Use of clostridial neurotoxin variants for treating neurological disorders

Abstract

The present invention relates to a polypeptide for promoting neuron growth or neuron repair to treat a subject's neurological disorder, wherein the polypeptide comprises: a clostridial neurotoxin light chain (L-chain) or a fragment thereof; and / or a fragment of a clostridial neurotoxin heavy chain (H-chain). Other polypeptides for promoting neuron growth or neuron repair to treat a subject's neurological disorder, as well as corresponding methods and uses are also provided.

Description

[0001] The present invention relates to the treatment of neurological disorders.

[0002] Neurological disorders include neuronal damage, neurodegenerative disorders, sensory disorders, and autonomic nerve disorders.

[0003] Neuronal injuries, such as spinal cord injury (SCI), result in the degeneration of damaged axons, which prevents normal sensory, motor, and autonomic functions. Recovery can occur through endogenous mechanisms, such as regeneration of damaged axons and lateral sprouting of undamaged axons, leading to the restoration of innervation of denervated targets. However, the regenerative capacity of damaged neurons in adult mammals, especially in the spinal cord, is limited, and patients may suffer from various disabilities, which greatly affect the quality of life.

[0004] Conventional therapies for neuronal injury include interleukin-6 (IL-6) and stem cell transplantation, however few are in the development stage for clinical use. Therefore, there is still a need for therapeutic agents for neuronal injury that can promote neuronal growth or repair.

[0005] Bacteria in the genus Clostridia produce highly virulent and specific protein toxins that poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include neurotoxins produced by C. tetani (TeNT) and neurotoxins produced by C. botulinum (BoNT) serotypes AG and X (see WO2018 / 009903 A2), as well as neurotoxins produced by C. baratii and C. butyricum.

[0006] Among the clostridial neurotoxins, some of the most potent are known. For example, botulinum neurotoxin has a median lethal dose (LD ) for mice ranging from 0.5 to 5 ng / kg, depending on the serotype. 50 ) values. Both tetanus toxin and botulinum toxin act by inhibiting the function of affected neurons, particularly by inhibiting the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts on the central nervous system.

[0007] In nature, clostridial neurotoxins are synthesized as single-chain polypeptides that are modified post-translationally by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, often called the activation site, which is located between the cysteine ​​residues that provide the interchain disulfide bond. It is this two-chain form that is the active form of the toxin. The two chains are called the heavy chain (H-chain) (whose molecular weight is approximately 100 kDa) and the light chain (L-chain) (whose molecular weight is approximately 50 kDa). The H chain contains the N-terminal translocation component (H N domain) and the C-terminal targeting component (H C domain). The cleavage site is located between the L-chain and the translocation domain components. C After the domain binds to its target neuron and internalizes the bound toxin into the cell via endosomes, H N The domain translocates the L-chain across the endosomal membrane and into the cytosol, while the L-chain provides the protease function. Also known as non-cytotoxic protease).

[0008] Non-cytotoxic proteases act by proteolytically cleaving intracellular transporters called SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin). The acronym SNARE is derived from the term soluble NSF attachment receptor ( S oluble N SF A ttachment Re ceptor), where NSF stands for N-ethylmaleimide sensitive factor ( N -ethylmaleimide- S ensitive F The SNARE proteins are components of the intracellular vesicle fusion complex and are therefore secreted molecules by vesicle transport from the cell. The protease function is a zinc-dependent endopeptidase activity that exhibits high substrate specificity for SNARE proteins. Therefore, once delivered to the desired target cell, the non-cytotoxic protease is able to inhibit cellular secretion from the target cell. The L-chain protease of the clostridial neurotoxin is a non-cytotoxic protease that cleaves SNARE proteins.

[0009] Given the ubiquity of SNARE proteins, Clostridial neurotoxins, such as botulinum toxin, have been successfully used in a variety of therapeutic applications.

[0010] WO 2016 / 170501 A1 describes the use of catalytically active full-length BoNT / A (containing an L-chain and a complete H-chain, including H N and H Cdomain) to treat paralysis caused by spinal cord injury. WO 2016 / 170501 A1 teaches that each functional domain of BoNT / A is essential for the observed therapeutic effect, including the H-chain binding and translocation ability and the L-chain non-cytotoxic protease activity. As described above, the full-length clostridial neurotoxin is very potent and specific safety procedures need to be adopted when handling the toxin. In addition, it is believed that the spread of the toxin from the target tissue is responsible for adverse side effects that may be life-threatening in extreme cases. This may be a particular concern when clostridial neurotoxin therapeutics (such as BoNT therapeutics) are used at high doses, concentrations, and injection volumes. Reported adverse effects of commercial BoNT / A therapies associated with this problem include weakness, generalized muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and dyspnea. Swallowing and breathing difficulties can be life-threatening, and deaths have been reported to be associated with the spread of the toxin's effects. Therefore, a safer therapeutic agent for promoting neuronal growth or repair is needed.

[0011] Due to their size, the use of full-length Clostridial neurotoxins (~150 kDa) or their complete H-chains (~100 kDa) is associated with an increased risk of eliciting an immune response in subjects treated with the polypeptides. C domain) results in binding of the polypeptide to the Clostridial neurotoxin target receptor, which may be associated with undesirable off-target effects in subjects to whom the polypeptide is administered.

[0012] The present invention overcomes one or more of the above-identified problems.

[0013] The inventors of the present invention surprisingly discovered that a fragment of a Clostridial neurotoxin L-chain and / or a Clostridial neurotoxin H-chain (e.g., a translocation domain (H N ) or receptor binding domain (H C )) polypeptides promote neuronal growth or repair, thereby finding utility in the treatment of neurological disorders. Advantageously, this allows the use of non-toxic (or substantially non-toxic) fragments of clostridial neurotoxins, which are smaller in size (compared to full-length H-chains or full-length clostridial neurotoxins) and are less likely to elicit an immune response in a subject to which the fragments are administered. In addition, the non-toxic (or substantially non-toxic) fragments are less expensive to manufacture and / or simpler to manufacture than the full-length clostridial neurotoxins. In addition, the non-toxic (or substantially non-toxic) fragments constitute more defined therapeutic agents than the full-length clostridial toxins, and given the shorter polypeptide length, the likelihood of, for example, cysteine ​​shuffling between domains is reduced.

[0014] Therefore, in one aspect, the present invention provides a polypeptide for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises:

[0015] Clostridial neurotoxin light chain (L-chain) or fragments thereof; and / or

[0016] Fragments of the heavy chain (H-chain) of Clostridial neurotoxin.

[0017] In a related aspect, a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject is provided, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises:

[0018] Clostridial neurotoxin L-chain or fragments thereof; and / or

[0019] Fragments of the H-chain of Clostridial neurotoxins.

[0020] In another aspect, there is provided a use of a polypeptide in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises:

[0021] Clostridial neurotoxin L-chain or fragments thereof; and / or

[0022] Fragments of the H-chain of Clostridial neurotoxins.

[0023] In one aspect, the invention provides a polypeptide for use in treating a neurological disorder in a subject, wherein the polypeptide comprises:

[0024] Clostridial neurotoxin light chain (L-chain) or fragments thereof; and / or

[0025] Fragments of the heavy chain (H-chain) of Clostridial neurotoxin.

[0026] In a related aspect, a method for treating a neurological disorder in a subject is provided, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises:

[0027] Clostridial neurotoxin L-chain or fragments thereof; and / or

[0028] Fragments of the H-chain of Clostridial neurotoxins.

[0029] In another aspect, there is provided a use of a polypeptide in the preparation of a medicament for treating a neurological disorder in a subject, wherein the polypeptide comprises:

[0030] Clostridial neurotoxin L-chain or fragments thereof; and / or

[0031] Fragments of the H-chain of Clostridial neurotoxins.

[0032] In one embodiment, the polypeptide of the invention comprises a Clostridial neurotoxin L-chain. Preferably, the L-chain is catalytically inactive.

[0033] Thus, in one aspect, the present invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a catalytically inactive Clostridial neurotoxin L-chain.

[0034] In a related aspect, the invention provides a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a catalytically inactive Clostridial neurotoxin L-chain.

[0035] In another aspect, the present invention provides use of a polypeptide comprising a catalytically inactive Clostridial neurotoxin L-chain in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject.

[0036] In one aspect, the invention provides a polypeptide for use in treating a neurological disorder in a subject, wherein the polypeptide comprises a catalytically inactive Clostridial neurotoxin L-chain.

[0037] In a related aspect, the invention provides a method for treating a neurological disorder in a subject, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a catalytically inactive Clostridial neurotoxin L-chain.

[0038] In another related aspect, the present invention provides use of a polypeptide comprising a catalytically inactive Clostridial neurotoxin L-chain in the preparation of a medicament for treating a neurological disorder in a subject.

[0039] The inventors of the present invention have shown for the first time that the catalytic activity of the Clostridial neurotoxin L-chain is not necessary for promoting neuronal growth or neuronal repair. Thus, the present invention allows for the provision of safer (less toxic) therapeutic agents.

[0040] The active clostridial neurotoxin L-chain has non-cytotoxic protease activity. Specifically, the active clostridial neurotoxin L-chain has endopeptidase activity and is capable of cleaving proteins of the exocytic fusion apparatus in target cells. The proteins of the exocytic fusion apparatus are preferably SNARE proteins, such as SNAP-25, synaptobrevin / VAMP, or syntaxin.

[0041] The term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L-chain means that the L-chain does not substantially exhibit non-cytotoxic protease activity, preferably the term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L-chain means that the L-chain does not exhibit non-cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin L-chain is a chain that does not cleave a protein of the extracellular fusion apparatus in a target cell. The term "substantially free of non-cytotoxic protease activity" means that the clostridial neurotoxin L-chain has less than 5% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain, such as less than 2%, 1% or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain. The non-cytotoxic protease activity can be measured by incubating a test clostridial neurotoxin L-chain with a SNARE protein and comparing the amount of SNARE protein cleaved by the test clostridial neurotoxin L-chain compared to the amount of SNARE protein cleaved by the catalytically active clostridial neurotoxin L-chain under the same conditions. Conventional techniques, such as SDS-PAGE and Western blotting, can be used to quantify the amount of cleaved SNARE protein. Suitable in vitro assays are described in WO 2019 / 145577 A1, which is incorporated herein by reference.

[0042] Cell-based and in vivo assays can also be used to determine whether a Clostridial neurotoxin comprising an L-chain and a functional cell binding and translocation domain has non-cytotoxic protease activity. Assays such as digital abduction score (DAS), dorsal root ganglion (DRG) assays, spinal cord neuron (SCN) assays, and mouse phrenic nerve hemidiaphragm (PNHD) assays are routine in the art. Suitable assays for determining non-cytotoxic protease activity can be assays described in Donald et al. (2018), Pharmacol Res Perspect, e00446, 1-14, which are incorporated herein by reference.

[0043] The catalytically inactive L-chain may have one or more mutations that inactivate the catalytic activity. For example, the catalytically inactive BoNT / A L-chain may comprise mutations of active site residues, such as His223, Glu224, His227, Glu262, and / or Tyr366. Position numbers correspond to amino acid positions of SEQ ID NO:62 and may be determined by aligning the polypeptide with SEQ ID NO:62. Since the presence of a methionine residue at position 1 of SEQ ID NO:62 is optional, the skilled artisan will consider the presence / absence of a methionine residue when determining amino acid residue numbering. For example, where SEQ ID NO:62 includes a methionine, position numbering will be as defined above (e.g., His223 will be His223 of SEQ ID NO:62). Alternatively, where a methionine is not present in SEQ ID NO:62, the amino acid residue numbering should be altered by -1 (e.g., His223 will be His222 of SEQ ID NO:62). Similar considerations apply when the methionine at position 1 of other polypeptide sequences described herein is present / absent, and the skilled artisan will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0044] In a particularly preferred embodiment, the polypeptide of the present invention may comprise a modified BoNT / A or fragment thereof (preferably BoNT / AH C 86, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1230, ASN 1231, ASN 1232, ASN 1233, ASN 1234, ASN 1236, ASN 1237, ASN 1238, ASN 1239, ASN 1240, ASN 1241, ASN 1242, ASN 1243, ASN 1244, ASN 1245, ASN 1246, ASN 1250, ASN 1251, ASN 1252, ASN 1253, ASN 1254, ASN 1255, ASN 1256, ASN 1257, ASN 1258, ASN 1260, ASN 1261, ASN 1262, ASN 1263 1242, ASN 1243, SER 1274, and THR 1277. Such modified BoNT / A or fragments thereof may demonstrate reduced or absent side effects compared to the use of known BoNT / A. The increased tissue retention properties of the modified BoNT / A of the present invention may also provide increased potency and / or duration of action, and may allow for reduced dosages used (or increased dosages without any additional side effects) compared to known Clostridial toxin therapeutics, thereby providing further advantages.

[0045] Modifications can be modifications when compared to an unmodified BoNT / A as set forth in SEQ ID NO:62, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:62. Because the presence of a methionine residue at position 1 of SEQ ID NO:62 (and SEQ ID NOs corresponding to the modified BoNT / A polypeptides or fragments thereof described herein) is optional, the skilled artisan will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, when SEQ ID NO:62 includes a methionine, the position numbering will be as defined above (e.g., ASN 886 will be ASN 886 of SEQ ID NO:62). Alternatively, when a methionine is not present in SEQ ID NO:2, the amino acid residue numbering should be altered by -1 (e.g., ASN 886 will be ASN 885 of SEQ ID NO:62). Similar considerations apply when a methionine is present / absent at position 1 of other polypeptide sequences described herein, and the skilled artisan will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0046] The above-mentioned modified amino acid residues are surface-exposed amino acid residues.

[0047] The modified BoNT / A or fragment thereof can comprise modifications at one or more amino acid residues selected from ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277.

[0048] When used in the context of a modified BoNT / A or fragments thereof, the term "one or more amino acid residues" preferably means at least 2, 3, 4, 5, 6, or 7 of the specified amino acid residues. Thus, a modified BoNT / A can comprise at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications at the specified amino acid residues. A modified BoNT / A or fragments thereof can comprise 1-30, 3-20, or 5-10 amino acid modifications. More preferably, when used in the context of a modified BoNT / A or fragments thereof, the term "one or more amino acid residues" refers to all of the specified amino acid residues.

[0049] Preferably, other than the one or more amino acid modifications at the designated amino acid residues, the modified BoNT / A or fragment thereof does not contain any further amino acid modifications when compared to SEQ ID NO: 62.

[0050] Modification selected from:

[0051] i. replacing acidic surface exposed amino acid residues with basic amino acid residues;

[0052] ii. replacing acidic surface exposed amino acid residues with uncharged amino acid residues;

[0053] iii. replacing uncharged surface-exposed amino acid residues with basic amino acid residues;

[0054] iv. inserting a basic amino acid residue; and

[0055] v. Deletion of acidic surface exposed amino acid residues.

[0056] The modifications as described above result in a modified BoNT / A or fragment thereof having an increased positive surface charge and an increased isoelectric point compared to the corresponding unmodified BoNT / A or fragment thereof.

