is capable of binding neuronal ganglioside GT1 b and synaptic vesicle protein SV2.
  • BoNT/Nbd type A activity includes any functional BoNT/Nbd type A.
  • a person of skill in the art is readily aware of how to identify polypeptides having BoNT/Nbd type A activity using routine experiments known in the art. A suitable experiment for identifying functional
  • BoNT/Nbd type A polypeptides is summarised in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013.
  • the polypeptide having BoNT/Nbd type A activity comprises the amino acid sequence shown in SEQ I D NO: 1 , or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 1 .
  • the term "variant" also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ I D NO: 1 , or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein.
  • a functional variant of SEQ ID NO: 1 may therefore be a conservative amino acid sequence variant of SEQ ID NO: 1 , wherein the variant has BoNT/Nbd type A activity.
  • Non-functional variants are amino acid sequence variants of SEQ I D NO: 1 that do not have BoNT/Nbd type A activity.
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 1 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • BoNT/Nbd type A A summary of the critical and non-critical amino acids in BoNT/Nbd type A is provided in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ I D NO: 1 .
  • a polypeptide having BoNT/Nbd type A activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ I D NO: 1 , or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ I D NO: 1), or portions or fragments thereof.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type B activity” refers to a polypeptide that retains the functional binding capacity of a BoNT type B binding domain i.e. it is capable of binding neuronal gangliosides and synaptic vesicle protein synaptotagmin.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type B activity” includes any functional BoNT/Nbd type B.
  • the polypeptide having BoNT/Nbd type B activity comprises the amino acid sequence shown in SEQ I D NO: 2, or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:2.
  • the term "variant" also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein.
  • a functional variant of SEQ ID NO:2 may therefore be a conservative amino acid sequence variant of SEQ ID NO:2, wherein the variant has
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 2 that do not have BoNT/Nbd type B activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and nonfunctional allelic variants) are well known to a person of ordinary skill in the art. A summary of the critical and non-critical amino acids in BoNT/Nbd type B is provided in Botulinum Neurotoxins.
  • a polypeptide having BoNT/Nbd type B activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:2, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ I D NO:2), or portions or fragments thereof.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type C activity” refers to a polypeptide that retains the functional binding capacity of a BoNT type C binding domain i.e. it is capable of binding neuronal gangliosides.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type C activity” includes any functional BoNT/Nbd type C.
  • a person of skill in the art is readily aware of how to identify polypeptides having BoNT/Nbd type C activity using routine experiments known in the art. A suitable experiment for identifying functional BoNT/Nbd type C polypeptides is summarised in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013.
  • the polypeptide having BoNT/Nbd type C activity comprises the amino acid sequence shown in SEQ I D NO: 3, or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:3.
  • the term "variant” also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:3, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein.
  • a functional variant of SEQ ID NO:3 may therefore be a conservative amino acid sequence variant of SEQ ID NO:3, wherein the variant has
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 3 that do not have BoNT/Nbd type C activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:3 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and nonfunctional allelic variants) are well known to a person of ordinary skill in the art.
  • BoNT/Nbd type C A summary of the critical and non-critical amino acids in BoNT/Nbd type C is provided in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ I D NO:3.
  • a polypeptide having BoNT/Nbd type C activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:3, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:3), or portions or fragments thereof.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type D activity” refers to a polypeptide that retains the functional binding capacity of a BoNT type D binding domain i.e. it is capable of binding neuronal gangliosides.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type D activity” includes any functional BoNT/Nbd type D.
  • the polypeptide having BoNT/Nbd type D activity comprises the amino acid sequence shown in SEQ ID NO: 4, or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:4.
  • the term "variant” also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:4, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein.
  • a functional variant of SEQ ID NO:4 may therefore be a conservative amino acid sequence variant of SEQ ID NO:4, wherein the variant has
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 4 that do not have BoNT/Nbd type D activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:4 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and nonfunctional allelic variants) are well known to a person of ordinary skill in the art.
  • BoNT/Nbd type D A summary of the critical and non-critical amino acids in BoNT/Nbd type D is provided in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ I D NO:4.
  • a polypeptide having BoNT/Nbd type D activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:4, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ I D NO:4), or portions or fragments thereof.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type E activity” refers to a polypeptide that retains the functional binding capacity of a BoNT type E binding domain i.e. it is capable of binding synaptic vesicle protein SV2 and neuronal gangliosides.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type E activity” includes any functional BoNT/Nbd type E.
  • BoNT/Nbd type E polypeptides A suitable experiment for identifying functional BoNT/Nbd type E polypeptides is summarised in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013.
  • the polypeptide having BoNT/Nbd type E activity comprises the amino acid sequence shown in SEQ I D NO: 5, or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:5.
  • variants also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:5, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein.
  • a functional variant of SEQ ID NO:5 may therefore be a conservative amino acid sequence variant of SEQ ID NO:5, wherein the variant has
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 5 that do not have BoNT/Nbd type E activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:5 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and nonfunctional allelic variants) are well known to a person of ordinary skill in the art.
  • BoNT/Nbd type E A summary of the critical and non-critical amino acids in BoNT/Nbd type E is provided in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ ID NO:5.
  • a polypeptide having BoNT/Nbd type E activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:5, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:5), or portions or fragments thereof.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type F activity” refers to a polypeptide that retains the functional binding capacity of a BoNT type F binding domain i.e. it is capable of binding synaptic vesicle protein SV2 and neuronal gangliosides.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type F activity” includes any functional BoNT/Nbd type F.
  • BoNT/Nbd type F polypeptides A suitable experiment for identifying functional BoNT/Nbd type F polypeptides is summarised in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013.
  • the polypeptide having BoNT/Nbd type F activity comprises the amino acid sequence shown in SEQ ID NO: 6, or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:6.
  • variants also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:6, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein.
  • a functional variant of SEQ ID NO:6 may therefore be a conservative amino acid sequence variant of SEQ ID NO:6, wherein the variant has BoNT/Nbd type F activity.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 6 that do not have BoNT/Nbd type F activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:6 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and nonfunctional allelic variants) are well known to a person of ordinary skill in the art.
  • BoNT/Nbd type F A summary of the critical and non-critical amino acids in BoNT/Nbd type F is provided in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ ID NO:6.
  • a polypeptide having BoNT/Nbd type F activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:6, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:6), or portions or fragments thereof.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type G activity" refers to a polypeptide that retains the functional binding capacity of a BoNT type G binding domain i.e.
  • a polypeptide having "botulinum neurotoxin binding domain (BoNT/Nbd) type G activity" includes any functional BoNT/Nbd type G.
  • BoNT/Nbd type G activity includes any functional BoNT/Nbd type G.
  • a person of skill in the art is readily aware of how to identify polypeptides having BoNT/Nbd type G activity using routine experiments known in the art.
  • a suitable experiment for identifying functional BoNT/Nbd type G polypeptides is summarised in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013.
  • the polypeptide having BoNT/Nbd type G activity comprises the amino acid sequence shown in SEQ ID NO: 7, or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:7.
  • the term "variant” also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:7, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein.
  • a functional variant of SEQ ID NO:7 may therefore be a conservative amino acid sequence variant of SEQ ID NO:7, wherein the variant has
  • Non-functional variants are amino acid sequence variants of SEQ I D NO:7 that do not have BoNT/Nbd type G activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:7 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and non- functional allelic variants) are well known to a person of ordinary skill in the art.
  • BoNT/Nbd type G A summary of the critical and non-critical amino acids in BoNT/Nbd type G is provided in Botulinum Neurotoxins. Editors: Rummel, Andreas, Binz, Thomas (Eds.) Springer, Current Topics in Microbiology and Immunology, 2013. Accordingly, a person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ I D NO:7.
  • a polypeptide having BoNT/Nbd type G activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ I D NO:7), or portions or fragments thereof.