[0057] The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are made from specific sequences of amino acids (also called amino acid residues when in proteins). Each amino acid in the standard set of twenty has a different side chain (or R group), which means that each amino acid residue in a protein displays different chemical properties, such as charge and hydrophobicity. These properties can be affected by the surrounding chemical environment, such as temperature and pH. The overall chemical characteristics of a protein will depend on the sum of these different factors.

[0058] Certain amino acid residues (described in detail below) have ionizable side chains that can exhibit a charge depending on the surrounding pH. At a given pH, whether such a side chain is charged depends on the pKa of the associated ionizable moiety, where pKa is the negative logarithm of the acid dissociation constant (Ka) for a specific proton from a conjugate base.

[0059] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with a pKa value of approximately 4.1 (the exact pKa value may depend on temperature, ionic strength, and the microenvironment of the ionizable group). Therefore, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as "physiological pH"). These side chains will protonate and lose their charge at low pH values.

[0060] In contrast, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values ​​of about 10 to 12. Therefore, these side chains show a positive charge at a pH of 7.4. These side chains will deprotonate and lose their charge at high pH values.

[0061] Therefore, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and the surrounding pH. Changing the surrounding pH changes the overall charge on the protein. Therefore, for every protein, there is a given pH at which the number of positive and negative charges is equal and the protein exhibits no overall net charge. This point is called the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry with which the skilled person is familiar.

[0062] Therefore, the isoelectric point (pI) is defined as the pH value at which a protein exhibits a net charge of zero. An increase in pI means that a protein requires a higher pH value to exhibit a net charge of zero. Therefore, an increase in pI indicates an increase in the net positive charge of a protein at a given pH. Conversely, a decrease in pI means that a protein requires a lower pH value to exhibit a net charge of zero. Therefore, a decrease in pI indicates a decrease in the net positive charge of a protein at a given pH.

[0063] Methods for determining the pI of a protein are known in the art and will be familiar to the skilled artisan. For example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pI"). Such calculations can be performed using computer programs known in the art, such as ExPASy(R). https: / / web.expasy.org / compute_pi / ) is the preferred method for calculating pi according to the present invention. The same calculation technique / program should be used to compare pi values ​​between different molecules.

[0064] Where appropriate, the calculated pI of a protein ("observed pI") may be experimentally confirmed using the technique of isoelectric focusing. This technique uses electrophoresis to separate proteins according to their pI. Isoelectric focusing is typically performed using a gel with a fixed pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH where its net charge is zero, which point is the pI of the protein. The results provided by isoelectric focusing are generally relatively low resolution in nature, and therefore the inventors of the present invention believe that the results provided by the calculated pI (as described above) are more suitable for use.

[0065] Throughout the specification of the present invention, "pi" means "calculated pi" unless otherwise specified.

[0066] The pi of a protein can be increased or decreased by changing the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pi can be provided by reducing the number of acidic residues or by increasing the number of basic residues.

[0067] The modified BoNT / A or fragments thereof of the present invention can have a pi value that is at least 0.2, 0.4, 0.5, or 1 pi unit higher than an unmodified BoNT / A (e.g., SEQ ID NO: 62) or a fragment thereof. Preferably, the modified BoNT / A or fragment thereof can have a pi of at least 6.6, such as at least 6.8.

[0068] The following table lists the properties of the 20 standard amino acids:

[0069]

[0070]

[0071] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0072] At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) are negatively charged, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) are positively charged. Aspartic acid and glutamic acid are called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0073] The following amino acids are considered to be uncharged polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0074] The following amino acids are considered to be uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0075] In an amino acid insertion, additional amino acid residues (not normally present) are incorporated into a BoNT / A polypeptide sequence or fragment thereof, thereby increasing the total number of amino acid residues in the sequence. In an amino acid deletion, amino acid residues are removed from a Clostridial toxin amino acid sequence, thereby decreasing the total number of amino acid residues in the sequence.

[0076] Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A or fragment thereof. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence or fragment thereof is replaced with a different amino acid residue. As described above, the replacing amino acid residue can be one of the 20 standard amino acids. Alternatively, the replacing amino acid in an amino acid substitution can be a non-standard amino acid (an amino acid that is not part of the above 20 standard groups). For example, the replacing amino acid can be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine). Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an E. coli auxotrophic expression host.

[0077] In one embodiment, the substitution is selected from the group consisting of: acidic amino acid residues substituted by basic amino acid residues, acidic amino acid residues substituted by uncharged amino acid residues, and uncharged amino acid residues substituted by basic amino amino acid residues. In one embodiment, the substitution is acidic amino acid residues substituted by uncharged amino acid residues, acidic amino acid residues substituted by their corresponding uncharged amide amino acid residues (i.e., aspartic acid substituted by asparagine, glutamic acid substituted by glutamine).

[0078] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0079] Preferably, the modified BoNT / A or fragment thereof for use in the present invention comprises a CN The modified BoNT / A or fragment thereof preferably also has a pI of at least 6.6. The modified BoNT / A preferably comprises modifications of at least 4 amino acids selected from the group consisting of ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, and ASN 1052, wherein the modifications comprise amino acid substitutions with lysine residues or arginine residues. For example, the modified BoNT / A or fragment thereof may comprise modifications of at least 5 amino acids selected from the group consisting of ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229, wherein the modifications comprise amino acid substitutions with lysine residues or arginine residues.

[0080] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. For example, amino acid modifications can be introduced by modifying a DNA sequence encoding a polypeptide (e.g., encoding an unmodified BoNT / A or fragment thereof). This can be accomplished using standard molecular cloning techniques, such as by site-directed mutagenesis, in which short strands of DNA (oligonucleotides) encoding the desired amino acids are used to replace the original coding sequence using a polymerase, or by inserting / deleting portions of a gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized.

[0081] In one aspect, the present invention provides a polypeptide for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0082] In a related aspect, a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject is provided, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0083] In another related aspect, provided is the use of a polypeptide in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0084] In one aspect, the present invention provides a polypeptide for treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0085] In a related aspect, a method for treating a neurological disorder in a subject is provided, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0086] In another related aspect, provided is the use of a polypeptide in the preparation of a medicament for treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0087] In one embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 42. Preferably, the polypeptide used according to the present invention comprises a polypeptide sequence as shown in SEQ ID NO:42.

[0088] In one embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity with SEQ ID NO: 41. Preferably, the polypeptide used according to the present invention comprises a polypeptide sequence encoded by a nucleotide sequence as shown in SEQ ID NO: 41.

[0089] In one embodiment, the polypeptide used according to the present invention (e.g., comprising SEQ ID NO:42 or encoded by SEQ ID NO:41) may be part of a polypeptide having at least 70% sequence identity with SEQ ID NO:61 or 65. Thus, in one embodiment, the polypeptide used according to the present invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity with SEQ ID NO:61 or 65. Preferably, the polypeptide used according to the present invention may comprise (more preferably consist of) SEQ ID NO:61 or 65. In one embodiment, the polypeptide comprises a catalytically inactive L-chain (e.g., according to SEQ ID NO:65).

[0090] In one embodiment, the polypeptide used according to the present invention (e.g., comprising SEQ ID NO:42 or encoded by SEQ ID NO:41) can be encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:60. Thus, in one embodiment, the polypeptide used according to the present invention can be encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity with SEQ ID NO:60. Preferably, the polypeptide used according to the present invention can be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO:60. In one embodiment, the polypeptide comprises a catalytically inactive L-chain.

[0091] SEQ ID NO:42 is an example of a modified BoNT / A fragment, while SEQ ID NOs:61 and 65 are examples of modified BoNT / A polypeptides having catalytic activity and inactive activity, respectively. Such modified BoNT / A polypeptides and fragments are particularly preferred for use in the present invention. When compared to wild-type BoNT / A, the polypeptides set forth in SEQ ID NOs:42, 61, and 62 have a number of amino acid modifications (e.g., substitutions) that increase the isoelectric point of the polypeptide. Without wishing to be bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and the cell surface, thereby increasing retention and / or duration of action at the site of administration. Therefore, it is contemplated that the neuronal growth and / or repair properties of SEQ ID NOs:42, 61, and 65 will be improved compared to equivalent polypeptides lacking such modifications.

[0092] For the catalytically active modified BoNT / A polypeptides described above (e.g., SEQ ID NO:61), one way in which these favorable properties, which represent an increase in the therapeutic index, can be defined is in terms of the safety ratio of the modified BoNT / A. In this regard, the adverse effects of the clostridial neurotoxin (caused by diffusion of the toxin from the site of administration) can be experimentally assessed by measuring the percent weight loss in a relevant animal model (e.g., mice, where weight loss is measured within seven days of administration). Conversely, the desired targeting of the clostridial neurotoxin can be experimentally assessed by the digital abduction score (DAS) assay, a measure of muscle paralysis. The DAS assay can be performed by injecting 20 μl of the clostridial neurotoxin formulated in gelatin phosphate buffer into the gastrocnemius / soleus complex of mice and then assessing the digital abduction score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are suspended briefly by their tails to elicit a characteristic startle response in which they extend their hind limbs and abduct their hind digits. After clostridial neurotoxin injection, the degree of digital abduction is scored on a five-point scale (0 = normal to 4 = maximal reduction in digital abduction and leg extension).

[0093] The safety ratio of the clostridial neurotoxin can then be expressed as the ratio between the amount of toxin required to achieve a 10% reduction in body weight (measured at peak effect within the first seven days after dosing in mice) and the amount of toxin required for a DAS score of 2. A high safety ratio score is therefore desirable, indicating that the toxin is able to effectively paralyze the target muscle with few undesirable off-target effects. The catalytically active modified BoNT / A of the present invention can have a higher safety ratio than the equivalent unmodified (natural) botulinum toxin (e.g., SEQ ID NO: 62).

[0094] Thus, in one embodiment, a catalytically active modified BoNT / A of the invention has a safety ratio of at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), wherein the safety ratio is calculated as: -10% body weight change required for toxin dose (pg / mouse) divided by DAS ED 50 (pg / mouse) [ED50 = dose required to produce a DAS score of 2].

[0095] In one embodiment, a catalytically active modified BoNT / A of the invention has a safety ratio of at least 10. In one embodiment, a modified BoNT / A of the invention or a fragment thereof has a safety ratio of at least 15.

[0096] A polypeptide comprising at least 70% sequence identity to SEQ ID NO: 61 is described in WO 2015 / 004461 A1, which is herein incorporated by reference in its entirety.

[0097] In one embodiment, a polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, 61 or 65 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 or 60 comprises a substitution at one or more (preferably two or more, three or more, four or more, five or more or six or more, more preferably all) positions 930, 955, 991, 1026, 1052, 1229 and 886. The position numbers correspond to the positions of SEQ ID NO: 62 and can be determined by aligning the polypeptide sequence with SEQ ID NO: 62 (unmodified / wild-type BoNT / A). Since the presence of a methionine residue at position 1 of SEQ ID NO: 62 is optional, the skilled artisan will take into account the presence / absence of a methionine residue when determining the amino acid residue numbering. For example, where SEQ ID NO: 62 includes a methionine, the position numbering will be as defined above (e.g., position 886 will be ASN 886 of SEQ ID NO: 62). Alternatively, when methionine is absent in SEQ ID NO: 62, the amino acid residue numbering should be changed by -1 (e.g., position 886 would be ASN885 of SEQ ID NO: 62). Similar considerations apply to the presence / absence of methionine at position 1 of other polypeptide sequences described herein, and the skilled artisan will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0098] Preferably, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, 61 or 65 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 or 60 comprises lysine or arginine (more preferably lysine) at one or more of 930, 955, 991, 1026, 1052, 1229 and 886. In one embodiment, the polypeptide comprises lysine or arginine (more preferably lysine) at at least two, three, four, five, six or all positions of 930, 955, 991, 1026, 1052, 1229 and 886. Most preferably, the polypeptide comprises lysine or arginine (more preferably lysine) at all positions of all positions 930, 955, 991, 1026, 1052, 1229 and 886.

[0099] The polypeptides of the present invention promote neuronal growth and / or neuronal repair. Therefore, the polypeptides can be used to treat neurological disorders. As used herein, the term "neurological disorder" is a disorder that can be treated by promoting neuronal growth and / or repair in a subject.

[0100] Therefore, in one aspect, the present invention provides a method for promoting neuronal growth and / or neuronal repair, the method comprising administering to a subject a polypeptide comprising a clostridial neurotoxin light chain (L-chain) or a fragment thereof; and / or a fragment of a clostridial neurotoxin heavy chain (H-chain). In another aspect, the present invention provides a method for promoting neuronal growth and / or neuronal repair, the method comprising administering to a subject a polypeptide comprising a clostridial neurotoxin L-chain without catalytic activity. In another aspect, a method for promoting neuronal growth or neuronal repair is provided, the method comprising administering to a subject a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 41. In another aspect, a method for promoting neuronal growth or neuronal repair is provided, the method comprising administering to a subject a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 63.

[0101] The term "promoting neuronal growth and / or neuronal repair" may mean that the polypeptide of the present invention initiates neuronal growth and / or neuronal repair, for example, in the absence of neuronal growth and / or neuronal repair. In other embodiments, the term "promoting neuronal growth and / or neuronal repair" may mean that the polypeptide of the present invention increases the rate of neuronal growth and / or neuronal repair. The increase may be an increase when compared to the rate of neuronal growth and / or neuronal repair in the absence of the polypeptide of the present invention. In one embodiment, neuronal growth and / or neuronal repair allows the reconstruction of damaged neuronal circuits, thereby restoring activity and / or neuronal communication in a neuronal network or neuronal population. Therefore, the term "neuronal repair" used herein may cover the repair of specific neurons as well as the repair of neuronal circuits.

[0102] The term "neuronal growth and / or neuronal repair" may also include neuronal plasticity. Thus, in one embodiment, the polypeptides of the invention promote neuronal plasticity. As used herein, the term "neuronal plasticity" includes axonal sprouting, dendritic sprouting, neurogenesis (e.g., the generation of new neurons), maturation, differentiation, and / or synaptic plasticity (e.g., including changes in synaptic strength, activity, anatomical structure, and / or connectivity). In one embodiment, the polypeptides of the invention promote the establishment of functional synapses (e.g., at or near the site of injury).

[0103] In the presence of a polypeptide of the present invention, neuronal growth and / or repair may be increased by at least 10%, 20%, 30%, 40%, 50%, 60% or 70% (preferably at least 80%) compared to neuronal growth and / or repair in the absence of a polypeptide of the present invention or in the presence of an alternative polypeptide. In some embodiments, in the presence of a polypeptide of the present invention, neuronal growth and / or repair may be increased by at least 100%, 150% or 200% compared to neuronal growth and / or repair in the absence of a polypeptide of the present invention or in the presence of an alternative polypeptide.

[0104] In one embodiment, the polypeptide of the present invention promotes neuronal growth. As used herein, the term "neuronal growth" encompasses the growth of any part of a neuron, including the growth of axons and / or dendrites. The polypeptide of the present invention can increase neurite length, neurite number (e.g., neurite number of each cell), and / or can increase the length and / or number of projections from the cell body or cell membrane of a neuron. Preferably, the polypeptide of the present invention promotes the axonal growth of a neuron (e.g., a neuron of a subject). In other words, it is preferred that the polypeptide of the present invention increases axonal growth, such as increasing axonal sprouting. The axonal growth can promote connection and / or chemical communication between neurons.

[0105] The neurological disorder treated by the polypeptide of the present invention may be neuronal damage, neurodegenerative disorder, sensory disorder or autonomic disorder.

[0106] Neurological disorders can be neuronal damage. In one embodiment, neuronal damage can be nerve trauma, neuropathy (e.g., peripheral neuropathy), spinal cord injury, nerve severance, brain injury (e.g., traumatic brain injury), non-traumatic injury (e.g., stroke or spinal cord infarction), or brachial plexus injury, such as Erb's palsy or Klumpke's palsy.

[0107] In one embodiment, nerve trauma may be caused by scars and / or fractures. In the case of such nerve trauma, nerve endings are damaged. The polypeptides of the present invention advantageously allow the repair of said nerve endings or allow the repair of distal nerve endings allowing the treatment of nerve damage.

[0108] The neuronal damage may be paralysis, such as paralysis caused by spinal cord injury (eg, caused by compression, constriction and / or stretching). In one embodiment, the spinal cord injury is paraplegia or quadriplegia.

[0109] The neurological disorder can be a sensory disorder. In one embodiment, the sensory disorder is a sensory neuropathy, a sensory motor polyneuropathy, a diabetic neuropathy, pain, Brown-Sequard syndrome, Charcot-Marie-Tooth disease, or Devic syndrome. Preferably, the sensory disorder described herein is not pain. In other words, the neurological disorder described herein is preferably not pain.

[0110] The neurological disorder can be an autonomic disorder. In one embodiment, the autonomic disorder is autonomic neuropathy, multiple system atrophy, acute idiopathic polyneuropathy, dysautonomia, familial dysautonomia, diabetic autonomic failure, isolated autonomic failure, temperature regulation disorder, hyperhidrosis, neurally mediated syncope (vasovagal, urinary, coughing, swallowing and other situational forms), erectile dysfunction, orthostatic hypotension, postural tachycardia syndrome (PoTS) or Guillain-Barre syndrome.

[0111] Neurological disorder can be a neurodegenerative disorder. In one embodiment, the neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, Parkinson's disease related disorder, motor neuron disease, peripheral neuropathy, motor neuropathy, prion disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, monolimbic muscular atrophy, Friedreich's ataxia, Hallervorden-Spatz disease or frontotemporal lobar degeneration. Preferably, the neurodegenerative disorder is Parkinson's disease or motor neuron disease. Advantageously, due to its ability to promote neuronal growth (for example, including neuronal plasticity) and / or neuronal repair, and further due to its ability to rebuild damaged neuronal circuits, it is believed that the polypeptide of the present invention can be used for treating neurodegenerative disorders, thereby restoring activity and / or neuronal communication in a neuronal network or a neuronal population.

[0112] In view of its ability to promote neuronal growth and / or neuronal repair, the polypeptides of the present invention can be considered to be neurotrophic polypeptides. The neurons described herein can be one or more selected from the following: motor neurons (including autonomic neurons), sensory neurons, spinal cord interneurons and brain interneurons. Therefore, in one embodiment, the polypeptides of the present invention promote the growth and / or repair of motor neurons, sensory neurons and / or interneurons. Preferably, the polypeptides of the present invention promote the growth and / or repair of motor neurons.

[0113] As used herein, a "subject" may be a mammal, such as a human or other mammal. Preferably, a "subject" refers to a human subject.

[0114] As used herein, the term "disorder" also includes "disease." In one embodiment, the disorder is a disease.

[0115] As used herein, the term "treat" or "treating" includes prophylactic treatment (e.g., preventing the onset of a disorder) as well as corrective treatment (treating a subject already suffering from a disorder). Preferably, "treat" or "treating" as used herein refers to corrective treatment.

[0116] As used herein, the term "treat" or "treating" refers to a disorder and / or its symptoms.

[0117] Therefore, the polypeptide of the present invention can be administered to a subject in a therapeutically effective amount or a prophylactically effective amount. Preferably, the polypeptide of the present invention is administered to a subject in a therapeutically effective amount.

[0118] A "therapeutically effective amount" is any amount of a polypeptide that, when administered alone or in combination to a subject to treat a disorder (or a symptom thereof), is sufficient to effect such treatment of the disorder or a symptom thereof.

[0119] A "prophylactically effective amount" is any amount of a polypeptide when administered alone or in combination to a subject to inhibit or delay the onset or recurrence of a disorder (or its symptoms). In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of the disease. "Inhibiting" the onset means reducing the likelihood of an onset of the disease (or its symptoms), or completely preventing the onset of the disease.

[0120] The polypeptides of the present invention may be formulated in any suitable manner for administration to a subject, for example as part of a pharmaceutical composition. Thus, in one aspect, the present invention provides a pharmaceutical composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and / or salt. In some embodiments, the polypeptides of the present invention may be in single-chain form, while in other embodiments, the polypeptides may be in double-chain form, for example wherein the two chains are linked by a disulfide bridge. Preferably, the polypeptide is in double-chain form.

[0121] The polypeptides of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection can be in the form of solutions, suspensions or emulsions, or in the form of dry powders dissolved or suspended in a suitable carrier before use.

[0122] In the case of a polypeptide for local delivery, the polypeptide may be formulated as a cream (eg, for topical application) or for subcutaneous injection.

[0123] Local delivery means may include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of polypeptides can be delivered to the lungs and / or other nasal and / or bronchial or airway passages.

[0124] The polypeptides of the invention may be administered to a subject by intrathecal or epidural injection in the spine at the level of the spinal segments involved in the innervation of the affected organ.

[0125] The route of administration can be by laparoscope and / or local injection. In one embodiment, the polypeptide of the present invention is applied at or near the site of injury, preferably at the site of injury. For example, in the case where the injury is a spinal cord injury, the polypeptide can be applied intrathecally or intraspinal (preferably intrathecally). In one embodiment, the route of administration of the polypeptide of the present invention can be perineural, intraneural, intraspinal and / or intrathecal.

[0126] The dosage range for administration of the polypeptides of the present invention is the dosage range that produces the desired therapeutic and / or preventive effect. It should be understood that the desired dosage range depends on the exact nature of the clostridial neurotoxin or composition, the route of administration, the nature of the formulation, the age of the subject, the nature, extent or severity of the subject's condition, contraindications, if any, and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical procedures for optimization.

[0127] In one embodiment, the dosage of the polypeptide is a fixed dose. The fixed dose may be in the range of 50pg to 250ug, preferably 100pg to 100ug. In one embodiment, the fixed dose may be at least 50pg, 100pg, 500pg, 1ng, 50ng, 100ng, 500ng, 1ug ​​or 50ug. The dose may be a single fixed dose.

[0128] Fluid dosage forms are typically prepared using a polypeptide and a pyrogen-free sterile carrier. Depending on the carrier and concentration used, the clostridial neurotoxin can be dissolved or suspended in the carrier. When preparing the solution, the polypeptide can be dissolved in the carrier, the solution can be made isotonic by adding sodium chloride if necessary, and sterilized by filtering through a sterile filter using aseptic techniques before being filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the solution stability is sufficient, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers and / or local anesthetics can be dissolved in the carrier.

[0129] Dry powders dissolved or suspended in a suitable carrier prior to use can be prepared by filling pre-sterilized ingredients into sterile containers using aseptic techniques in a sterile area. Alternatively, the ingredients can be dissolved into suitable containers using aseptic techniques in a sterile area. The product is then freeze-dried and the container is aseptically sealed.

[0130] Parenteral suspensions suitable for the administration routes described herein are prepared in substantially the same manner, except that the sterile components are suspended in a sterile vehicle, rather than dissolved, and sterilization cannot be achieved by filtration. The components may be isolated under sterile conditions, or alternatively may be sterilized after isolation, for example by gamma irradiation.

[0131] Advantageously, a suspending agent (such as polyvinylpyrrolidone) is included in the composition to facilitate uniform distribution of the ingredients.

[0132] Administration according to the present invention may utilize a variety of delivery techniques including microparticle encapsulation or high pressure aerosol bombardment.

[0133] The polypeptide of the present invention may be a Clostridial neurotoxin or a fragment thereof, preferably a fragment thereof.

[0134] In one embodiment, the polypeptide of the present invention may be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60. In one embodiment, the polypeptide of the present invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60. Preferably, the polypeptide of the present invention can be encoded by a nucleotide sequence comprising any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 60.

[0135] In one embodiment, a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65. In one embodiment, a polypeptide of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65. Preferably, the polypeptide of the present invention may comprise a polypeptide sequence of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0136] In one embodiment, the invention encompasses the use of a full-length clostridial neurotoxin comprising a clostridial neurotoxin L-chain and a clostridial neurotoxin H-chain, with the proviso that the clostridial neurotoxin L-chain is catalytically inactive.

[0137] The term "Clostridial neurotoxin" includes toxins produced by Clostridium botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), Clostridium tetani (tetanus neurotoxin), Clostridium butyricum (botulinum neurotoxin serotype E), and Clostridium baratii (botulinum neurotoxin serotype F), as well as modified Clostridial neurotoxins or derivatives derived from any of the foregoing.

[0138] Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum in the form of large protein complexes consisting of the BoNT itself complexed with a number of accessory proteins. There are currently eight different classes of botulinum neurotoxins, namely: botulinum neurotoxin serotypes A, B, C1, D, E, F, G and X, all of which have similar structures and modes of action. The different BoNT serotypes can be distinguished by inactivation by specific neutralizing antisera, and this classification by serotype is correlated with the percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage amino acid sequence identity.

[0139] BoNTs are absorbed in the gastrointestinal tract and, once in the systemic circulation, bind to the presynaptic membrane of cholinergic nerve terminals, blocking the release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP); BoNT / C1, BoNT / A, and BoNT / E cleave synaptosomal-associated protein of 25 kDa (SNAP-25); and BoNT / C1 cleaves synaptobrevin. BoNT / X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin1.

[0140] Tetanus toxin is produced by Clostridium tetani as a single serotype. Clostridium butyricum produces BoNT / E, while Clostridium balatii produces BoNT / F.

[0141] The term "clostridial neurotoxin" is also intended to include modified clostridial neurotoxins and derivatives thereof, including but not limited to those described below. A modified clostridial neurotoxin or derivative may contain one or more amino acids that have been modified compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. For example, a modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may alter functional aspects of the toxin, such as biological activity or persistence. Therefore, in one embodiment, a clostridial neurotoxin of the invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.

[0142] The modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., a modified H C domain), wherein the modified heavy chain binds to target neural cells with a higher or lower affinity than a native (unmodified) Clostridial neurotoxin. C Such modifications in the domain may include modifying H C Residues in the ganglioside binding site or protein (SV2 or synaptotagmin) binding site of a domain that alter binding to a ganglioside receptor and / or protein receptor of a target neural cell. Examples of such modified Clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0143] The modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domains, which may alter or modify the SNARE protein specificity of the modified L-chain. Examples of such modified clostridial neurotoxins are described in WO 2010 / 120766 and US 2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0144] The modified clostridial neurotoxin can comprise one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified clostridial neurotoxin. For example, the modified clostridial neurotoxin can comprise a leucine- or tyrosine-based motif, wherein the motif increases or decreases the biological activity and / or biological persistence of the modified clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM (wherein x is any amino acid). Suitable tyrosine-based motifs include Yxx-Hy (wherein Hy is a hydrophobic amino acid). Examples of modified clostridial neurotoxins comprising leucine- and tyrosine-based motifs are described in WO 2002 / 08268, which is incorporated herein by reference in its entirety.

[0145] As described above, a modified clostridial neurotoxin (or clostridial neurotoxin fragment) can be a toxin comprising one or more modifications that increase the isoelectric point of the clostridial neurotoxin when compared to an equivalent unmodified clostridial neurotoxin lacking the one or more modifications. Suitable modified clostridial neurotoxins are described above and in WO 2015 / 004461 A1 and WO2016 / 110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 61 and 42 as described herein.

[0146] The term "clostridial neurotoxin" is intended to include hybrid and chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or a subtype thereof, and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, a hybrid clostridial neurotoxin may contain a complete light chain from a light chain of one clostridial neurotoxin subtype and a heavy chain from another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin may contain a portion of a heavy chain (e.g., a binding domain) of a clostridial neurotoxin subtype, while another portion of the heavy chain is from another clostridial neurotoxin subtype. Similarly or alternatively, a therapeutic element may comprise light chain portions from different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a method of delivering the therapeutic benefit of these clostridial neurotoxins to subjects who are immunoresistant to a given clostridial neurotoxin subtype, to subjects who may have a lower than average receptor concentration for a given clostridial neurotoxin heavy chain binding domain, or to subjects who may have protease-resistant variants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and synaptophysin). Hybrid and chimeric clostridial neurotoxins are described in US 8,071,110, which disclosure is incorporated herein by reference in its entirety. Thus, in one embodiment, a clostridial neurotoxin (or fragment thereof) of the invention is a hybrid clostridial neurotoxin or a chimeric clostridial neurotoxin.

[0147] In a particularly preferred embodiment, the polypeptide of the invention may be a chimeric clostridial neurotoxin comprising (preferably consisting of) a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (HC domain) or a portion thereof. A suitable chimeric and / or hybrid clostridial neurotoxin may be one taught in WO 2017 / 191315 A1, which is incorporated herein by reference. Such preferred sequences include SEQ ID NOs: 44, 63 and 64.

[0148] BoNT / A LH N Domains can covalently link BoNT / BH C The chimeric BoNT / A is also referred to herein as "BoNT / AB" or "BoNT / AB chimera."

[0149] LH N The C-terminal amino acid residue of the domain may correspond to the LH that separates BoNT / A N and HC domain 3 10 The first amino acid residue of the helix, while H C The N-terminal amino acid residue of the domain may correspond to the LH that separates BoNT / B N and H C Domain 3 10 The second amino acid residue of the helix.

[0150] The article refers to "LH that separates BoNT / A N and HC domain 3 10 The first amino acid residue of the helix is ​​the N and H C Domain 3 10 N-terminal residue of the helix.

[0151] The article mentions "LH that separates BoNT / B N and HC domain 3 10 The second amino acid residue of the helix is ​​the N and H C Domain 3 10 The amino acid residue following the N-terminal residue of the helix.

[0152] "3 10 A "helix" is a secondary structure found in proteins and peptides along with α-helices, β-sheets, and inversions. 10The amino acids in the helix are arranged in a right-handed helical structure, in which each complete turn is completed by three residues and ten atoms, which separate the intramolecular hydrogen bonds between them. Each amino acid corresponds to a 120° turn in the helix (i.e., there are three residues per turn of the helix), and the translation along the helical axis is (=0.2 nm) and has 10 atoms in a ring that forms hydrogen bonds. Most importantly, the NH group of the amino acid forms a hydrogen bond with the C=O group of the amino acid three residues ahead; this repeated i+3→i hydrogen bond defines the 3 10 Spiral. 3 10 The helix is ​​a standard concept in structural biology with which the skilled person is familiar.

[0153] This 3 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of the four residues. As used herein, the term "separating LH N and H C Domain 3 10 The "helix" is composed of these six residues.