  • a polypeptide having "tetanus neurotoxin binding domain (TeNT/Nbd) activity” refers to a polypeptide that retains the functional binding capacity of a TeNT binding domain i.e. it is capable of binding neuronal ganglioside GT1 b and nidogens.
  • a polypeptide having "tetanus neurotoxin binding domain (TeNT/Nbd) activity” includes any functional TeNT/Nbd.
  • the polypeptide having TeNT/Nbd activity comprises the amino acid sequence shown in SEQ I D NO: 8, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:8.
  • variant also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:8, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO:8 may therefore be a conservative amino acid sequence variant of SEQ ID NO:8, wherein the variant has
  • Non-functional variants are amino acid sequence variants of SEQ I D NO: 8 that do not have TeNT/Nbd activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:8 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and nonfunctional allelic variants) are well known to a person of ordinary skill in the art.
  • a polypeptide having TeNT/Nbd activity may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ I D NO:8, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:8), or portions or fragments thereof.
  • the neurotoxins provided herein comprise a SNARE peptidase domain.
  • SNARE peptidase domain refers to protein domain within a polypeptide, wherein the protein domain hydrolyses one more SNAREs.
  • the protein domain functions as a protease enzyme that performs proteolysis on its substrate, wherein the substrate is a SNARE.
  • enzyme domain is used herein as alternative terminology to describe a (SNARE) peptidase domain.
  • the SNARE peptidase domain of the neurotoxin is a botulinum SNARE peptidase domain.
  • other SNARE peptidase domains may alternatively be present, for example other Clostridial SNARE peptidase domains such a tetanus SNARE peptidase domain (or functional allelic variants, fragments or portions thereof).
  • such alternative SNARE peptidase domains could also function as the "toxin" component once in the target cell, and thus could also be used to form a functional neurotoxin as defined herein.
  • a "botulinum SNARE peptidase domain” refers to a SNARE peptidase domain as found in naturally occurring botulinum neurotoxins (e.g. any one of botulinum neurotoxin types A, B, C, D, E, F and G), and functional derivatives (or variants) thereof. It includes functional allelic variants, fragments or portions thereof.
  • a SNARE peptidase domain from any one of botulinum neurotoxin types A, B, C, D, E, F and G could be present in the neurotoxins presented herein, and could function as the "toxin" component of the neurotoxin once in the target cell.
  • a "botulinum SNARE peptidase domain” therefore refers to a polypeptide that retains the functional peptidase activity of a botulinum SNARE peptidase domain i.e. it is capable of catalysing the proteolysis of at least one of three SNAREs, namely VAMP/synaptobrevin, SNAP25 or syntaxin.
  • a person of skill in the art is readily aware of how to identify a botulinum SNARE peptidase domain polypeptide using routine experiments known in the art. A suitable experiment for identifying functional botulinum SNARE peptidase domains is summarised in Darios, F. et al.
  • a botulinum SNARE peptidase domain comprises the amino acid sequence shown in SEQ I D NO: 12, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 12.
  • variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 12.
  • variants also encompasses homologues.
  • the term "botulinum SNARE peptidase domain” includes isozymes and allozymes of a polypeptide comprising the amino acid sequence shown in SEQ I D NO: 12.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 12, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • a functional variant of SEQ ID NO: 12 may therefore be a conservative amino acid sequence variant of SEQ ID NO:2, wherein the variant has botulinum SNARE peptidase activity.
  • Non-functional variants are amino acid sequence variants of SEQ I D NO: 12 that do not have botulinum SNARE peptidase activity. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 12 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g.
  • a SNARE peptidase domain according to the invention may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 12, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 12), portions or fragments thereof.
  • SEQ I D No: 12 The amino acid sequence shown in SEQ I D No: 12 is that of a Botulinum neurotoxin type A SNARE peptidase domain.
  • other Botulinum neurotoxin SNARE peptidase domains may also be used, e.g. that of Botulinum neurotoxin type B (which is shown as part of the sequence of SEQ I D No: 14.
  • Variants of the SNARE peptidase domain of BoNT type B are equally covered, as for the SNARE peptidase domain of BoNT type A (SEQ ID NO: 12), and therefore the paragraphs above apply equally for SEQ ID NO: 12 and the SNARE peptidase domain portion of SEQ I D NO: 14).
  • the neurotoxins provided herein also comprise a translocation domain.
  • a translocation domain refers to protein domain within a polypeptide, wherein the protein domain facilitates endocytosis of the neurotoxin into the cytosol of the target cell.
  • the translocation domain is typically derived from the same origin as the SNARE peptidase domain (as the SNARE peptidase domain and translocation domain of each neurotoxin are typically inseparable in terms of structure and function).
  • a BoNT type A SNARE peptidase domain is typically used with a BoNT type A translocation domain; a BoNT type B SNARE peptidase domain is typically used with a BoNT type B translocation domain; a BoNT type C SNARE peptidase domain is typically used with a BoNT type C translocation domain; a BoNT type D SNARE peptidase domain is typically used with a BoNT type D translocation domain; a BoNT type E SNARE peptidase domain is typically used with a BoNT type E translocation domain; a BoNT type F SNARE peptidase domain is typically used with a BoNT type F translocation domain; BoNT type G SNARE peptidase domain is typically used with a BoNT type G translocation domain; and a TeNT SNARE peptidase domain is typically used with a TeNT translocation domain.
  • the translocation domain of the invention may be a botulinum translocation domain.
  • a "botulinum translocation domain” refers to a translocation domain as found in naturally occurring botulinum neurotoxins (e.g. any one of botulinum neurotoxin types A, B, C, D, E, F and G), and functional derivatives (or variants) thereof. It includes functional allelic variants, fragments or portions thereof.
  • a "botulinum translocation domain” therefore refers to a polypeptide that retains the functional translocation activity of a botulinum translocation domain i.e. it is capable of facilitating endocytosis of the neurotoxin into the cytosol of the target cell.
  • a person of skill in the art is readily aware of how to identify botulinum translocation domain polypeptides using routine experiments known in the art. A suitable experiment for identifying functional botulinum translocation domain polypeptides is summarised in Bade, S. et al. Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J. Neurochem.
  • the translocation domain polypeptide comprises the amino acid sequence shown in SEQ I D NO: 13, or functional variants (or functional fragments) thereof.
  • Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 13.
  • the term "variant” also encompasses homologues.
  • Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 13, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • a functional variant of SEQ ID NO: 13 may therefore be a conservative amino acid sequence variant of SEQ ID NO: 13, wherein the variant is capable of facilitating endocytosis of the neurotoxin into the cytosol of a target cell.
  • Non-functional variants are amino acid sequence variants of SEQ I D NO: 13 that are not capable of facilitating endocytosis of the neurotoxin into the cytosol of the target cell.
  • Non- functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 13 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • Methods for identifying functional and non-functional variants e.g. functional and non-functional allelic variants
  • a translocation domain according to the invention may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 13, or portions or fragments thereof.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 13), or portions or fragments thereof.
  • SEQ I D No: 13 The amino acid sequence shown in SEQ I D No: 13 is that of a Botulinum neurotoxin type A translocation domain.
  • other Botulinum neurotoxin translocation domains may also be used, e.g. that of Botulinum neurotoxin type B (which is shown as part of the sequence of SEQ ID No: 14.
  • Variants of the translocation domain of BoNT type B are equally covered, as for the translocation domain of BoNT type A (SEQ ID NO: 13), and therefore the paragraphs above apply equally for SEQ I D NO: 13 and the translocation domain portion of SEQ ID NO: 14).