[0154] By performing structural analysis and sequence alignment, the LH N and H C Domain 3 10 Spiral. This 3 10 The helix is ​​at its N-terminal end (i.e., at the end of LH N The C-terminal part of the domain is surrounded by an α-helix, and at its C-terminus (i.e., at the H C The N-terminal part of the 3 10 The first (N-terminal) residue of a helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0155] Separation of LH N and H C Domain 3 10 Helices can be determined, for example, from publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0156] Publicly available computer modeling and alignment tools can also be used to determine the segregation of LH in other neurotoxins. N and H C Domain 310 The positions of the helices are determined by, for example, the homology modeling servers LOOPP (Learn, Observe and Export Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / Modeling Y Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wisart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ) and many other tools / services listed in the Internet Resources for Molecular and Cellular Biologists (http: / / molbiol-tools.ca / ). In particular, “H N / H CN “The region surrounding the junction is highly conserved structurally, making it an ideal region for superimposing different serotypes.

[0157] For example, the following method can be used to determine the presence of this 3 10 The sequence of the spiral:

[0158] 1. Based on the BoNT / A1 crystal structure (3BTA.pdb), the structural homology modeling tool LOOP (http: / / loopp.org) was used to obtain the predicted structures of other BoNT serotypes;

[0159] 2. Edit the structure (pdb) file thus obtained to include only H CN The N-terminus of the domain and the approximately 80 residues preceding it (which are H N domain), thereby retaining the highly conserved "H N / H CN "area;

[0160] 3. Use the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) to superimpose each serotype onto the 3BTA.pdb structure;

[0161] 4. Check the superimposed pdb files and add BoNT / A1 H C The first 3 of the domain 10 Helical mapping followed by identification of corresponding residues in other serotypes;

[0162] 5. Align the other BoNT serotype sequences with Clustal Omega to check if the corresponding residues are correct.

[0163] LH determined by this method N , H C and 3 10 Examples of helical domains are presented below:

[0164]

[0165] Using structural analysis and sequence alignment, it was discovered that the LH N and H C Domain 3 10 The β-strand following the helix is ​​conserved in all botulinum and tetanus neurotoxins and separates LH from N and H C Domain 3 10 The first residue of the helix begins at residue 8 (eg, for BoNT / A1, at residue 879).

[0166] The BoNT / AB chimera can contain H from BoNT / B C Domain covalently linked to LH from BoNT / A N Domain,

[0167] ·LH N The C-terminal amino acid residue of the domain corresponds to the H C The eighth amino acid residue from the N-terminus of the β-strand from the beginning (N-terminus) of the domain, and

[0168] · Among them, H C The N-terminal amino acid residue of the domain corresponds to the H C The seventh amino acid residue from the N-terminus of the β-strand where the domain begins (N-terminus).

[0169] The BoNT / AB chimera may contain an H C Domain covalently linked to BoNT / A LH N Domain,

[0170] ·LH N The C-terminal amino acid residue of the domain corresponds to the LH N The C-terminal amino acid residue of the α-helix at the end of the domain (C-terminus), and

[0171] · Among them, H C The N-terminal amino acid residue of the domain corresponds to the LH residue immediately adjacent to the LH residue of BoNT / B.N The C-terminal amino acid residue of the C-terminal amino acid residue of the α-helix at the end (C-terminus) of the domain.

[0172] The rationale for the design process of the BoNT / AB chimera was to try to ensure that the secondary structure was not affected, thereby minimizing any changes to the tertiary structure and the function of each domain. 10 The four central amino acid residues of the helix ensure the optimal conformation of the chimeric neurotoxin, thereby allowing the chimeric neurotoxin to fully exert its function.

[0173] LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 62, or a polypeptide sequence having at least 70% sequence identity thereto. N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 62, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the LH from a BoNT / A N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:62.

[0174] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:52, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:52, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H from a BoNT / B C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:52.

[0175] Preferably, the BoNT / AB chimera comprises BoNT / A LH N Domains and BoNT / BH C More preferably, LH N The domain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 62), and the H C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO: 52).

[0176] Preferably, BoNT / BH C Domain in H CCThe subdomain also comprises at least one amino acid residue substitution, addition, or deletion that has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence. CC Suitable amino acid residue substitutions, additions or deletions in the subdomains have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both incorporated herein by reference).

[0177] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains include substitution mutations selected from the following: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0178] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains further include a combination of two substitution mutations selected from the following: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1191Y, or E1191V and W1178Q.

[0179] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains also include a combination of three substitution mutations, which are E1191M, S1199W and W1178Q.

[0180] Preferably, BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomain include a combination of two substitution mutations, which are E1191M and S1199Y.

[0181] The modification can be a modification when compared to an unmodified BoNT / B as set forth in SEQ ID NO:52, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:52. Since the presence of a methionine residue at position 1 of SEQ ID NO:52 is optional, the skilled artisan will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, where SEQ ID NO:52 includes a methionine, the position numbering will be as defined above (e.g., E1191 would be E1191 of SEQ ID NO:52). Alternatively, where a methionine is not present in SEQ ID NO:52, the amino acid residue numbering should be altered by -1 (e.g., E1191 would be E1190 of SEQ ID NO:52). Similar considerations apply when a methionine is present / absent at position 1 of other polypeptide sequences described herein, and the skilled artisan will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0182] Therefore, in one aspect, the present invention provides a polypeptide for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0183] In a related aspect, a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject is provided, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:63 or 64.

[0184] In a further related aspect, provided is the use of a polypeptide in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0185] In one aspect, the present invention provides a polypeptide for treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0186] In a related aspect, a method for treating a neurological disorder in a subject is provided, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0187] In yet another related aspect, provided is a use of a polypeptide in the preparation of a medicament for treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0188] In one embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity with SEQ ID NO: 63 or 64. Preferably, the polypeptide used according to the present invention comprises (more preferably consists of) the polypeptide sequence shown in SEQ ID NO: 63 or 64.

[0189] Preferably, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:63 comprises a catalytically inactive L chain, such as SEQ ID NO:64.

[0190] Chimeric and / or hybrid Clostridial neurotoxins for use in the present invention may comprise a portion of a BoNT / A polypeptide and a portion of a BoNT / B polypeptide, examples of which include the polypeptide described herein as SEQ ID NO:44.

[0191] Suitable chimeric Clostridial neurotoxins may include BoNT / FA. Indeed, in particularly preferred embodiments, polypeptides of the invention may include BoNT / FA or fragments thereof. Catalytically inactive forms of BoNT / FA are described herein as SEQ ID NOs: 26 and 34. Suitable fragments of BoNT / FA are also described herein as SEQ ID NOs: 28, 30, and 32.

[0192] The term "Clostridial neurotoxin" may also include newly discovered members of the botulinum neurotoxin protein family expressed by non-Clostridial microorganisms, such as the toxin encoded by Enterococcus that has the closest sequence identity to BoNT / X, the toxin encoded by Weissella oryzae called BoNT / WO (NCBI Ref Seq: WP_027699549.1), which cleaves VAMP2 at W89-W90, the toxin encoded by Enterococcus faecium (GenBank: 0T022244.1), which cleaves VAMP2 and SNAP25, and the toxin encoded by Chryseobacterium pipero (NCBI Ref. Seq: WP_034687872.1).

[0193] The polypeptides of the present invention may lack the functional H of the clostridial neurotoxin C domain, and also lacks any functionally equivalent exogenous ligand targeting moiety (TM).

[0194] Thus, in particularly preferred embodiments, the clostridial neurotoxins of the invention are not retargeted clostridial neurotoxins. In retargeted clostridial neurotoxins, the clostridial neurotoxin is modified to include an exogenous ligand referred to as a targeting moiety (TM). The TM is selected to provide binding specificity to a desired target cell, and as part of the retargeting process, the native binding moiety of the clostridial neurotoxin (e.g., H C Domain or H CC domain). Retargeting techniques are described, for example, in EP-B-0689459; WO 1994 / 021300; EP-B-0939818; US 6,461,617; US 7,192,596; WO 1998 / 007864; EP-B-0826051; US ​​5,989,545; US 6,395,513; US 6,962,703; WO 1996 / 033273; EP-B-0996468; US 7,052,702; WO 1999 / 017806; EP-B-1107794; US 6,632,440; WO 2000 / 010598; WO 2001 / 21213; WO 2006 / 059093; WO 2000 / 62814; WO 2000 / 04926; WO 1993 / 15766; WO 2000 / 61192; and WO 1999 / 58571; all of which are incorporated herein by reference in their entirety.

[0195] As mentioned above, the (full-length) clostridial neurotoxin is formed by two polypeptide chains, a heavy chain (H chain) with a molecular weight of about 100 kDa and a light chain (L chain) with a molecular weight of about 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and the N-terminal translocation component (H N domain).

[0196] The clostridial neurotoxin may be selected from the group consisting of BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Preferably, the clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from the group consisting of BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X.

[0197] In one embodiment, the clostridial neurotoxin may be BoNT / A. A reference BoNT / A sequence is shown as SEQ ID NO:51. In another embodiment, the clostridial neurotoxin may be BoNT / B. A reference BoNT / B sequence is shown as SEQ ID NO:52. In another embodiment, the clostridial neurotoxin may be BoNT / C. A reference BoNT / C sequence is shown as SEQ ID NO:53. In another embodiment, the clostridial neurotoxin may be BoNT / D. A reference BoNT / D sequence is shown as SEQ ID NO:54. In another embodiment, the clostridial neurotoxin may be BoNT / E. A reference BoNT / E sequence is shown as SEQ ID NO:55. In another embodiment, the clostridial neurotoxin may be BoNT / F. A reference BoNT / F sequence is shown as SEQ ID NO:56. In another embodiment, the clostridial neurotoxin may be BoNT / G. A reference BoNT / G sequence is shown as SEQ ID NO:57. In another embodiment, the clostridial neurotoxin may be TeNT. A reference TeNT sequence is shown as SEQ ID NO:58. In another embodiment, the Clostridial neurotoxin can be a BoNT / X. A reference BoNT / X sequence is shown as SEQ ID NO:59.

[0198] In one embodiment, a polypeptide of the invention comprises a fragment of BoNT / A or a fragment of BoNT / F. In another embodiment, a polypeptide of the invention comprises a catalytically inactive L chain of BoNT / A or BoNT / F.

[0199] In embodiments where the polypeptides described herein have a tag (eg, a His-tag) and / or a linker for purification, the tag and / or linker is optional.

[0200] Suitable full-length Clostridial neurotoxins are described herein.

[0201] In one embodiment, a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, or 65, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. In one embodiment, a polypeptide of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, or 65, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. Preferably, the polypeptide of the present invention may comprise a polypeptide sequence comprising any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65, provided that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive.

[0202] In one embodiment, the polypeptide of the present invention may be a polypeptide encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25, 33 or 60, provided that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. In one embodiment, the polypeptide of the present invention is a polypeptide encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25, 33 or 60, provided that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. Preferably, the polypeptide of the present invention is a polypeptide encoded by a nucleotide sequence comprising any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25, 33 or 60, provided that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive.

[0203] In one embodiment, a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. In one embodiment, a polypeptide of the invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. Preferably, a polypeptide of the invention comprises any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive.

[0204] In one embodiment, the polypeptide of the invention is a full-length clostridial neurotoxin selected from the group consisting of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT.

[0205] In one embodiment, the polypeptide of the present invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63. In one embodiment, the polypeptide of the present invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63. In one embodiment, the polypeptide of the present invention may comprise a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63. Preferably, the polypeptide of the present invention may comprise (more preferably consist of) a polypeptide sequence comprising any one of SEQ ID NOs: 52-59, 61 or 63.

[0206] In particularly preferred embodiments, the polypeptide of the invention is not a full-length catalytically active Clostridial neurotoxin, eg, is not a full-length catalytically active BoNT / A.

[0207] A polypeptide of the invention may comprise (or consist of) a fragment of a Clostridial neurotoxin, such as a fragment of any of the full-length Clostridial neurotoxins described herein.

[0208] In one embodiment, a polypeptide of the invention may comprise a fragment of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, or 65. In one embodiment, a polypeptide of the invention may comprise a fragment of a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, or 65. Preferably, the polypeptide of the present invention may comprise a fragment of a polypeptide sequence comprising any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0209] In one embodiment, the polypeptide of the invention comprises (or consists of) a clostridial neurotoxin L chain or a fragment thereof. A fragment of a clostridial neurotoxin L-chain may have ≤400, ≤350, ≤300, ≤250, ≤200, ≤150, ≤100, or ≤50 amino acid residues of a clostridial neurotoxin L-chain. In one embodiment, a fragment of a clostridial neurotoxin L chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of a clostridial neurotoxin L chain. For example, a fragment of a clostridial neurotoxin L chain may have 20-400, 50-300, or 100-200 amino acid residues of a clostridial neurotoxin L chain.

[0210] Examples of L-chain reference sequences include:

[0211] Botulinum neurotoxin type A: amino acid residues 1-448

[0212] Botulinum neurotoxin type B: amino acid residues 1-440

[0213] Botulinum neurotoxin type C1: amino acid residues 1-441

[0214] Botulinum neurotoxin type D: amino acid residues 1-445

[0215] Botulinum neurotoxin type E: amino acid residues 1-422

[0216] Botulinum neurotoxin type F: amino acid residues 1-439

[0217] Botulinum neurotoxin type G: amino acid residues 1-441

[0218] Tetanus neurotoxin: amino acid residues 1-457

[0219] For the recently identified BoNT / X, the L-chain is reported to correspond to amino acids 1-439 thereof, with the L-chain boundaries potentially varying by approximately 25 amino acids (eg, 1-414 or 1-464).

[0220] The reference sequences identified above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US 2007 / 0166332 (incorporated herein by reference in its entirety) cites a slightly different Clostridium sequence:

[0221] Botulinum neurotoxin type A: amino acid residues M1-K448

[0222] Botulinum neurotoxin type B: amino acid residues M1-K441

[0223] Botulinum neurotoxin type C1: amino acid residues M1-K449

[0224] Botulinum neurotoxin type D: amino acid residues M1-R445

[0225] Botulinum neurotoxin type E: amino acid residues M1-R422

[0226] Botulinum neurotoxin type F: amino acid residues M1-K439

[0227] Botulinum neurotoxin type G: amino acid residues M1-K446

[0228] Tetanus neurotoxin: amino acid residues M1-A457

[0229] Suitable Clostridial neurotoxin L chains are described herein.

[0230] The Clostridial neurotoxin L-chain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 6, 24, 32 or 40, or a fragment thereof. In one embodiment, the Clostridial neurotoxin L-chain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NO: 6, 24, 32 or 40, or a fragment thereof. Preferably, the Clostridial neurotoxin L chain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NO: 6, 24, 32 or 40, or a fragment thereof.

[0231] The clostridial neurotoxin L-chain may be a chain encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 5, 23, 31 or 39 or a fragment thereof. In one embodiment, the clostridial neurotoxin L-chain is a chain encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NO: 5, 23, 31 or 39 or a fragment thereof. Preferably, the clostridial neurotoxin L-chain is a chain encoded by a nucleotide sequence comprising any one of SEQ ID NO: 5, 23, 31 or 39 or a fragment thereof.