  • the SNARE peptidase domain and the translocation domain are from the same botulinum neurotoxin. Preferably, they are joined via a disulphide bond as in a naturally occurring botulinum neurotoxin. If the SNARE peptidase domain and the translocation domain are joined by a peptide bond between the amino acid chains, preferably, there is a nicking site in the amino acid sequence between the SNARE peptidase domain and the translocation domain which is recognised by a protease to cause cleavage of the amino acid sequence between the two parts. In one embodiment, the nicking site is a thrombin site which can be cleaved by thrombin. As discussed in more detail elsewhere herein, the SNARE peptidase domain and the translocation domain may be attached to a polypeptide helix (linker) derived from a SNAP protein.
  • linker polypeptide helix
  • a "naturally-occurring" polypeptide refers to an amino acid sequence that occurs in nature.
  • a “non-essential” (or “non-critical”) amino acid residue is a residue that can be altered from the wild-type sequence of (e.g. , the sequence of SEQ ID NOs:1 to 22) without abolishing or, more preferably, without substantially altering a biological activity, whereas an “essential” (or “critical”) amino acid residue results in such a change.
  • amino acid residues that are conserved are predicted to be particularly non-amenable to alteration, except that amino acid residues within the hydrophobic core of domains can generally be replaced by other residues having approximately equivalent hydrophobicity without significantly altering activity.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. , lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g. , tyrosine, phenylalanine, tryptophan, histidine.
  • a nonessential (or non-critical) amino acid residue in a protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • a "biologically active portion" of protein or a protein portion with “biological activity” includes a fragment of protein that participates in an interaction between molecules and non-molecules.
  • Biologically active portions of proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the protein, e.g. , the amino acid sequences shown in SEQ ID NO: 1 to 22, which include fewer amino acids than the full length protein, and exhibit at least one activity of the encoded protein.
  • biologically active portions comprise a domain or motif with at least one activity of the protein, e.g., the biologically active portion may retain one of the following activities (as appropriate); BoNT/Nbd type A activity, BoNT/Nbd type B activity, BoNT/Nbd type C activity, BoNT/Nbd type D activity, BoNT/Nbd type E activity, BoNT/Nbd type F activity, BoNT/Nbd type G activity, or TeNT/Nbd activity.
  • activities is used to mean the functional activity of each binding domain (i.e. its binding capacity).
  • a biologically active portion of protein can be a polypeptide that is, for example, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acids in length of SEQ I D NO:1 to 22.
  • sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the N BLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410).
  • gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the polypeptides described herein can have amino acid sequences sufficiently or substantially identical to the amino acid sequences of SEQ ID NO:1 to 22.
  • the terms "sufficiently identical” or “substantially identical” are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g.
  • amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity.
  • amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91 %, 92%, 93%,
  • a neurotoxin comprising a SNARE peptidase domain, translocation domain, first neuronal binding domain, and second neuronal binding domain as described above may be generated using any suitable technique. Suitable techniques are readily identifiable by a person of skill in the art. For example, a neurotoxin comprising a SNARE peptidase domain, a translocation domain, a first neuronal binding domain, and a second neuronal binding domain may be generated as a (recombinant) single polypeptide chain (e.g. fusion protein). Methods for generating such proteins are well known in the art.
  • FIG. 8 the structure of native botulinum type A (PDB 3BTA, 4JRA and 2VU9) is shown in Figure 8. It binds SV2 protein (SV2) and ganglioside GT1 b in the neuronal cell membrane via its (first) neuronal binding domain (Nbd) (Benoit, R. M., Frey, D., Hilbert, M., Kevenaar, J. T., Wieser, M. M., Stirnimann, C. U., McMillan, D., Ceska, T., Lebon, F., Jaussi, R., Steinmetz, M. O., Schertler, G. F. X., Hoogenraad, C.
  • SV2 protein SV2 protein
  • Nbd neuronal binding domain
  • the translocation domain and the SNARE peptidase domain are labelled as Td and Lc (Light chain), respectively.
  • the free C-terminal amino acid Leu1295 can be used for adding a peptide sequence which comprises a second neuronal binding domain (and e.g. also comprises a flexible spacer peptide described elsewhere herein).
  • the neurotoxins described herein comprise a SNARE peptidase domain, translocation domain, first neuronal binding domain, and second neuronal binding domain within a single polypeptide chain.
  • the neurotoxin comprises a spacer peptide that spatially separates the first neuronal binding domain from the second neuronal binding domain within the neurotoxin.
  • the spacer peptide is a flexible spacer peptide. The spacer peptide may be located between the first neuronal binding domain and the second neuronal binding domain when the neurotoxin is generated as a (recombinant) single polypeptide chain (e.g.
  • the order of the domains in the single polypeptide chain may therefore be SNARE peptidase domain, translocation domain, first neuronal binding domain, [optional (flexible) spacer peptide)], and second neuronal binding domain (N-terminus to C-terminus).
  • the (flexible) spacer peptide sequence is preferably at least 31 amino acids in length (e.g. is from 31 to 66, or 39 to 66 amino acids in length).
  • a neurotoxin protein complex may be generated using the stapling reaction described in US 2011/0038892.
  • the neurotoxin of the invention may therefore comprise an irreversible helical bundle protein complex of a SNARE peptidase domain, translocation domain, first neuronal binding domain and second neuronal binding domain.
  • the neurotoxin of the invention also comprises a (flexible) spacer peptide sequence (e.g. of at least 31 amino acids in length (e.g. 31 to 66 or 39 to 66 amino acids in length) which functions to spatially separate the two neuronal binding domains within the neurotoxin structure (see Fig. 8; also described in detail elsewhere herein).
  • the "stapling method" described in detail below comprises generating different fusion polypeptides (e.g. a fusion protein comprising a synaptobrevin linker and a first neuronal binding domain; a fusion protein comprising a syntaxin linker and a second neuronal binding domain; and a fusion protein comprising a SNARE peptidase domain, a translocation domain and a SNAP linker) and performing the stapling reaction described elsewhere herein to generate a neurotoxin complex comprising the SNARE peptidase domain, translocation domain, first neuronal binding domain and second neuronal binding domain irreversibly linked together by formation of a SNARE complex helical bundle.
  • fusion polypeptides e.g. a fusion protein comprising a synaptobrevin linker and a first neuronal binding domain; a fusion protein comprising a syntaxin linker and a second neuronal binding domain; and a fusion protein comprising a SNARE
  • a linker e.g. "linker 1” may be attached to a first neuronal binding domain (e.g. by expressing the linker and first neuronal binding domain as a fusion protein in a suitable host, or by synthetically generating the fusion protein).
  • a complementary linker e.g. "linker 2” (preferably with a flexible spacer peptide) may be attached to a second neuronal binding domain (e.g. by expressing the linker (with optional flexible spacer peptide sequence) and second neuronal binding domain as a fusion protein in a suitable host, or by synthetically generating the fusion protein).
  • a third fusion protein may then be generated (e.g.
  • SNARE peptidase domain comprising the SNARE peptidase domain, translocation domain and a third linker (e.g. a "SNAP” linker such as a "SNAP25” linker).
  • the stapling reaction may then be performed, to irreversibly link the SNARE peptidase domain, translocation domain, first neuronal binding domain and second neuronal binding domain together to form a neurotoxin as described above.
  • linkerl (also known as the synaptobrevin linker herein) comprises the amino acid sequence of SEQ ID NO:9. This sequence represents the polypeptide helix (amino acids 25 to 84) of synaptobrevin, which forms a SNARE complex with
  • the first neuronal binding domain is attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO:9.
  • An alternative "linker 1" comprises the amino acid sequence of SEQ ID NO: 15 .
  • the sequence of the linker i.e. in this case linker 1 may have at least about 80% sequence identity with the sequence of the selected SNARE domain/motif or the portion thereof (e.g. it may have at least about 80% sequence identity with the sequence of SEQ ID NO:9 or SEQ I D NO: 15). More preferably, the sequence identity should be at least about 85%, and even more preferably at least about 90%. In one embodiment, the sequence identity may be at least about 95%, at least about 98% or even 100%.
  • This linker can have a flexible spacer peptide to improve bioactivity.
  • Iinker2 (also known as the syntaxin linker herein) comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 17 (SEQ ID NO: 17 is an example of a "Iinker2" with a flexible spacer peptide).
  • SEQ ID NO: 10 represents the polypeptide helix (amino acids 195 to 253) of a specific syntaxin, syntaxin 3, which forms a SNARE complex with complementary polypeptide helices of other appropriate SNARE proteins.
  • the second neuronal binding domain is attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:17.
  • the sequence of the linker i.e.
  • linker 2 may have at least about 80% sequence identity with the sequence of the selected SNARE domain/motif or the portion thereof (e.g. it may have at least about 80% sequence identity with the sequence of SEQ ID NO: 10 or SEQ ID NO:17). More preferably, the sequence identity should be at least about 85%, and even more preferably at least about 90%. In one embodiment, the sequence identity may be at least about 95%, at least about 98% or even 100%.
  • either or both linkers can carry a flexible spacer peptide (e.g. of at least 31 amino acids in length, e.g. 31 to 66 or 39 to 66 aa in length) to improve bioactivity.
  • a "SNAP25" linker (also known as the SNAP linker herein) linker comprises the amino acid sequence of SEQ ID NO:1 1.
  • SEQ ID NO: 11 represents the SNAP25 sequence, which forms a SNARE complex with complementary polypeptide helices of other appropriate SNARE proteins. Accordingly, in one embodiment, the SNARE peptidase domain and the translocation domain are attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.
  • this linker may also comprise the amino acid sequence of other appropriate SNAP proteins.
  • the SNARE peptidase domain, translocation domain and SNAP linker are all present within a single polypeptide chain.
  • the sequence of linker i.e. in this case the SNAP linker
  • the sequence identity may be at least about 85%, and even more preferably at least about 90%.
  • the sequence identity may be at least about 95%, at least about 98% or even 100%.
  • the neurotoxin of the invention may therefore comprise an irreversible helical bundle protein complex of a SNARE peptidase domain, translocation domain, first neuronal binding domain and second neuronal binding domain, wherein:
  • the first neuronal binding domain is attached to an amino acid sequence comprising a linker having at least about 80% sequence identity with the sequence of SEQ ID NO:9 or SEQ ID NO:15;
  • the second neuronal binding domain is attached to an amino acid sequence comprising a linker having at least about 80% sequence identity with the sequence of SEQ I D NO: 10 or SEQ ID NO: 17;
  • the SNARE peptidase domain and the translocation domain are attached to an amino acid sequence comprising a linker having at least about 80% sequence identity with the sequence of SEQ ID NO: 1 1.
  • the neurotoxin of the invention further comprises a spacer peptide sequence that spatially separated the first and second neuronal binding domains (e.g. the binding domains are spatially separated by inclusion of a spacer peptide into one or both linkers).
  • fusion proteins By inserting a flexible spacer peptide of suitable length into at least one of the fusion proteins (e.g. in between the syntaxin linker and the second neuronal binding domain in the appropriate fusion protein described above), the inventors were able to generate neurotoxins wherein the two binding domains were kept at a favourable distance from each other, with optimal bioactivity.
  • the rational design of fusion proteins (including spacer peptides) used for the construction of the neurotoxins described herein required great consideration.
  • the average length of spacer peptides that connect protein domains in natural multi-domain proteins has previously been calculated to be 6-16 residues, with the spacer peptides being grouped into small, medium, and large spacers with average length of 4.5 ⁇ 0.7, 9.1 ⁇ 2.4, and 21.0 ⁇ 7.6 residues, respectively (see Chen et al 2013, Advanced Drug Delivery Reviews, 65, pp 1357 to 1369).
  • Spacer peptides which connect protein domains to each other can adopt various structures (flexible or rigid) and exert diverse functions to fulfil the optimal joining of fusion proteins (see Table 2, Chen et al, 2013).
  • the flexible spacer peptides used in the invention are unusually long with amino acid lengths being from at least 31 amino acids (e.g. 31 to 66 or 39 to 66 aa). This allows for proper folding and optimal biological activity of the fusion proteins.
  • a "spacer peptide” refers to a peptide sequence that is used to spatially separate two protein domains in the final neurotoxin structure.
  • the spacer peptide may join the two protein domains together (e.g. it may be located between the first neuronal binding domain and the second neuronal binding domain of the neurotoxin when it is generated as a single polypeptide chain).
  • the spacer peptide may be present in a fusion protein that is used in the stapling reaction described herein, wherein the spacer peptide is located in between (i.e. joins together) the "linker" used in the stapling mechanism (e.g.
  • the spacer peptide spatially separates the attached binding domain from the stapling mechanism and thus spatially separates the attached binding domain from the other "stapled” binding domain in the final neurotoxin structure.
  • spacer peptide spacer peptide sequence
  • spacer peptide region are used interchangeably herein.
  • Spacer peptides may serve to connect protein domains by joining the carboxyl terminus of one protein moiety to the amino terminus of the next protein moiety without compromising cooperative inter-domain interactions or biological activity.
  • the rational design of suitable spacer peptides is described in detail in Chen et al., 2013, Advanced Drug Delivery Reviews 65 (2013) 1357-1369. Methods for designing appropriate spacer peptides are therefore well known in the art.
  • the spacer peptide of the invention is a flexible spacer peptide.
  • the properties of flexible spacer peptides also known as flexible linkers in the art are described in detail in the art (see for example Chen et al., 2013, Advanced Drug Delivery Reviews 65 (2013) 1357-1369).
  • flexible spacer peptides are usually applied when the joined domains require a certain degree of movement or interaction.
  • Flexible spacer peptides in natural proteins can vary in length from 5 up to 20 amino acids and are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains. The length of the flexible spacer peptides can be adjusted to allow for proper folding or to achieve optimal biological activity of the fusion proteins.
  • the spacer peptide of the invention may be a (flexible) spacer peptide of any appropriate length, for example at least 25 amino acids in length, at least 30, at least 31 , at least 35, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 66 amino acids in length (or any ranges therein between).
  • the spacer peptide is of 31 to 66 (e.g. 39 to 66) amino acids in length.
  • spacer peptides include a spacer peptide comprising any one of the following the amino acid sequences:
  • GASGGGGASSAGGGSSAGSGSSGGGAAAGSG (SEQ ID NO:20) GASGGGGASSAGGGSSAGSGSSGGGAAAGSGASGSASGS (SEQ ID NO: 21 ; see also Figure 31); or GASGGGGASSAGGGSSAGSGSSGGGAAAGSGSGASGGATAATGASGGGGASSAGGGS SAGSGSSGG (SEQ ID NO: 22; see also Figure 25).
  • the spacer peptides of the invention are exemplified by the amino acid sequences shown in SEQ ID NO:20, SEQ ID NO:21 , and SEQ ID NO:22.
  • methods for modifying these spacer peptides, or for generating other appropriate spacer peptides are well known in the art (see examples of appropriate sequences discussed in Chen et al. 2013, cited elsewhere herein).
  • Spacer peptides of the invention therefore include modifications of the spacer peptides shown in SEQ ID Nos: 20 to 22; e.g.
  • spacer peptides with at least 50% identity to these sequences; at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99% or 100% sequence identity to the spacer peptides shown in SEQ ID Nos: 20 to 22.
  • Appropriate modifications include conservative (or non-conservative) amino acid substitutions, deletions or additions.
  • the neurotoxins described herein may also be further modified, for example by the addition of organic molecules.
  • Cy3-maleimide may be conjugated to the free cysteines of a SNAP25 linker.
  • the linker may immediately adjacent to the corresponding neurotoxin domain, or alternatively, there may be one or more amino acids between the linker and its corresponding neurotoxin domain.
  • one or more amino acids may be needed between the linker and its corresponding domain in order to retain the domain's structural integrity (and/or function) in the resultant neurotoxin complex. Identifying whether or not one or more amino acids is required or optimal is well within the capabilities of a person of skill in the art. Suitable amino acid sequences for this purpose are well known to those skilled in the art.