[0232] In one embodiment, the polypeptide of the present invention comprises a fragment of a clostridial neurotoxin H-chain (or consists of it). The fragment of the clostridial neurotoxin H-chain may have ≤800, ≤700, ≤600, ≤500, ≤400, ≤350, ≤300, ≤250, ≤200, ≤150, ≤100 or ≤50 amino acid residues of the clostridial neurotoxin H-chain. In one embodiment, the fragment of the clostridial neurotoxin H-chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of the clostridial neurotoxin H-chain. For example, the fragment of the clostridial neurotoxin H-chain may have 20-800, 30-600, 40-400, 50-300 or 100-200 amino acid residues of the clostridial neurotoxin H-chain.

[0233] The H-chain of the clostridial neurotoxin consists of two structural / functional domains: the translocation domain (H N ) and receptor binding domain (H C ).

[0234] In one embodiment, the polypeptide of the present invention comprises a clostridial neurotoxin translocation domain or a fragment thereof (or consists of it). A fragment of a clostridial neurotoxin translocation domain may have ≤400, ≤350, ≤300, ≤250, ≤200, ≤150, ≤100 or ≤50 amino acid residues of a clostridial neurotoxin translocation domain. In one embodiment, a fragment of a clostridial neurotoxin translocation domain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin translocation domain. For example, a fragment of a clostridial neurotoxin translocation domain may have 20-400, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin translocation domain.

[0235] The translocation domain is a fragment of the H-chain of a clostridial neurotoxin that is approximately equivalent to the amino-terminal half of the H-chain, or a domain that corresponds to this fragment in a complete H-chain. CFunction can be achieved by missing H C The amino acid sequence can be removed by removing it (at the DNA synthesis level, or by nuclease or protease treatment at the post-synthetic level). Alternatively, H C Functional Inactivation. Thus, in some embodiments, the H-chain may be unable to bind to the binding site on the target cell to which the native Clostridial neurotoxin (ie, the holotoxin) binds.

[0236] Examples of suitable (reference) translocation domains include:

[0237] Botulinum neurotoxin type A - amino acid residues (449-871)

[0238] Botulinum neurotoxin type B - amino acid residues (441-858)

[0239] Botulinum neurotoxin type C - amino acid residues (442-866)

[0240] Botulinum neurotoxin type D - amino acid residues (446-862)

[0241] Botulinum neurotoxin type E - amino acid residues (423-845)

[0242] Botulinum neurotoxin type F - amino acid residues (440-864)

[0243] Botulinum neurotoxin type G - amino acid residues (442-863)

[0244] Tetanus neurotoxin - amino acid residues (458-879)

[0245] The reference sequences identified above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US 2007 / 0166332 (incorporated herein by reference) cites a slightly different Clostridium sequence:

[0246] Botulinum neurotoxin type A - amino acid residues (A449-K871)

[0247] Botulinum neurotoxin type B - amino acid residues (A442-S858)

[0248] Botulinum neurotoxin type C - amino acid residues (T450-N866)

[0249] Botulinum neurotoxin type D - amino acid residues (D446-N862)

[0250] Botulinum neurotoxin type E - amino acid residues (K423-K845)

[0251] Botulinum neurotoxin type F - amino acid residues (A440-K864)

[0252] Botulinum neurotoxin type G - amino acid residues (S447-S863)

[0253] Tetanus neurotoxin - amino acid residues (S458-V879)

[0254] In the context of the present invention, various Clostridial neurotoxins H comprising a translocation domain N The active fragments can be used in various aspects of the present invention. In one embodiment, these active fragments can promote the release of non-cytotoxic proteases (e.g., Clostridial L-chains) from intracellular vesicles into the cytoplasm of target cells and thus participate in the overall cellular mechanism for executing the proteolytic cleavage of the substrate by the Clostridial neurotoxin. N The length of the region is about 410-430 amino acids and contains the translocation domain. Studies have shown that the H from the heavy chain of the clostridial neurotoxin N The entire length of the region is not necessary for the translocation activity of the translocation domain. N A region comprising a translocation domain having, for example, a length of at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. Other aspects of this embodiment may include a Clostridial neurotoxin H N Regions comprising a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, and at most 425 amino acids.

[0255] For further details on the genetic basis of toxin production in Clostridium botulinum and Clostridium tetani, see Henderson et al. (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0256] Terminology N Including the naturally occurring neurotoxin H N parts, and modified H having amino acid sequences that do not exist in nature and / or synthetic amino acid residues N In one embodiment, the modified H N Some still exhibit the above translocation function.

[0257] In a preferred embodiment, the polypeptide of the invention comprises a Clostridial neurotoxin receptor binding domain (H C ) or a fragment thereof (or consisting thereof). Clostridial neurotoxin receptor binding domain (H C) may have a Clostridial neurotoxin receptor binding domain (H C ) of ≤350, ≤300, ≤250, ≤200, ≤150, ≤100 or ≤50 amino acid residues. In one embodiment, the Clostridial neurotoxin receptor binding domain (H C ) fragment has a Clostridial neurotoxin receptor binding domain (H C ). For example, the clostridial neurotoxin receptor binding domain (H C ) may have a Clostridial neurotoxin receptor binding domain (H C ) of 20-350, 50-300 or 100-200 amino acid residues.

[0258] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include:

[0259] BoNT / A-N872-L1296

[0260] BoNT / B-E859-E1291

[0261] BoNT / C1-N867-E1291

[0262] BoNT / D-S863-E1276

[0263] BoNT / E-R846-K1252

[0264] BoNT / F-K865-E1274

[0265] BoNT / G-N864-E1297

[0266] TeNT-I880-D1315

[0267] For the recently identified BoNT / X, H C The domain corresponds to amino acids 893-1306 thereof, with domain boundaries potentially varying by about 25 amino acids (eg, 868-1306 or 918-1306).

[0268] The Clostridial neurotoxin H-chain may further comprise a translocation facilitating domain. Such domains facilitate delivery of the L-chain into the cytoplasm of target cells and are described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is incorporated herein by reference.

[0269] For example, the translocation facilitating domain may comprise a Clostridial neurotoxin H CNdomain or a fragment or variant thereof. In more detail, the Clostridial neurotoxin H CN The translocation facilitating domain can have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, the clostridial neurotoxin H CN The translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include:

[0270] Botulinum neurotoxin type A - amino acid residues (872-1110)

[0271] Botulinum neurotoxin type B - amino acid residues (859-1097)

[0272] Botulinum neurotoxin type C - amino acid residues (867-1111)

[0273] Botulinum neurotoxin type D - amino acid residues (863-1098)

[0274] Botulinum neurotoxin type E - amino acid residues (846-1085)

[0275] Botulinum neurotoxin type F - amino acid residues (865-1105)

[0276] Botulinum neurotoxin type G - amino acid residues (864-1105)

[0277] Tetanus neurotoxin - amino acid residues (880-1127)

[0278] Depending on the serotype / subtype, the above sequence positions may vary slightly and the appropriate (reference) Clostridial neurotoxin H CN Further examples of domains include:

[0279] Botulinum neurotoxin type A - amino acid residues (874-1110)

[0280] Botulinum neurotoxin type B - amino acid residues (861-1097)

[0281] Botulinum neurotoxin type C - amino acid residues (869-1111)

[0282] Botulinum neurotoxin type D - amino acid residues (865-1098)

[0283] Botulinum neurotoxin type E - amino acid residues (848-1085)

[0284] Botulinum neurotoxin type F - amino acid residues (867-1105)

[0285] Botulinum neurotoxin type G - amino acid residues (866-1105)

[0286] Tetanus neurotoxin - amino acid residues (882-1127)

[0287] Suitable Clostridial neurotoxins H are described herein C Structural domain.

[0288] Clostridial neurotoxin H C The domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48 or 50, or a fragment thereof. In one embodiment, the Clostridial neurotoxin H C The domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NO: 8, 22, 30, 38, 42, 44, 46, 48 or 50, or a fragment thereof. Preferably, the Clostridial neurotoxin H C The domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NO: 8, 22, 30, 38, 42, 44, 46, 48 or 50, or a fragment thereof.

[0289] Clostridial neurotoxin H C The domain may be a domain encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47 or 49, or a fragment thereof. In one embodiment, the Clostridial neurotoxin H C The domain is a domain encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NO: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof. Preferably, the Clostridial neurotoxin H C The domain is a domain encoded by a nucleotide sequence comprising any one of SEQ ID NO: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof.

[0290] In one embodiment, the clostridial neurotoxin H used in the present invention C The variant BoNT / A HC domain is a variant BoNT / A HC domain. C The domain may comprise modifications of one or more amino acid residues selected from Y1117, F1252, H1253, and L1278. For example, a variant BoNT / AH CThe domain may comprise one or more (preferably two or more) of the following modifications Y1117V, F1252Y, H1253K and L1278F or L1278H.

[0291] In one embodiment, the variant BoNT / AH C The domains comprise the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278F; or Y1117V, F1252Y, H1253K, and L1278H.

[0292] Preferably, variant BoNT / AH C The domains comprise the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278H.

[0293] The modification can be a modification when compared to an unmodified BoNT / A as set forth in SEQ ID NO:62, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:62. Since the presence of a methionine residue at position 1 of SEQ ID NO:62 is optional, the skilled artisan will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, where SEQ ID NO:62 comprises a methionine, the position numbering will be as defined above (e.g., Y1117 will be aligned with Y1117 of SEQ ID NO:62). Alternatively, where a methionine is not present in SEQ ID NO:62, the amino acid residue numbering should be altered by -1 (e.g., Y1117 will be aligned with Y1116 of SEQ ID NO:52). Similar considerations apply when a methionine at position 1 of other polypeptide sequences described herein is present / absent, and the skilled artisan will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0294] Variant BoNT / AH C The domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46, 48, or 50, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. In one embodiment, the variant BoNT / AH C The domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 46, 48, or 50, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. In one embodiment, the variant BoNT / AH CThe domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46, 48, or 50, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. Preferably, the variant BoNT / H C The domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NO: 46, 48 or 50 or a fragment thereof.

[0295] Variant BoNT / AH C The domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46 or 50, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. In one embodiment, the variant BoNT / AH C The variant BoNT / A HC domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 46 or 50, or a fragment thereof, with the proviso that the variant BoNT / A HC domain comprises a modification as described above. In one embodiment, the variant BoNT / A HC domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 46 or 50, or a fragment thereof. C The domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46 or 50, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. Preferably, the variant BoNT / H C The domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NO: 46 or 50 or a fragment thereof.

[0296] Variant BoNT / AH C The domain may be a domain encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 45, 47, or 49, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. In one embodiment, the variant BoNT / AH C A domain is a domain encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 45, 47 or 49, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. In one embodiment, the variant BoNT / AH CThe domain is a domain encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45, 47, or 49, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. Preferably, the variant BoNT / H C The domain is a domain encoded by any one of SEQ ID NO: 45, 47 or 49, or a fragment thereof.

[0297] Variant BoNT / AH C The domain may be a domain encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 45 or 49 or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. In one embodiment, the variant BoNT / AH C A domain is a domain encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 45 or 49, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. In one embodiment, the variant BoNT / AH C The domain is a domain encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45 or 49, or a fragment thereof, provided that the variant BoNT / AH C The domain comprises modifications as described above. Preferably, the variant BoNT / H C The domain is a domain encoded by any one of SEQ ID NO: 45 or 49 or a fragment thereof.

[0298] Any of the above-described facilitating domains may be combined with any of the above-described translocation domain peptides suitable for use in the present invention. Thus, for example, a non-Clostridium facilitating domain may be combined with a non-Clostridium translocation domain peptide or with a Clostridial translocation domain peptide. Alternatively, a Clostridial neurotoxin H CN The translocation facilitating domain can be combined with a non-Clostridial translocation domain peptide. Alternatively, the Clostridial neurotoxin H CN The facilitating domain can be combined with a Clostridial translocation domain peptide, examples of which include:

[0299] Botulinum neurotoxin type A - amino acid residues (449-1110)

[0300] Botulinum neurotoxin type B - amino acid residues (442-1097)

[0301] Botulinum neurotoxin type C - amino acid residues (450-1111)

[0302] Botulinum neurotoxin type D - amino acid residues (446-1098)

[0303] Botulinum neurotoxin type E - amino acid residues (423-1085)

[0304] Botulinum neurotoxin type F - amino acid residues (440-1105)

[0305] Botulinum neurotoxin type G - amino acid residues (447-1105)

[0306] Tetanus neurotoxin - amino acid residues (458-1127)

[0307] In some embodiments, the Clostridial neurotoxins of the invention may lack the functional H C In one embodiment, the clostridial neurotoxin preferably lacks the last 50 C-terminal amino acids of the clostridial neurotoxin holotoxin. In another embodiment, the clostridial neurotoxin preferably lacks the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of the clostridial neurotoxin holotoxin. Alternatively, H C Binding activity can be eliminated / reduced by mutagenesis - for example, referring to BoNT / A for convenience, modification of one or two amino acid residues in the ganglioside binding pocket (W1266 to L and Y1267 to F) results in H C The region loses its receptor binding function. Similar mutations can be made to non-serotype A clostridial peptide components, such as constructs based on C. botulinum B (W1262 to L and Y1263 to F) or C. botulinum E (W1224 to L and Y1225 to F) with mutations. Other mutations in the active site achieve H C The same elimination of receptor binding activity, such as Y1267S in botulinum toxin type A and the corresponding highly conserved residues in other clostridial neurotoxins. Details of this and other mutations are described in Rummel et al. (2004) (Molecular Microbiol. 51: 631-634), which is incorporated herein by reference.

[0308] H of natural clostridial neurotoxin C The peptide contains approximately 400-440 amino acid residues and consists of two functionally distinct domains of approximately 25 kDa each, the N-terminal region (usually referred to as the H CN peptide or domain) and the C-terminal region (often referred to as H CCpeptide or domain). This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4:788-792; Herreros J (2000) Biochem. J. 347:199-204; Halpern J (1993) J. Biol. Chem. 268:15, pp. 11188-11192; Rummel A (2007) PNAS 104:359-364; Lacey DB (1998) Nat. Struct. Biol. 5:898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25:90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7:1751-1759; and Rummel A (2004) Mol. Microbiol. 51 (3), 631-643. In addition, it has been well documented that the C-terminal region (H CC ) (which constitutes the C-terminal 160-200 amino acid residues) is responsible for the binding of the clostridial neurotoxin to its natural cell receptor (i.e. the nerve endings at the neuromuscular junction) - a fact also confirmed by the above-mentioned publication. Therefore, throughout this specification, references to the lack of a functional heavy chain H C The presence of a peptide (or domain) that renders the heavy chain incapable of binding to a cell surface receptor to which the native Clostridial neurotoxin binds means that the Clostridial heavy chain simply lacks a functional H CC peptide. In other words, H CC A peptide region may be partially or completely deleted, or otherwise modified (eg, by conventional chemical or proteolytic treatment) to reduce its native binding ability to nerve endings at the neuromuscular junction.