  • the term "attached” is also used to describe the interaction between e.g. two or more domains within a single chain polypeptide (e.g. a polypeptide comprising a SNARE peptidase domain, a translocation domain and a third linker - for use in the stapling reaction described above; or a neurotoxin comprising a SNARE peptidase domain, a translocation domain, a first neuronal binding domain and a second neuronal binding domain within a single polypeptide chain (as exemplified in Figure 8).
  • the term “attached” also refers to direct or indirect attachment (i.e. one or more amino acids may be present between the recited domains).
  • Identifying whether or not one or more amino acids is required or optimal is well within the capabilities of a person of skill in the art. Suitable amino acid sequences for this purpose are well known to those skilled in the art.
  • the stapling technique described generally in US201 1038892 and utilized herein allows the controlled assembly of a stable SNARE complex formed of distinct functional units. The advantage of having a stable complex is that it can be used in relatively harsh conditions without the risk of the complex dissociating. Since the SNARE complex is well studied and is well known to those skilled in the art, a skilled person would be able to establish whether particular proteins are suitable for use in the present invention and how to manipulate these proteins and their sequences to produce the polypeptide helices of the invention so that they can form a stable SNARE complex.
  • the SNARE complex is formed from the following proteins: SNAP-25; syntaxin; and synaptobrevin. These proteins, as well as other SNARE proteins, contain SNARE motifs or SNARE domains which are the portions of the proteins which are involved in forming the SNARE complex. These SNARE domains or motifs are helices which pack together to form the SNARE complex. Generally, only a portion of the SNARE proteins is involved in SNARE complex formation; not the entire SNARE protein. For example, syntaxin has a C-terminal trans-membrane domain, a SNARE domain and an N-terminal regulatory domain, also known as the head domain. Obviously, only the SNARE domain is involved in forming the SNARE complex.
  • SNARE motif and "SNARE domain” are well known to those skilled in the art. Further, the SNARE motifs and SNARE domains of the various different SNARE proteins are also well known to a skilled person (Jahn R and Scheller R H (2006); Sieber et al. (2006); Besteiro (2006)).
  • the polypeptide helices (linkers) used by the inventors to generate the neurotoxins provided herein are based on specific SNARE domains or motifs of the SNARE proteins that form the SNARE complex, i.e. a SNAP protein; syntaxin; and synaptobrevin.
  • the SNAP protein linker can be any SNAP protein which can form part of a SNARE complex.
  • the skilled person is aware of the various SNAP proteins which can form part of a SNARE complex.
  • the SNAP protein may be SNAP-25A, SNAP-25B, SNAP-23 (also known as syndet), or SNAP-29.
  • the SNAP protein is not a-SNAP.
  • the SNAP protein is SNAP-25, i.e. SNAP-25A or SNAP-25B.
  • the syntaxin protein from which the polypeptide helix is derived can be any syntaxin protein which can form part of a SNARE complex.
  • the syntaxin protein may be selected from syntaxin 1A, syntaxin 1 B, syntaxin 2 (also known as epimorphin), syntaxin 3 and syntaxin 4, syntaxin 5, syntaxin 6, syntaxin 7, syntaxin 8, syntaxin 10, syntaxin 11 , syntaxin 13, syntaxin 17 or syntaxin 18.
  • the syntaxin protein is syntaxin 1A or 3.
  • the synaptobrevin protein (or homolog thereof) from which the polypeptide helix (linker) is derived can be any synaptobrevin protein or homolog which can form part of a SNARE complex.
  • the skilled person is aware of the various synaptobrevin proteins and homologs which can form part of a SNARE complex.
  • Synaptobrevin is a member of the vesicle- associated membrane protein (VAMP) family.
  • VAMP proteins are known to be able to form SNARE complexes and, therefore, may be suitable for providing the basis upon which a polypeptide helix can be derived.
  • homologs of synaptobrevin are VAMP proteins which can form part of a SNARE complex.
  • synaptobrevin protein or homolog thereof may be selected from synaptobrevin 1 , synaptobrevin 2, synaptobrevin 3 (also known as cellubrevin) and synaptobrevin 7 (also known as TI-VAMP).
  • the polypeptide helix is derived from a synaptobrevin protein.
  • the synaptobrevin protein is synaptobrevin 1 , 2 or 3.
  • the organism from which the SNARE proteins originate can be any suitable organism in which SNARE complexes are utilised.
  • the proteins may originate from:
  • the SNARE proteins may be derived from yeast (Rossi G et al. (1997)).
  • the organism from which the SNARE proteins originate may depend on the application of the complexing system. For example, for medical applications, the SNARE proteins preferably originate from humans.
  • the polypeptide helices (linkers) used in the stapling method are derived from the SNARE proteins which form the SNARE complex.
  • the helices of the SNARE motif or domain of the SNARE proteins which form the SNARE complex are generally about 50-60 amino acids in length.
  • the four polypeptide helices of the invention are derived from the SNARE motif or SNARE domain of the SNARE proteins.
  • the term "derived from” means that the sequence of the polypeptide helix is substantially the same as the sequence of the SNARE domain/motif or a portion thereof so that it is capable of forming a stable SNARE complex.
  • the sequence of the polypeptide helix should have at least about 80% sequence identity with the sequence of the selected SNARE domain/motif or the portion thereof. More preferably, the sequence identity should be at least about 85%, and even more preferably at least about 90%. In one embodiment, the sequence identity may be at least about 95%, at least about 98% or even 100%. However, in some embodiments, it may be preferable for the sequence of the polypeptide helix to differ from the sequence of the selected SNARE domain/motif or the portion thereof. This may be beneficial in terms of expression of the protein, purification of the protein or down-stream applications. For example, and without limitation, this may include the addition of histidine residues at either end of the sequence to enable purification, or incorporation of additional lysine or cysteine residues for functional attachment of the peptides to surfaces etc.
  • US20110038892 may be used in to generate the neurotoxins provided herein and are specifically incorporated herein in their entirety.
  • the average length of spacer peptides connecting protein domains in natural multi-domain proteins was calculated to be 6-16 residues with the spacer peptides grouped into small, medium, and large with average length of 4.5 ⁇ 0.7, 9.1 ⁇ 2.4, and 21.0 ⁇ 7.6 residues, respectively (see Chen et al 2013, Advanced drug Delivery reviews 65 (2013) pp 1357-1369).
  • Spacer peptides which connect protein domains can adopt various structures (flexible or rigid) and exert diverse functions to fulfil the optimal joining of fusion proteins (see Table 2, of Chen et al., 2013).
  • the flexible spacer peptides useful in the context of this invention are unusually long, with amino acid length being at least 31 amino acids (e.g. from 31 to 66 or from 39 to 66 aa).
  • the length of the flexible spacer peptides can be further adjusted to allow for proper folding or to achieve optimal biological activity of the fusion proteins.
  • neurotoxin efficacy can be improved by modifying the neurotoxin protein to include two neuronal binding domains.
  • these novel molecules are more effective in cleaving their SNARE substrate (e.g. SNAP25) in target host cells compared to equivalent neurotoxin molecules with a single neuronal (receptor) binding domain.
  • SNARE substrate e.g. SNAP25
  • the resultant neurotoxin can be used to more effectively target specific neuronal populations.
  • compositions e.g. a pharmaceutical composition
  • Such compositions may be used for cosmetic (i.e. non- therapeutic) or therapeutic purposes as outlined below.
  • a neurotoxin as provided herein may thus be part of a composition (e.g. a pharmaceutical composition) that comprises the neurotoxin and one or more other components.
  • a composition may be a composition that comprises a neurotoxin of the invention and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
  • pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, i.e. , the material may be administered to an individual along with the selected neurotoxin without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • Excipients are natural or synthetic substances formulated alongside an active ingredient
  • a neurotoxin as provided herein
  • Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life.
  • Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art.
  • suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation.
  • Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.
  • Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
  • Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
  • compositions provided herein may be used for cosmetic (i.e. non-therapeutic) or therapeutic purposes as outlined below.
  • compositions of the invention may advantageously be used to prevent, regulate or reduce skin wrinkling in a subject.
  • such uses are considered “cosmetic” and/or “non-therapeutic”.
  • a method of preventing, regulating or reducing skin wrinkling in a subject is therefore provided, wherein the method comprises administering a composition of the invention to the subject.
  • such methods are considered “cosmetic” and/or “non- therapeutic”.
  • the terms “cosmetic” and “non-therapeutic” are used interchangeably and are intended to refer to interventions performed with the intention of addressing (e.g.
  • a composition of the invention may be used for preventing, regulating or reducing skin wrinkling in a subject. Suitable methods for administering a composition for this purpose are well known in the art, and include but are not limited to injection (e.g. of botulinum neurotoxin type A).
  • a composition of the invention may be used for correcting an external appearance distorted due to excessive neuromuscular activity in a subject.
  • excessive neuromuscular activity refers to an increase in neuromuscular activity compared to the norm.
  • Examples of distorted external appearances that may be corrected using a composition of the invention include muscle spasms and muscle tics.
  • a composition of the invention may be used for preventing, regulating or reducing sweating due to excessive neuronal activity in a subject.
  • excessive neuronal activity refers to an increase in neuronal activity compared to the norm.
  • Examples of excessive neuronal activity that may result in sweating that may be prevented, reduced or regulated using a composition of the invention include focal hyperhidrosis of the palms, armpits and/or soles.
  • compositions of the invention may also be used for therapeutic purposes.
  • therapeutic is intended to refer to “treatment” of a subject.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom.
  • disease and “disorder” are used interchangeably.
  • subject refers to an individual, e.g., a human, pig, horse, mouse, cow, rat etc having or at risk of having a specified condition, disorder or symptom.
  • the subject may be a patient i.e. a subject in need of treatment in accordance with the invention.
  • the subject may have received treatment for the condition, disorder or symptom.
  • the subject has not been treated prior to treatment in accordance with the present invention.
  • compositions provided herein may be used for treating or preventing a condition, disorder or symptom which is alleviated by the inhibition of neural terminals.
  • the skilled person will be fully aware of the diseases or conditions which are alleviated by the inhibition of neural terminals since the use of botulinum toxin A has been in widespread use for medicinal and cosmetic therapies for a number of years (see, for example, Jankovic (2004) Botulinum in clinical practice. J Neurol Neurosurg Psychiatry 75 951-957).
  • some of the diseases or conditions which are alleviated by the inhibition of neural terminals are selected from the group consisting of: excessive sweating, salivation, dystonias, gastrointestinal disorders, urinary disorders, facial spasms, strabismus, cerebral palsy, stuttering, chronic tension headaches, hyperlacrymation, hyperhidrosis, spasms of the inferior constrictor of the pharynx, spastic bladder, pain, migraine, and cosmetic treatments such as reducing wrinkles, brow furrows, etc.
  • compositions of the invention may therefore be used to treat or prevent a neurological condition, disorder or symptom in a subject.
  • Methods of treating or preventing a neurological condition, disorder or symptom are also provided, comprising administering a composition of the invention to a subject. Accordingly, in vivo methods of treatment are provided, which may be prophylactic and/or therapeutic.
  • treating or preventing a "neurological condition, disorder and/or symptom" is intended to include treating or preventing cholinergic controlled secretions, pain, a neurological disorder or condition in a subject, conditions or diseases resulting from involuntary spasms, muscle spasticity, an allergy, strabismus, occupational cramps, anal fissures, migraine headaches, brusism, and any combinations thereof.
  • the pain is selected from the group consisting of: pain associated with neuromuscular disorders, pain associated with arthritis, pain associated with trigeminal neuralgia, headache pain, inflammatory nociceptive pain, and neuropathic pain.
  • the neuropathic pain is selected from the group consisting of: cancer pain, post-operative neuropathic pain, allodynia, post-herpetic neuralgia bone pain, peripheral neuropathy
  • the pain associated smooth muscle disorder is selected from the group consisting of: achalasia, and spasms in the sphincters of the cardiovascular arteriole, circulatory system-affiliated pain, gastrointestinal system, urinary, gall bladder, rectum and other visceral pain, irritable bowel syndrome.
  • the condition or disease resulting from involuntary muscle spasms is selected from the group consisting of: hemifacial spasms, blepharospasm, laryngeal dysphoria, head dystonias, neck dystonias, limb dystonias, and rectal spasms.
  • the cholinergic controlled secretion is selected from the group consisting of: lacrimation, salivation, mucus secretion, gastrointestinal secretion and hyperhidrosis.
  • BoNT type A The botulinum neurotoxin type A (BoNT type A; or BoNT/A) has proven to be of great medical importance due to its ability to cause a very long neuromuscular paralysis upon local injections of minute amounts (1 pM concentration) (Montecucco, C. et al. (2009)). Over the last 30 years, BoNT/A and other botulinum neurotoxins have been successfully exploited for medicinal and cosmetic purposes. These toxins silence neuromuscular junctions and also can block neurotransmitter release from many types of neurons.
  • BoNT/A a form of BoNT/A that has been used successfully for cosmetic treatment. Since the paralysis of neuromuscular junctions is reversible, the sustained relaxation of muscles requires repeat injections every three to four months.
  • BoNT/A can block innervation of not only striated muscles but also of smooth muscles. Furthermore, the cholinergic junctions of the autonomous nervous system that control sweating, salivation and other types of secretion are as sensitive to BoNT/A and BoNT/B as are the neuromuscular junctions. Therefore, botulinum-based treatments have recently expanded to include a dazzling array of nearly a hundred conditions from dystonias to gastrointestinal and urinary disorders.
  • BoTox A common complaint regarding the treatment of migraine with commercially available BoTox is that it takes too long to take effect. Often taking a week for a noticeable reduction in headaches to occur. Pertinently, the Botulinum constructs described herein begin to cleave cellular SNAREs four times faster than their native Botulinum toxin counterparts (Fig. 28 and 30).
  • BoNT/A has the longest paralysing effect among the seven immunologically distinct serotypes of BoNTs (A-G), thus underpinning the usefulness of specifically BoNT/A in the treatment of neurological disorders.
  • All BoNTs are synthesised by the bacteria as single polypeptide chains with a molecular mass of 150 kDa. Following bacterial death and lysis, the toxins are 'nicked' by bacterial proteases to yield the 50 kDa light and the 100 kDa heavy chains that are kept together by a disulphide bond. The two chains, still linked through the disulphide bond, traverse the intestinal epithelial cells by transcytosis, enter the bloodstream and eventually bind to peripheral cholinergic nerve terminals.
  • BoNTs The extreme toxicity of BoNTs indicates that the peripheral nerve endings carry molecules that can serve as BoNTs' high-affinity receptors. Indeed, several synaptic vesicle proteins have been shown to act as receptors for BoNTs. While the heavy chains are responsible for BoNTs' binding to nerve terminals, the light chains are potent endopeptidases that attack the vesicle fusion machinery and therefore have to get inside the nerve terminal. BoNTs accomplish this task by hijacking the vesicle endocytosis route. As the pH of the recycling vesicle's interior drops, the BoNTs undergo major conformational changes.
  • Td and HN translocating domains of the heavy chains
  • BoNT/A and BoNT/E proteolyse SNAP-25 cleave VAMP on the synaptic vesicles.
  • BoNTs B, D, F and G cleave VAMP on the synaptic vesicles.
  • SNAP-25 shortened by only nine amino acids by BoNT/A retains its ability to interact with the plasma membrane syntaxin and vesicular synaptobrevin but cannot mediate the normal vesicle fusion process.