[0309] Thus, in one embodiment, the Clostridial neurotoxin H of the invention N The peptide lacks the C-terminal peptide portion of the clostridial neurotoxin (H CC ) and therefore lacks the H of the native Clostridial neurotoxin C For example, in one embodiment, the C-terminally extended Clostridium H N The peptide lacks the C-terminal 40 amino acid residues, or the C-terminal 60 amino acid residues, or the C-terminal 80 amino acid residues, or the C-terminal 100 amino acid residues, or the C-terminal 120 amino acid residues, or the C-terminal 140 amino acid residues, or the C-terminal 150 amino acid residues, or the C-terminal 160 amino acid residues of the heavy chain of the Clostridium neurotoxin. In another embodiment, the Clostridium H NThe peptide lacks the entire C-terminal peptide portion of the Clostridial neurotoxin (H CC ), and therefore lacks the H of the native Clostridial neurotoxin C For example, in one embodiment, Clostridium H N The peptide lacks the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of the heavy chain of the clostridial neurotoxin. As another example, the clostridial H N The peptide lacks a Clostridium H selected from the following CC Reference sequence:

[0310] Botulinum neurotoxin type A - amino acid residues (Y1111-L1296)

[0311] Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

[0312] Botulinum neurotoxin type C - amino acid residues (Y1112-E1291)

[0313] Botulinum neurotoxin type D - amino acid residues (Y1099-E1276)

[0314] Botulinum neurotoxin type E - amino acid residues (Y1086-K1252)

[0315] Botulinum neurotoxin type F - amino acid residues (Y1106-E1274)

[0316] Botulinum neurotoxin type G - amino acid residues (Y1106-E1297)

[0317] Tetanus neurotoxin - amino acid residues (Y1128-D1315).

[0318] The reference sequences identified above should be considered as a guide, as slight variations may occur depending on the subserotype.

[0319] In a preferred embodiment, the polypeptide of the invention comprises (or consists of) a fragment of a clostridial neurotoxin L-chain or a fragment thereof and a fragment of a clostridial neurotoxin H-chain. For example, a polypeptide may comprise a clostridial neurotoxin L-chain or a fragment thereof and a clostridial neurotoxin translocation domain (H N Preferably, the polypeptide does not further comprise a Clostridial neurotoxin receptor binding domain (H C ) or Clostridial neurotoxin receptor binding domain (H CC). Thus, in one embodiment, the polypeptides of the invention lack the Clostridial neurotoxin receptor binding domain (H CC ) The C-terminal portion of ). Advantageously, such polypeptides lack endogenous Clostridial neurotoxin receptor binding ability and therefore exhibit fewer off-target effects in subjects to which the polypeptides are administered.

[0320] In one embodiment, the polypeptide of the present invention consists essentially of a clostridial neurotoxin L-chain or a fragment thereof and / or a fragment of a clostridial neurotoxin H-chain. As used in this context, the term "consisting essentially of" means that the polypeptide does not further comprise one or more amino acid residues that confer additional functionality to the polypeptide, for example when administered to a subject. In other words, a polypeptide "consisting essentially of a clostridial neurotoxin L-chain or a fragment thereof and / or a fragment of a clostridial neurotoxin H-chain" may further comprise one or more amino acid residues (for amino acid residues of a clostridial neurotoxin L-chain or a fragment thereof and / or a fragment of a clostridial neurotoxin H-chain), but the one or more additional amino acid residues do not confer additional functionality to the polypeptide, for example when administered to a subject. Additional functionality may include enzymatic activity, binding activity and / or any physiological activity.

[0321] In one embodiment, in addition to any clostridial neurotoxin sequence, the polypeptide may also include a non-clostridial neurotoxin sequence. The non-clostridial neurotoxin sequence preferably does not destroy the ability of the polypeptide of the present invention to promote neuronal growth or neuronal repair. Preferably, the non-clostridial neurotoxin sequence is not a sequence with catalytic activity (eg, enzymatic activity). Preferably, the non-clostridial sequence is not a sequence that binds to a cell receptor. In other words, it is most preferred that the non-clostridial sequence is not a ligand for a cell receptor. The cell receptor may be a protein cell receptor, such as an intrinsic membrane protein. Examples of cell receptors can be found in the IUPHAR Guide to Pharmacology Database, Version 2019.4, available at https: / / www.guidetopharmacology.org / download.jsp#db_reports. Non-clostridial neurotoxin sequences may include tags that facilitate purification, such as His tags. Preferably, any clostridial neurotoxin sequence included in the polypeptide consists of a clostridial neurotoxin L-chain or a fragment thereof and / or a fragment of a clostridial neurotoxin H-chain. In one embodiment, the clostridial neurotoxin sequence included in the polypeptide may consist of a clostridial neurotoxin L-chain. In one embodiment, the clostridial neurotoxin sequence contained in the polypeptide may consist of a clostridial neurotoxin translocation domain. In one embodiment, the clostridial neurotoxin sequence contained in the polypeptide may consist of a clostridial neurotoxin receptor binding domain. In one embodiment, the clostridial neurotoxin sequence contained in the polypeptide may consist of a clostridial neurotoxin L-chain and a clostridial neurotoxin translocation domain.

[0322] Suitable polypeptides comprising (or consisting of) a Clostridial neurotoxin L-chain and a translocation domain are described herein.

[0323] The clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and a translocation domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4, 20, 28 or 36, or a fragment thereof. In one embodiment, the clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and a translocation domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 4, 20, 28 or 36, or a fragment thereof. Preferably, the clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and a translocation domain comprises a polypeptide sequence comprising (more preferably consisting of) any one of SEQ ID NOs: 4, 20, 28 or 36, or a fragment thereof.

[0324] The clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and a translocation domain may be a clostridial neurotoxin encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3, 19, 27 or 35, or a fragment thereof. In one embodiment, the clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and a translocation domain is a clostridial neurotoxin encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 3, 19, 27 or 35, or a fragment thereof. Preferably, the clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and a translocation domain is a clostridial neurotoxin encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 3, 19, 27 or 35, or a fragment thereof.

[0325] The polypeptides of the present invention may be free of complexing proteins present in naturally occurring Clostridial neurotoxin complexes.

[0326] The polypeptides of the present invention may be produced using recombinant nucleic acid technology. Thus, in one embodiment, the polypeptide (as described above) is a recombinant polypeptide.

[0327] In one embodiment, a nucleic acid (eg, DNA) comprising a nucleic acid sequence encoding a polypeptide is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator.

[0328] In a preferred embodiment, the vector has a promoter selected from:

[0329]

[0330] In another preferred embodiment, the vector has a promoter selected from:

[0331]

[0332] Any suitable method known in the art can be used to prepare nucleic acid molecules. Therefore, chemical synthesis techniques can be used to prepare nucleic acid molecules. Alternatively, molecular biology techniques can be used to prepare nucleic acid molecules of the present invention.

[0333] The DNA constructs of the present invention are preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

[0334] Depending on the final host cell (eg, E. coli) expression system to be employed, the above nucleic acid sequence information is optionally modified with respect to codon preference.

[0335] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0336] A polypeptide of the invention (particularly any Clostridial neurotoxin portion thereof) may exist as a single chain or as a double chain.

[0337] The present invention provides a method for producing a single-chain polypeptide having a light chain and a heavy chain, the method comprising expressing a nucleic acid as described herein in an expression host, lysing the host cells to provide a host cell homogenate containing the single-chain polypeptide, and isolating the single-chain polypeptide. In one aspect, the present invention provides a method for activating a polypeptide as described herein, the method comprising contacting the polypeptide with a protease that hydrolyzes a peptide bond in an activation loop of the polypeptide, thereby converting the (single-chain) polypeptide into a corresponding double-chain polypeptide (e.g., wherein the light chain and the heavy chain are linked together by a disulfide bond).

[0338] Therefore, the present invention provides a double-chain polypeptide obtainable by the method of the present invention.

[0339] Embodiments directed to the various therapeutic uses of the invention are intended to apply equally to the therapeutic methods, polypeptides of the invention, and vice versa.

[0340] Sequence homology

[0341] Any of a variety of sequence alignment methods can be used to determine percent identity, including but not limited to global methods, local methods, and hybrid methods, such as segment methods. Protocols for determining percent identity are routine procedures within the scope of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up the scores for each residue pair and by applying gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22 (22) Nucleic Acids Research 4673-4680 (1994); and iterative improvement, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264 (4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences.Non-limiting methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8 (5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262 (5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20 (9) Bioinformatics: 1428-1435 (2004).

[0342] Therefore, the sequence identity percentage is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In brief, as shown below, two amino acid sequences are aligned using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra) to optimize the alignment score (amino acids are represented by standard single letter codes).

[0343] The "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of % sequence identity can also take into account the number of spaces that need to be introduced to optimize the alignment of two or more sequences, as well as the length of each space. Specific mathematical algorithms familiar to those skilled in the art (e.g., BLAST) can be used to compare sequences and determine the percent identity between two or more sequences.

[0344] Determining alignment scores for sequence identity

[0345]

[0346] The percent identity is then calculated as:

[0347]

[0348] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably insignificant, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect polypeptide folding or activity; small deletions, typically 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as an amino terminal methionine residue, a small linker peptide of up to about 20-25 residues or an affinity tag.

[0349] Conservative amino acid substitution

[0350] Alkaline: Arginine

[0351] Lysine

[0352] Histidine

[0353] Acid: Glutamic acid

[0354] Aspartic acid

[0355] Polarity: Glutamine

[0356] Asparagine

[0357] Hydrophobicity: Leucine

[0358] Isoleucine

[0359] Valine

[0360] Aromatic: Phenylalanine

[0361] Tryptophan

[0362] Tyrosine

[0363] Small: Glycine

[0364] Alanine

[0365] Serine

[0366] Threonine

[0367] Methionine

[0368] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can replace the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace polypeptide amino acid residues. The polypeptides of the present invention can also contain non-naturally occurring amino acid residues.

[0369] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methyl-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second approach, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). The non-natural amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring substances by in vitro chemical modification. Chemical modification can be used in combination with site-directed mutagenesis to further expand the scope of substitution (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0370] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids and unnatural amino acids can be substituted for amino acid residues in the polypeptides of the invention.

[0371] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). The sites of biological interaction can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identification of essential amino acids can also be inferred from homology analysis with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0372] A variety of amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed in Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclose a method of simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0373] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th Edition, John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991), provide the skilled artisan with a general dictionary of many of the terms used in the present disclosure.

[0374] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction, respectively.

[0375] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0376] In this article, amino acid names, three-letter abbreviations or single-letter abbreviations are used to refer to amino acids. As used herein, the term "protein" includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codons of amino acid residues can be used. The 3-letter codons of amino acids are defined according to the Joint Committee on Biochemical Nomenclature (JCBN) of IUPAC IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0377] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described and may vary accordingly. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0378] In the case of providing a range of values, it is understood that, unless the context clearly indicates otherwise, between the upper and lower limits of the range, each intermediate value, to one tenth of the lower limit unit, is specifically included in the present disclosure. Each smaller range between any specified value or intermediate value in a specified range and any other specified value or intermediate value in the specified range is included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the range or excluded in the range, and each range including one of the upper and lower limits in the smaller range, excluding none, or including both is also included in the present disclosure, subject to any explicitly excluded limits in the described range. In the case where the described range includes one or two limits, the range excluding any one or two of those included limits is also included in the present disclosure.

[0379] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents and reference to "a clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0380] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0381] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0382] Figure 1 Shown are the neurotrophic effects of different recombinantly expressed catalytically inactive BoNT serotypes compared to the positive control brain-derived neurotrophic factor (BDNF) in the motor neuron-like cell line NSC34. *p<0.05 relative to untreated control, one-way ANOVA followed by Dunnett's multiple comparison test. Data are mean ± standard error of three independent experiments, each performed in six replicate wells.

[0383] Figure 2 The neurotrophic effects of botulinum neurotoxin serotype A fragment and the effects of recombinantly expressed catalytically inactive BoNT / A in the motor neuron-like cell line NSC34 are shown. BDNF was used as a positive control. *p<0.05 relative to untreated control, one-way ANOVA followed by Dunnett's multiple comparison test. Data are mean ± standard error of three independent experiments, each performed in six replicate wells.

[0384] Figure 3 The neurotrophic effects of negative control and recombinantly expressed catalytically inactive BoNT / A (BoNT / A(0)) in the motor neuron-like cell line NSC34 are shown. BDNF was used as a positive control. *p<0.05 relative to untreated control, one-way ANOVA followed by Dunnett's multiple comparison test. Data are mean ± standard error of three independent experiments, each performed in six replicate wells.

[0385] Figure 4 Shown are the results of a horizontal ladder test of mice administered vehicle control (PBS) or 100 pg, 100 ng, or 50 ug of rBoNT / A(0).

[0386] Figure 5 Shown are: (A) Immunohistochemistry using an antibody that binds to neurofilament 200 (NF200) 4 weeks after administration of vehicle (PBS) (left panel) or 100 ng rBoNT / A (0) (right panel); and (B) Immunohistochemistry using an antibody that binds to MAP1B 4 weeks after administration of vehicle (PBS) (left panel) or 100 ng rBoNT / A (0) (right panel). Lesion sites are indicated by * (and for Figure 5 B, indicated by white arrows).

[0387] Figure 6 (A) catalytically inactive BoNT / A (0), (B) BoNT / A light chain plus translocation domain fragment (LH N / A), (C) BoNT / A light chain (LC / A, i.e., L / A), and (D) BoNT / A receptor binding domain (H C / A) Effect on the number of neurites per cell. BoNT or BoNT fragments were compared to BSA (negative control), BDNF (positive control) and tested at concentrations of 0.1 nM, 1 nM, and 10 nM. *p<0.05 relative to BSA control, one-way ANOVA followed by Dunnett's post hoc test. Data are mean ± s.e. mean.

[0388] Figure 7 (A) catalytically inactive BoNT / FA (0), (B) BoNT / FA light chain plus translocation domain fragment (LH N / FA), (C) BoNT / FA light chain (LC / FA, i.e., L / FA), and (D) BoNT / FA receptor binding domain (H / FA). CFigure 2 Effect of 1:100 μg / mL of BoNT on the number of neurites per cell. BoNT or BoNT fragments were compared to BSA (negative control), BDNF (positive control) and tested at concentrations of 0.1 nM, 1 nM, and 10 nM. *p<0.05 relative to BSA control, one-way ANOVA followed by Dunnett's post hoc test. Data are mean ± s.e. mean.

[0389] Figure 8 (A) BoNT / F light chain plus translocation domain fragment (LH N / F), (B) BoNT / F light chain (LC / F, i.e., L / F), and (C) BoNT / F receptor binding domain (H C Effect of BDNF (negative control) on the number of neurites per cell. BoNT or BoNT fragments were compared with BSA (negative control), BDNF (positive control) and tested at concentrations of 0.1 nM, 1 nM and 10 nM. *p<0.05 relative to BSA control, one-way ANOVA followed by Dunnett's post hoc test. Data are mean ± s.e. mean.

[0390] Fig. 9 The rH of the cation is shown C Effect of mrHC / A on the number of neurites per cell. Cationic BoNT fragments were compared with BSA (negative control), BDNF (positive control) and tested at concentrations of 0.1 nM, 1 nM and 10 nM. *p<0.05 relative to BSA control, one-way ANOVA followed by Dunnett's post hoc test. Data are mean ± s.e. mean.