  • BoNTs botulinum neurotoxins
  • BoNTs can stop the neurotransmitter release of not only acetylcholine but also glutamate, glycine, noradrenaline, dopamine, serotonin, ATP and various neuropeptides (Ashton et al. (1988), Capogna, M. et al. (1997), Sanchez-Prieto, J. et al. (1987), Verderio, C. et al. (2004), Luvisetto, S. et al. (2004), and Costantin, L. et al. (2005)).
  • Botulinum-2xNbd/A shows a lack of paralysis, likely due to the extension in size of the molecule caused by the addition of the rigid SNARE complex, which would not be present in a recombinantly produced single molecule that only comprised the LC, TD and 2 binding domains (as in Fig. 8).
  • Botulinum-2xNbd/A has over 12x increased efficacy of SNAP25 cleavage, the most relevant indicator of botulinum enhanced activity, in comparison to native BoNT/A ( Figure 4, Figure 28).
  • compositions described herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be topical, intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, transdermal, epidural, intrathecal, percutaneous, or by infusion.
  • compositions described herein may be in any form suitable for the above modes of administration.
  • suitable forms for parenteral injection include a sterile solution, suspension or emulsion
  • suitable forms for topical administration include an ointment or cream
  • suitable forms for rectal administration include a suppository.
  • the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery.
  • compositions described herein are for administration in an effective amount.
  • An "effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response.
  • the effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient/subject.
  • the suitable dosage of the neurotoxin for a given patient/subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the neurotoxin for example severity and type of disorder, condition or symptom, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors.
  • the dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient/subject.
  • Effective dosages may be determined by either in vitro or in vivo methods.
  • compositions of the present invention are advantageously presented in unit dosage form.
  • the invention is directed to a neurotoxin comprising a SNARE peptidase domain, a translocation domain, a first neuronal binding domain, and a second neuronal binding domain, wherein the first and second binding domains are each selected from the group consisting of:
  • the first and second binding domains are spatially separated in the neurotoxin due to the presence of a (flexible) spacer peptide.
  • novel neurotoxin molecules of the invention include two neuronal (receptor) binding domains (preferably separated by a (flexible) spacer peptide). Surprisingly, these novel molecules have improved neurotoxin activity within target cells. The presence of two neuronal (receptor) binding domains (preferably separated by a (flexible) spacer peptide) increases the efficacy of delivery of the molecule into target cells.
  • the invention can also be applied to the delivery of cargo molecules attached to the neuronal binding domain. Accordingly, in one aspect, the invention provides a molecule comprising a cargo molecule, a first neuronal binding domain, and a second neuronal binding domain, wherein the first and second binding domains each selected from the group consisting of:
  • the first and second binding domains are spatially separated in the neurotoxin due to the presence of a (flexible) spacer peptide.
  • the first and second neuronal binding domains are the same.
  • the polypeptide of (i) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO: 1 , optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide of (ii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:2, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.
  • the polypeptide of (iii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:3, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:3.
  • the polypeptide of (iv) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:4.
  • the polypeptide of (v) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO:5, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:5.
  • the polypeptide of (vi) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO:6, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:6.
  • the polypeptide of (vii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO:7, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:7.
  • the polypeptide of (viii) comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ I D NO:8, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:8.
  • the first neuronal binding domain is attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO:9.
  • the second neuronal binding domain is attached to an amino acid sequence comprising the amino acid sequence of SEQ I D NO:10.
  • the cargo molecule is attached to an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 1.
  • the cargo molecule, first neuronal binding domain, and second neuronal binding domain are comprised within a single polypeptide chain.
  • the invention provides a composition comprising a molecule according to the invention, and a pharmaceutically acceptable excipient, adjuvant, diluent or carrier.
  • the invention provides the use of a molecule according to the invention for use in therapy and/or diagnosis.
  • the exact nature of the therapy and/or diagnosis will depend on the identity of the cargo molecule.
  • Figure 6 provides a non-limiting example of a molecule comprising a cargo molecule, a first neuronal binding domain and a second neuronal binding domain, wherein the first and second binding domains are each selected from the group consisting of: (i) a polypeptide having botulinum neurotoxin binding domain (BoNT/Nbd) type A activity;
  • the cargo molecule of the molecule comprises Cy3.
  • the cargo molecule may comprise any other e.g. enzymatic, therapeutic or imaging moiety that is suitable for delivery to target cells (neuronal cells).
  • the cargo molecule By selecting specific neuronal binding domains (and binding domain combinations), the cargo molecule can be targeted to a specific neuronal population.
  • the invention therefore provides a new mechanism to improve selective delivery of cargo molecules (e.g. organic molecules, therapeutic nucleic acids, or therapeutic peptides) to target cells.
  • cargo molecules e.g. organic molecules, therapeutic nucleic acids, or therapeutic peptides
  • the cargo molecule may be any domain (also referred to as "moiety") which a skilled person might want to deliver to the target cells.
  • the cargo molecule/moiety may be selected from a small molecule, a polymer containing a small molecule, a polypeptide, a protein, a nucleic acid or derivative, and a particle or nanoparticle.
  • the cargo moiety may be:
  • a small molecule or a polymer containing a small molecule such as:
  • an affinity tag e.g. biotin
  • a therapeutic e.g. a toxin or a drug
  • a reactive group for further/downstream cross-linking, polymerisation and further derivatisation e.g. an amino group, carboxyl group, sulfhydryl group, guanidine group, phenolic group, thioether group, imidazol group, indol group, etc. ;
  • a spontaneously reactive group suitable for further modification e.g. a maleimide or derivative for cross-linking to SH groups, or any other chemistry suitable for cross-linking
  • a molecule for direct attachment to surfaces e.g. an SH— containing molecule for attachment to metal surfaces;
  • an imaging reagent e.g. a fluorescent or absorbent moiety for UV, VIS, IR, Raman, NMR, MRI, PET, X-ray or other imaging;
  • a biologically relevant ligand e.g. for receptor binding/targeting
  • a biologically relevant substrate e.g. a phosphorylation or other PTM site
  • a biologically relevant molecule e.g. a lipid or carbohydrate
  • a protective group or molecule e.g. PEG;
  • a metal-chelating compound 2. a polypeptide or protein such as:
  • polypeptide or protein containing a functional site e.g. a protease digestion site
  • a targeting functional peptide e.g. for different organelle targeting, nuclear targeting (for transfection), intracellular targeting (for drug delivery), etc. ;
  • a peptide affinity tag e.g. Flag, Myc, VSV, HA, 6x His, 8xhis, poly-His, etc.;
  • polypeptide or protein capable of forming a protein-protein interaction, e.g. PDZ, SH2/3; an antibody, antibody fragment, antibody mimic, RNA- or peptide-based aptamer, or another affinity reagent (proteinous or non-proteinous);
  • an enzyme e.g. for research, diagnostics (the complexing system can be used to immobilise enzymes for some applications) and therapeutic applications, for nucleic acid synthesis or amplification including promoters, polymerases, restriction endonucleases, or other modifying enzymes;
  • nucleic acid or derivative such as:
  • DNA, RNA, or PNA for detection, immobilisation, hybridisation, synthesis priming, synthesis and amplification, labelling, signal detection and signal amplification, transcription and translation;
  • a particle or nanoparticle such as:
  • a metallic particle or nanoparticle e.g. gold or silver for staining or labelling
  • a semiconductor particle or nanoparticle e.g. quantum dots for labelling and detection
  • a polymer micro or nanoparticle e.g. resins, gels, etc.
  • the first cargo moiety is an enzymatic, therapeutic or imaging moiety.
  • the enzymatic, therapeutic or imaging agent may be any suitable agent.
  • the imaging agent can be any agent which can be attached to a helix and which allows the position of the helix to be imaged, for example, a GFP fluorescent tag, fluorescently labelled peptides, and MRI contrast agents.
  • the enzymatic agent can be any enzyme or functional portion thereof.
  • the enzymatic, therapeutic or imaging agent is an enzymatic agent.
  • the therapeutic agent can be an organic drug.
  • GST glutathione-S-transferase
  • Linking two binding domains involved attaching complementary linkers to botulinum binding domains in the first instance.
  • the final linking reaction was based on the formation of the ternary SNARE complex which is irreversible in extracellular environment.
  • the SNARE complex forms within 60 min upon mixing three required components in 1 :1 :1 ratio at 20°C.
  • the three components are based on synaptobrevinA AMP, syntaxin and SNAP25 sequences (Darios, F. et al. Proc Natl Acad Sci U S A 107, 18197-18201).
  • the linkerl sequence is based on the sequence of rat VAMP2 (2-84, SEQ ID: 15) and consists of the amino acids:
  • Nbd/T Fusion of the linkerl with the tetanus binding domain (Nbd/T) was made by inserting the DNA sequence for VAMP2 (2-84) into the Xhol site of the pGEX-KG vector and the DNA sequence for the tetanus binding domain (856 - 1315) into the Sad site. Nbd/T was replaced with botulinum binding domains as shown below.
  • the Iinker2 was based on the SNARE helix of rat syntaxin 3 (195-253, SEQ ID: 10) and consisted of the amino acids:
  • Linker2-Nbd/T was designed by inserting the DNA sequence for rat syntaxin 3 (195-253) into the Xbal site of the pGEX-KG vector and the DNA sequence of Nbd/T into the Sad site. Nbd/T was replaced with botulinum binding domains as required. Syntaxin 1 SNARE helix is poorly expressed in bacteria forcing us to use the Syntaxin 3 SNARE helix in fusion proteins.
  • the enzymatic portion of the botulinum type A1 neurotoxin consisting of its light chain and translocation domain fused to SNAP25 was prepared as previously described (Darios, F. et al. Proc Natl Acad Sci U S A 107, 18197-18201).
  • the Iinker2 was utilised as a staple to link the enzymatic portion of the botulinum type A1 neurotoxin with any clostridial binding domain (Darios, F. et al. Proc Natl Acad Sci U S A 107, 18197-18201).
  • the staple is based on the Syntaxin 1 SNARE helix prepared as a chemically synthetic peptide
  • Fusion proteins containing SNAP25, linkerl and Iinker2 components were mixed at an equimolar ratio in Buffer A and were left at 20°C for 1 hour to allow formation of the SNARE complex. Irreversible assembly of protein complexes was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were run at 4°C and protein were visualised by Coomassie Blue staining. 3. Recombinant preparation the BoNT/C molecule containing two binding domains separated by a flexible spacer peptide
  • BoNT-2xNbd/C was designed by inserting the DNA sequence for the 39 aa spacer peptide, followed by the DNA sequence for BoNT/C 867-1291 at the 3' end of the DNA sequence for the full BoNT/C, described previously (Rummel, A., Hafner, K., Mahrhold, S., Darashchonak, N., Holt, M., Jahn, R., Beermann, S., Karnath, T., Bigalke, H. & Binz, T. J Neurochem.
  • Double- and single-liganded constructs containing botulinum type A or botulinum type C enzyme were compared for the cleavage of intraneuronal SNAP25 which can be detected as a minute shift in the molecular weight of SNAP25 in SDS-PAGE gels followed by
  • Immunoblotting can be performed using total SNAP25 antibody which recognizes both the intact and cleaved SNAP25, and a custom-made antibody raised against the cleaved end of SNAP25 (TRIDEANQ, SEQ ID: 18) which recognizes only the botulinum cleaved product.
  • TIDEANQ cleaved end of SNAP25
  • the uncleaved SNAP25 serves as a useful internal loading control, therefore the inventors used the total SNAP25 antibody in ex-vivo cell experiments (Fig. 3).
  • SDS-PAGE loading buffer 56 mM sodium dodecyl sulfate, 0.05 M Tris-HCI, pH 6.8, 1.6 mM EDTA, 6.25% glycerol, 0.0001% bromophenol blue, 10 mM MgCI2, 26 U/mL benzonase. Plates were then shaken at 900 rpm for 10 min. Samples were boiled for 3 min at 95°C and then run on 12% Novex SDS- PAGE gels (Invitrogen).
  • FIG. 4 Further examples for superior neuronal activity of double-liganded constructs utilising botulinum binding domains are in Fig. 4.
  • neuroblastoma cells originated from sympathetic nervous system (Fig. 5).
  • Double- and single-liganded constructs containing botulinum type C enzyme were compared for the ability to cause cell death to differentiated human neuroblastoma cells originated from sympathetic nervous system via cleavage of SNAP25 and syntaxin, (Rust, A., Leese, C, Binz, T. & Davletov, B. Oncotarget l, 33220-33228, (2016)).
  • the inventors prepared fluorescent tetanus double-liganded construct and compared it to single-liganded construct for binding mouse motor neurons.
  • cysteine-rich SNAP25 protein can be exploited.
  • the native SNAP25B DNA was inserted into the BamHI site of the pGEX-KG plasmid. After SNAP25 expression in E.coli, Cy3-maleimide was conjugated to the free cysteines of SNAP25 at 8 fold molar excess. Conjugated SNAP25-Cy3 was then purified by gel filtration.
  • Nbd/T with linkerl
  • Coverslips were washed 3 times in PBS, then incubated with the appropriate fluorescently conjugated secondary antibodies diluted in 5% BSA for 1 h at room temperature. Finally, coverslips were washed 3 times with PBS, once with water and then mounted using Mowiol-488. Coverslips were imaged with an invert Zeiss LSM 780 confocal microscope using a 63X Plan-Apochromat oil immersion objective with an NA of 1.4. Immunofluorescence staining was quantified using ImageJ. 7. Increased SNAP25 cleavage observed in the rat spinal cord following injection of double- liganded botulinum enzyme
  • Rat spinal cord sections were incubated with anti-cleaved SNAP-25 antibody (1 :10000 diluted in PBS) and left overnight on a rocker at room temperature. The following day, the sections underwent three PBS washes before incubation with biotinylated secondary antibodies (goat anti-rabbit; 1 :400; Vector Stain) for 90 minutes at room temperature as part of a Tyramide Signal Amplification (TSA) protocol. After additional three washes, sections were incubated in ABC complex (1 :125; Vector Stain, ABC elite kit, Vector Labs) for 30mins at room temperature. Again after three PBS washes, sections were incubated with biotinylated tyramide (1 :75; TSA Stain Kit; Perkin Elmer) for 7 minutes.
  • biotinylated secondary antibodies goat anti-rabbit; 1 :400; Vector Stain
  • TSA Tyramide Signal Amplification
  • SNAP-25 cleavage was visible in the ipsilateral ventral horn of the lumbar spinal cord, following injection of both botulinum-2xNbd/T and botulinum-1x Nbd/T (Fig. 7).
  • lumbar spinal cord sections prepared from botulinum-2xNbd/T injected rats showed a much greater medial spread of SNAP-25 cleavage with the dense staining reaching the central canal and additional projections decussating to the contralateral spinal cord.
  • the area positively stained for SNAP-25 cleavage was calculated as a percentage of the total area of the spinal cord section using a thresholding technique. Intraplantar injection of botulinum- 2xNbd/T resulted in an approximately three times larger area of SNAP-25 cleavage in comparison to botulinum-1xNbd/T.

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Abstract

La présente invention concerne des neurotoxines comprenant un domaine de peptidase SNARE, un domaine de translocation, un premier domaine de liaison neuronale (Nbd) et un second domaine de liaison neuronale (Nbd). L'invention concerne également des compositions (par exemple, des compositions pharmaceutiques) comprenant lesdites neurotoxines, et leurs utilisations.
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