[0391] Fig.10 (A) toxHC / A YH (ie rH C / A variant Y1117V H1253K) and (B) toxHC / A YFHL (L to H) (i.e., rH C Effect of variants Y1117V F1252Y H1253K L1278H) on the number of neurites per cell. Variant BoNT fragments were compared to BSA (negative control), BDNF (positive control) and tested at concentrations of 0.1 nM, 1 nM, and 10 nM. *p<0.05 relative to BSA control, one-way ANOVA followed by Dunnett's post hoc test. Data are mean ± s.e. mean.

[0392] Sequence Listing

[0393] Where an initial Met amino acid residue or the corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue / codon is optional.

[0394] SEQ ID NO: 1 - Nucleotide sequence of recombinant catalytically inactive BoNT / A (rBoNT / A(0))

[0395] SEQ ID NO:2—Polypeptide sequence of rBoNT / A(0)

[0396] SEQ ID NO:3-rLH N / A nucleotide sequence (light chain plus translocation domain only).

[0397] SEQ ID NO:4-rLH N / A polypeptide sequence

[0398] SEQ ID NO:5-nucleotide sequence of rL / A (light chain only)

[0399] SEQ ID NO:6-peptide sequence of rL / A

[0400] SEQ ID NO:7-rH C / A nucleotide sequence

[0401] SEQ ID NO:8-rH C / A polypeptide sequence

[0402] SEQ ID NO: 9—Nucleotide sequence of rBoNT / B (0)

[0403] SEQ ID NO: 10—Polypeptide sequence of rBoNT / B(0)

[0404] SEQ ID NO: 11 - Nucleotide sequence of rBoNT / C (0)

[0405] SEQ ID NO: 12 - polypeptide sequence of rBoNT / C (0)

[0406] SEQ ID NO: 13 - Nucleotide sequence of rBoNT / E(0)

[0407] SEQ ID NO: 14 - polypeptide sequence of rBoNT / E(0)

[0408] SEQ ID NO: 15—Nucleotide sequence of rBoNT / F(0)

[0409] SEQ ID NO: 16 - polypeptide sequence of rBoNT / F(0)

[0410] SEQ ID NO: 17 - Nucleotide sequence of rBoNT / A(0) (His-tagged)

[0411] SEQ ID NO: 18 - Polypeptide sequence of rBoNT / A(0) (His-tagged)

[0412] SEQ ID NO:19-rLH N / A nucleotide sequence (His-tagged)

[0413] SEQ ID NO:20-rLH N / A polypeptide sequence (His-tagged)

[0414] SEQ ID NO:21-rH C / A nucleotide sequence (His-tagged)

[0415] SEQ ID NO:22-rH C / A polypeptide sequence (His-tagged)

[0416] SEQ ID NO:23 - Nucleotide sequence of rLc / A (His-tagged)

[0417] SEQ ID NO:24 - polypeptide sequence of rLc / A (His-tagged)

[0418] SEQ ID NO:25—Nucleotide sequence of rBoNT / FA (0) (His-tagged)

[0419] SEQ ID NO:26 - Polypeptide sequence of rBoNT / FA (0) (His-tagged)

[0420] SEQ ID NO:27-rLH N / FA nucleotide sequence (His-tagged)

[0421] SEQ ID NO:28-rLH N / FA polypeptide sequence (His-tagged)

[0422] SEQ ID NO:29-rH C / FA nucleotide sequence (His-tagged)

[0423] SEQ ID NO:30-rH C / FA polypeptide sequence (His-tagged)

[0424] SEQ ID NO:31 - Nucleotide sequence of rLC / FA (His-tagged)

[0425] SEQ ID NO:32 - polypeptide sequence of rLC / FA (His-tagged)

[0426] SEQ ID NO:33—Nucleotide sequence of rBoNT / F(0) (His-tagged)

[0427] SEQ ID NO:34 - Polypeptide sequence of rBoNT / F(0) (His-tagged)

[0428] SEQ ID NO:35-rL H Nucleotide sequence of N / F (His tag)

[0429] SEQ ID NO:36-rL H N / F peptide sequence (His-tagged)

[0430] SEQ ID NO:37-rH C / F nucleotide sequence (His-tagged)

[0431] SEQ ID NO:38-rH C / F polypeptide sequence (His-tagged)

[0432] SEQ ID NO:39—Nucleotide sequence of rLC / F (His-tagged)

[0433] SEQ ID NO:40 - polypeptide sequence of rLC / F (His-tagged)

[0434] SEQ ID NO:41 - cationic rH C / A nucleotide sequence (His-tagged)

[0435] SEQ ID NO:42—Cationic rH C / A polypeptide sequence (His-tagged)

[0436] SEQ ID NO:43-rH C / AB nucleotide sequence (His-tagged)

[0437] SEQ ID NO:44-rH C / AB peptide sequence (His-tagged)

[0438] SEQ ID NO:45-rH C Nucleotide sequence of / A variant Y1117V H1253K (His-tagged)

[0439] SEQ ID NO:46-rH C / A polypeptide sequence of variant Y1117V H1253K (His-tagged)

[0440] SEQ ID NO:47-rH C Nucleotide sequence of / A variant Y1117V F1252Y H1253K L1278F (His-tagged)

[0441] SEQ ID NO:48-rH C / A polypeptide sequence of variant Y1117V F1252Y H1253K L1278F (His-tagged)

[0442] SEQ ID NO:49-rH C Nucleotide sequence of / A variant Y1117V F1252Y H1253K L1278H (His-tagged)

[0443] SEQ ID NO:50-rH C / A variant Y1117V F1252Y H1253K L1278H polypeptide sequence (His-tagged)

[0444] SEQ ID NO:51 - Polypeptide sequence of BoNT / A - Uniprot P10845

[0445] SEQ ID NO:52 - Polypeptide sequence of BoNT / B - Uniprot P10844

[0446] SEQ ID NO:53 - Polypeptide sequence of BoNT / C - Uniprot P18640

[0447] SEQ ID NO:54 - Polypeptide sequence of BoNT / D - Uniprot P19321

[0448] SEQ ID NO:55 - Polypeptide sequence of BoNT / E - Uniprot Q00496

[0449] SEQ ID NO:56 - Polypeptide sequence of BoNT / F - Uniprot A7GBG3

[0450] SEQ ID NO:57 - Polypeptide sequence of BoNT / G - Uniprot Q60393

[0451] SEQ ID NO:58 - TeNT polypeptide sequence - Uniprot P04958

[0452] SEQ ID NO:59 - Polypeptide sequence of BoNT / X

[0453] SEQ ID NO:60—Nucleotide sequence of mrBoNT / A

[0454] SEQ ID NO:61 - polypeptide sequence of mrBoNT / A

[0455] SEQ ID NO:62 - Unmodified BoNT / A1 polypeptide sequence

[0456] SEQ ID NO:63—Polypeptide sequence of mrBoNT / AB

[0457] SEQ ID NO:64—Polypeptide sequence of mrBoNT / AB(0)

[0458] SEQ ID NO:65—Polypeptide sequence of MrBoNT / A(0)

[0459] SEQ ID NO: 1—Nucleotide sequence of rBoNT / A(0)

[0460]

[0461] SEQ ID NO:2—Polypeptide sequence of rBoNT / A(0)

[0462]

[0463] SEQ ID NO:3-rLH N / A nucleotide sequence

[0464]

[0465] SEQ ID NO:4-rLH N / A polypeptide sequence

[0466] MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEAHHHHHHHHHH

[0467] SEQ ID NO:5-nucleotide sequence of rL / A

[0468]

[0469] SEQ ID NO:6-peptide sequence of rL / A

[0470] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLGLEAHHHHHHHHHH

[0471] SEQ ID NO:7-rH C / A nucleotide sequence

[0472]

[0473] SEQ ID NO:8-rH C / A polypeptide sequence

[0474] MHHHHHHKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0475] SEQ ID NO: 9—Nucleotide sequence of rBoNT / B (0)

[0476]

[0477] SEQ ID NO: 10—Polypeptide sequence of rBoNT / B(0)

[0478]

[0479] SEQ ID NO: 11 - Nucleotide sequence of rBoNT / C (0)

[0480]

[0481] SEQ ID NO: 12 - polypeptide sequence of rBoNT / C (0)

[0482]

[0483] SEQ ID NO: 13 - Nucleotide sequence of rBoNT / E(0)

[0484]

[0485] SEQ ID NO: 14 - polypeptide sequence of rBoNT / E(0)

[0486]

[0487] SEQ ID NO: 15—Nucleotide sequence of rBoNT / F(0)

[0488]

[0489] SEQ ID NO: 16-peptide sequence of rBoNT / F(0)

[0490]

[0491] SEQ ID NO: 17—Nucleotide sequence of rBoNT / A(0) (His-tag)

[0492]

[0493] SEQ ID NO: 18 - polypeptide sequence of rBoNT / A (0) (His-tag)

[0494]

[0495] SEQ ID NO:19-rLH N / A nucleotide sequence (His-tag)

[0496]

[0497] SEQ ID NO:20-rLH N / A polypeptide sequence (His-tag)

[0498] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTENLYFQGASHHHHHHHH

[0499] SEQ ID NO:21-rH C / A nucleotide sequence (His-tag)

[0500] SEQ ID NO:22-rH C / A polypeptide sequence (His-tag)

[0501] MHHHHHHENLYFQGKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0502] SEQ ID NO:23—Nucleotide sequence of rLC / A (His-tag)

[0503]

[0504] SEQ ID NO:24—peptide sequence of rLC / A (His-tag)

[0505] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEENLYFQGASHHHHHHHH

[0506] SEQ ID NO:25—Nucleotide sequence of rBoNT / FA (0) (His-tag)

[0507]

[0508] SEQ ID NO:26—Polypeptide sequence of rBoNT / FA (0) (His-tag)

[0509]

[0510] SEQ ID NO:27-rLH N / FA nucleotide sequence (His-tag)

[0511]

[0512] SEQ ID NO:28-rLH N / FA peptide sequence (His-tag)

[0513] MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLEEIKNAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLTTNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEYATAFNDISIASHPSLFIKDPALILMHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAKDSNGVYSVDLNKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNIGALSINYQGQNIGSDINSIKKLQGQGVSVRVVRLCSNSNTKNSLC ITVNNRDLFFIASQESYGENTINTYKEIDDTTTLDPSFEDILDKVILNFNEQVIPQMPNRNVSTDIQKDNYIPKYDYNRTTDIIDSYEVGRNYNTFFYLNAQKFSPNESNITLTSFDTGLLEGSKVYTFFSSDFINNINKPVQALLFIEWVKQVIRDFTTEATKTSTVDKLKDISLVVPYIGLANLIGDEIYKQHFAEVELVGAGLLLEFSPEFLIPT LLIFTIKGYLTGSIRDKDKIIKTLDNALNVRDQKWKELYRWVVSKWLTTINTQFNKRKEQMYKALKNQATAIKKIIENKYNNYTTDEKSKIDSSYNINEIERTLNEKINLAMKNIEQFITESSIAYLINIINNETIQKLKSYDDLVRRYLLGYIRNHSSILGNSVEELNSKVNNHLDNGIPFELSSYTNDSLIRYFNKNYGEENLYFQGASHHHHHHH

[0514] SEQ ID NO:29-rH C / FA nucleotide sequence (His-tag)

[0515]

[0516] SEQ ID NO:30-rH C / FA peptide sequence (His-tag)

[0517] MLKYNCILNIKYEMDRDKLVDSSGYRSRINIGTGVKFSEIDKNQVQLSNLESSKIEVILNNGVIYNSMYENFSTSFWIRIPKYFRNINNEYKIISCMQNNSGWEVSLNFSNMNSKIIWTLQDTEGIKKTVVFQYTQNINISDYINRWIFVTITNNRLSNSKIYINGRLINEESISDLGNIHASNNIMFKLDGCRDPHRYIWIKYFNLFDKELNKKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYLDVNNVGIRGYMYLKGPRGRIVTTNIYLNSTLYMGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSAVEIPDVGNLSQVVVMKSENDQGIRNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIGKASRTFGCSWEFIPVDDGWGESSLENLYFQGASHHHHHHHH

[0518] SEQ ID NO:31 - Nucleotide sequence of rLC / FA (His-tag)

[0519] SEQ ID NO:32 - polypeptide sequence of rLC / FA (His-tag)

[0520] MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLEEIKNAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLTTNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEYATAFNDISIASHSPSLFIKDPALILMHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAKDSNGVYSVDLNKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNIGALSINYQGQNIGSDINSIKKLQGQGVVSRVVRLCSNSENLYFQGASHHHHHHHH

[0521] SEQ ID NO:33—Nucleotide sequence of rBoNT / F(0) (His-tag)

[0522]

[0523] SEQ ID NO:34 - Polypeptide sequence of rBoNT / F(0) (His-tag)

[0524]

[0525] SEQ ID NO:35-rL H Nucleotide sequence of N / F (His-tag)

[0526]

[0527] SEQ ID NO:36-rL H N / F peptide sequence (His-tag)

[0528] MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTVNVKSSIILNLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYETFNDISGGYNSTESFIADPAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAP PRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLNNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLANIGNEVQKENFKEAFELLGAGILLEFVPELLIPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERFITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKENLYFQGASHHHHHHH

[0529] SEQ ID NO:37-rH C / F nucleotide sequence (His-tag)

[0530]

[0531] SEQ ID NO:38-rH C / F peptide sequence (His-tag)

[0532] MIKDNSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDCIRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNSRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQENENLYFQGASHHHHHHHH

[0533] SEQ ID NO:39—Nucleotide sequence of rLC / F (His-tag)

[0534]

[0535] SEQ ID NO:40 - polypeptide sequence of rLC / F (His-tag)

[0536] MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTVNVKSSIILNLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYETFNDISGGYNSTESFIADPAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSENLYFQGASHHHHHHH

[0537] SEQ ID NO:41 - Cationic rH C / A nucleotide sequence (His-tag)

[0538] SEQ ID NO:42—Cationic rH C / A polypeptide sequence (His-tag)

[0539] MIINTSILNLRYESKHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLKLAAALEHHHHHH

[0540] SEQ ID NO:43-rH C / AB nucleotide sequence (His-tag)

[0541]

[0542] SEQ ID NO:44-rH C / AB peptide sequence (His-tag)

[0543] MILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEKLAAALEHHHHHH

[0544] SEQ ID NO:45-rH C Nucleotide sequence of / A variant Y1117VH1253K (His-tag)

[0545]

[0546] SEQ ID NO:46-rH C / A polypeptide sequence of variant Y1117VH1253K (His-tag)

[0547] MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFKQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLHHHHHHHHHH

[0548] SEQ ID NO:47-rH C Nucleotide sequence of / A variant Y1117VF1252YH1253KL1278F (His-tag)

[0549]

[0550] SEQ ID NO:48-rH C / A polypeptide sequence of variant Y1117VF1252YH1253KL1278F (His-tag)

[0551] MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGYKQFNNIAKLVASNWYNRQIERSSRTFGCSWEFIPVDDGWGERPLHHHHHHHHHH

[0552] SEQ ID NO:49-rH C Nucleotide sequence of / A variant Y1117VF1252YH1253KL1278H (His-tag)

[0553]

[0554] SEQ ID NO:50-rH C / A polypeptide sequence of variant Y1117VF1252YH1253KL1278H (His-tag)

[0555] MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGYKQFNNIAKLVASNWYNRQIERSSRTHGCSWEFIPVDDGWGERPLHHHHHH

[0556] SEQ ID NO:51 - Polypeptide sequence of BoNT / A-UniProt P10845

[0557]

[0558] SEQ ID NO:52 - Polypeptide sequence of BoNT / B-UniProt P10844

[0559]

[0560] SEQ ID NO:53 - Polypeptide sequence of BoNT / C-UniProt P18640

[0561] SEQ ID NO:54 - Polypeptide sequence of BoNT / D-UniProt P19321

[0562]

[0563] SEQ ID NO:55 - Polypeptide sequence of BoNT / E-UniProt Q00496

[0564] SEQ ID NO:56 - Polypeptide sequence of BoNT / F-UniProt A7GBG3

[0565]

[0566] SEQ ID NO:57 - Polypeptide sequence of BoNT / G-UniProt Q60393

[0567]

[0568] SEQ ID NO:58 - polypeptide sequence of TeNT-UniProt P04958

[0569]

[0570] SEQ ID NO:59 - Polypeptide sequence of BoNT / X

[0571]

[0572] SEQ ID NO:60 - Nucleotide sequence of mrBoNT / A

[0573]

[0574] SEQ ID NO:61 - polypeptide sequence of mrBoNT / A

[0575]

[0576] SEQ ID NO:62—Unmodified BoNT / A1 polypeptide sequence

[0577]

[0578] SEQ ID NO:63 - polypeptide sequence of mrBoNT / AB

[0579]

[0580] SEQ ID NO:64 - polypeptide sequence of mrBoNT / AB(0)

[0581]

[0582] SEQ ID NO:65 - polypeptide sequence of mrBoNT / A(0)

[0583] Example

[0584] Example 1

[0585] Multiple catalytically inactive BoNT serotypes increase total neurite length compared to untreated control cells

[0586] Materials and methods

[0587] Five catalytically inactive (i.e., endopeptidase-inactive) botulinum neurotoxin (BoNT) serotypes, corresponding to serotypes A, B, C, E, and F, were recombinantly expressed in E. coli and are denoted rBoNT / A(0), rBoNT / B(0), rBoNT / C(0), rBoNT / E(0), and rBoNT / F(0). As a result of the catalytic inactivity, these molecules are unable to cleave their respective (SNARE) protein substrates.

[0588] Motor neuron-like hybrid cell line (NSC34 cells) (Tebu-Bio, Cedarlane Laboratories, France) were cultured on poly-D-lysine coated black multiwells at 5000 cells / well and cultured in DM EM supplemented with 10% FCS and penicillin / streptomycin. After plating, cells were differentiated into motor neurons by exposure to 1 μM retinoic acid and low serum for 4 days, and then treated with 3 different concentrations: 0.1, 1 and 10 nM of rBoNT / A(0), rBoNT / B(0), rBoNT / C(0), rBoNT / E(0) and rBoNT / F(0) for 4 days and fixed with paraformaldehyde 4%-sucrose 4%. Brain-derived neurotrophic factor (BDNF) (commercially available from ReproTech EC Ltd, London, UK) 1 ng / ml was used as a positive control for neuronal growth. Cells were fixed with paraformaldehyde 4%-sucrose 4% and then stained with appropriate antibodies. In particular, anti-βIII tubulin mAb (Promega G7121) was diluted (1:1000) in 1×PBS+2%BSA+0.3%TritonX-100, and the plate was incubated at 37°C for 3 hours. Then Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody (Life Tech catalog number A-11001) (1:2000 in 1×PBS+2%BSA+0.3%TritonX-100) was applied at 37°C for 1h. Cell nuclei were stained with DAPI. Image analysis: 6 images per well were taken with an ArrayScan XTI HCA reader (Thermo Fisher Scientific) with a 10× objective. All analyses were performed using Image J software (open source software from NIH, Maryland, USA). Three independent experiments were performed. Each independent experiment contained 6 replicates.

[0589] result

[0590] Cells were exposed to different catalytically inactive BoNT serotypes for 4 days ( Figure 1 ). Figure 1 The average neurite outgrowth of NSC34 cells exposed to three different concentrations is shown. The figure presents the average of three independent experiments. The average neurite outgrowth data confirm that rBoNT / A(0) increased the neurite length per NSC34 cell when compared to untreated controls, similar to the positive control BDNF. rBoNT / B(0), rBoNT / C(0), rBoNT / E(0), and rBoNT / F(0) were also found to increase the neurite length per NSC34 cell.

[0591] Therefore, these data demonstrate that the neurotrophic properties of BoNT / A can also be extrapolated to other BoNT serotypes.

[0592] Example 2

[0593] BoNT L-chain and LH N Increased total neurite length relative to controls

[0594] Materials and methods

[0595] Catalytically inactive botulinum toxin rBoNT / A (0) was recombinantly expressed in E. coli. Fragments of BoNT / A were also expressed in E. coli and expressed as light chain (L / A), light chain and translocation domain (LH N / A) and the cell binding domain fragment of the heavy chain (H C As in Example 1, NSC34 cells were exposed to BoNT / A fragments and full-length rBoNT / A (0).

[0596] result

[0597] Figure 2 Shown are the results of exposure to three different concentrations of rBoNT / A(0), rL / A, and rLH N / A and rH C The average neurite outgrowth of NSC34 cells was 1.447 W / m2 / dL. The figure presents the average of three independent experiments.

[0598] With rH C When compared to untreated controls, rL / A and rLH N rL / A increased neurite length per NSC34 cell at every concentration, similar to the positive control BDNF. N The / A fragments are neurotrophic because both lack the Clostridial toxin receptor binding domain (present in rH C / A).

[0599] Example 3

[0600] Other proteins administered at concentrations similar to BoNT / A(0) or its fragments did not increase neurite outgrowth material and method

[0601] NSC34 cells were differentiated and then cultured for 4 days under the following experimental conditions: (1) Untreated cell control: cells were subjected to the same number of manipulations as compound treated cells, i.e., washing / feeding, however, untreated control cells were only exposed to growth medium, (2) BDNF-positive assay control, 1 ng / ml, (3) 3 doses (0.1, 1 and 10 nM) of BoNT / A(0), (4) Negative assay controls (protein controls): 1. A7030, Sigma, bovine serum albumin (BSA), 2. NBP1-37082, Bio-techne, recombinant human annexin A4 protein, 3. U-100AT, Bio-techne, recombinant plant ubiquitin protein, 4. E. coli expression lysate, which does not contain botulinum neurotoxin or its fragments. All negative control proteins were tested at a final concentration of 1.5 μg / ml. This concentration corresponds to 10 nM of BoNT / A(0). Protein solutions were in PBS, except for annexin 4-20 mM Tris-HCl buffer (pH 8.0) containing 20% ​​glycerol, 0.2 M NaCl. All protein solutions were 1 mg / l. Cells were stained with 1:1000 anti-βIII tubulin dilution in 1× PBS-4% BSA-0.3% Triton XI00 and secondary antibody anti-mouse AlexaFluor 488; DAPI was used as a nuclear stain. All raw images of β3-tubulin signals were processed using NeurphologyJ (an Image J Macro, NIH, Maryland, USA).

[0602] result

[0603] Cells were exposed to different experimental conditions. Figure 3 The mean neurite length in NSC34 cells is shown. The figure presents the average of three independent experiments. The data of mean neurite growth confirm that rBoNT / A(0) increases the neurite length per NSC34 cell when compared to untreated controls, similar to the positive control BDNF. In contrast, none of the other "negative control" conditions increased neurite length. This confirms that exposure to rL / A and rLH N / A (and various BoNT serotypes and rH C / A) and demonstrates that this effect is not caused by exposure of NSC34 cells to proteins or putative residual E. coli components present in botulinum toxin preparations.

[0604] Example 4

[0605] Treatment of neuronal damage in vivo

[0606] A study was designed to investigate the efficacy of catalytically inactive botulinum toxin rBoNT / A(0) in enhancing functional recovery and neural regeneration using an in vivo mouse dorsal column injury model. This model can be used to analyze the efficacy of molecules that cause local sprouting and / or long tract axon regeneration. It is well known that crush injury is a common condition in spinal cord injury, and therefore this model mimics most of the pathological changes that occur in the spinal cord after trauma (for details on the model and injury response, see Lagord et al., 2002; Molecular and Cellular Neuroscience 20:69; Esmaelli et al., 2014; Neural Regeneration Research 9:1653; Surey et al., 2014; Neuroscience 275C:62; Almutiri et al., 2018; Scientific Reports 8:10707).

[0607] Materials and methods

[0608] Spinal cord injury mouse model

[0609] Before surgery, C57 / BL mice were injected subcutaneously with buprenorphine at 1.8 ml / l. 2 The patients were anesthetized with 5% isoflurane in 4% paracentesis, and body temperature and heart rate were monitored throughout the surgery. After partial laminectomy at thoracic 8 (T8), ascending sensory, descending motor, and segmental proprioceptive axons (SPA) of the dorsal column (SDC) of the spinal cord were bilaterally compressed using 1 mm deep × 1 mm wide graduated clock forceps.

[0610] Drug administration

[0611] rBoNT / A(0) was administered by a single intrathecal 10 μl injection (into the CSF of the spinal canal) at the time of surgery at one of 3 doses (100 pg, 100 ng, and 50 μg / mouse). Treatment groups for each of the 3 doses were as follows:

[0612] 1. Vehicle (phosphate-buffered saline [PBS]), SDC injury plus an immediate single 10 μl intrathecal injection of vehicle; n = 6 mice.

[0613] 2. BoNT-treated, ie, SDC injury plus immediate single 10 μl intrathecal injection of one of three doses of BoNT (100 pg, 100 ng, and 50 μg / mouse); 3×n=6 / group; 18 mice.

[0614] Intrathecal injection of BoNT was performed as follows. Mice were placed in the prone position and injections were performed between the L5 and S1 vertebrae. The spinous processes were dissected and reflected rostrally to visualize the ligamentum flavum, and a 25G blunt needle was inserted into the ligamentum flavum at a 60° horizontal angle, and entry into the intrathecal space was confirmed by the presence of reflux of cerebrospinal fluid (CSF) and a "tail flick". The 10 μl injection was then slowly injected over 1 min, and CSF expression was promoted by gentle tail elevation.

[0615] End point of measurement

[0616] 1. Motor function was measured using the horizontal ladder walking test at baseline (before injury) and then again at 2d, 1w, 2w, 3w, and 4w after SDC injury.

[0617] 2. Qualitative histological assessment of sprouting and regeneration from motor and sensory neurons / axons at the 4W time point, i.e., axonal growth of short (<1mm) and long (~5mm) distances. Tissue sections stained for neurofilament 200 (NF200) detected mature axons. Phosphorylated MAP1b is present in growing axons and growth cones, where it maintains a dynamic balance between cytoskeletal components and regulates the stability and interaction of microtubules and actin to promote axon growth, neural connections and regeneration in the central nervous system. MAP1b staining reveals areas of active axon sprouting.

[0618] Horizontal ladder test

[0619] This tests motor function and is performed on a 0.6 meter long horizontal ladder with a width of 8 cm and a randomly adjusted rung with a variable gap of 1-2 cm. Before injury, mice were evaluated again at 2d, 1w, 2w, 3w and 4w after SDC injury to cross the ladder, and left and right hind paw sliding and total number of steps were recorded by individuals who did not know the treatment group. To calculate the average error rate, the number of slides was divided by the total number of steps.

[0620] Tissue preparation and cryosectioning

[0621] At 4 weeks after SDC injury, mice were perfused intracardially with 4% formaldehyde (Raymond A Lamb, Peterborough, UK), and dissected segments of the T8 cord containing the DC injury site (injury site + 5 mm on either side) together with the tibial cranial muscle were postfixed for 2 hr at room temperature, cryoprotected in a graded series of sucrose, blocked in optimal cutting temperature medium (OCT; Raymond A Lamb), and sectioned at 15 μm thickness using a Bright cryostat.

[0622] Immunohistochemistry

[0623] The sections were thawed at room temperature for 30 min and then washed twice in 0.1 M phosphate buffered saline, pH 7.4 (PBS, Raymond ALamb). The sections were then permeabilized in 0.1% Triton X-100 in PBS (Sigma) for 10 min and blocked in PBS containing 0.5% bovine serum albumin (BSA) and 0.1% Triton-X100 (both from Sigma) for 30 min at room temperature. The sections were then incubated with appropriate primary antibodies diluted in antibody dilution buffer (ADB, PBS containing 0.5% BSA and 0.05% Tween-20 (both from Sigma)) and incubated overnight at 4°C in a humidified chamber. The sections were then washed in PBS and incubated with appropriate fluorescently labeled secondary antibodies diluted in ADB. The sections were then washed in PBS and coverslips were mounted using Vectashield (Vector Laboratories, Peterborough, UK) containing DAPI. Negative controls were included in each round of treatment, in which the primary antibody was omitted, and these were used to set the background threshold level for image capture. Sections were viewed and images captured using an Axioplan 2 epifluorescence microscope equipped with an Axiocam HRc running Axiovision software.

[0624] The primary antibodies used were as follows:

[0625] Rabbit anti-NF200 Sigma, Poole, UK (1:300 dilution)

[0626] Rabbit MAP1b Abcam, Cambridge, UK (1:400 dilution)

[0627] The secondary antibodies used were as follows:

[0628] Alexa 488 anti-rabbit IgG Invitrogen, Paisley, UK (1:400 dilution)

[0629] Alexa 594 anti-rabbit IgG Invitrogen, Paisley, UK (1:400 dilution)

[0630] statistics

[0631] Functional data were statistically analyzed using SPSS 20 (IBM, USA). A normal distribution test was performed to determine the most appropriate statistical analysis to compare treatments. Statistical significance was determined at p < 0.05.

[0632] result

[0633] Figure 4 It was shown that administration of rBoNT / A(0) reduced the extent of dorsal column injury-induced motor deficits at day 2 when compared to vehicle control for both the 100 pg and 100 ng doses. Administration of rBoNT / A(0) significantly reduced dorsal column injury-induced motor deficits and the rate of recovery at 4 weeks when compared to vehicle control at all doses tested. Furthermore, the effect was more pronounced when rBoNT / A(0) was administered intrathecally than when administered intraspinal (data not shown).

[0634] Immunohistochemical evaluation used antibodies against neurofilament 200 (NF200), which is expressed in mature axons, and MAP1b, which revealed neurofilaments in the terminals of actively sprouting axons, indicating that axons were still actively sprouting around and within the lesion site.

[0635] Figure 5 A shows that many NF200-stained axons are visible around the lesion site in vehicle-treated animals, and few, if any, NF200+ axons are present within the lesion core of untreated animals. In contrast, many NF200-stained axons are visible around the lesion site in rBoNT / A(0)-treated animals, and many NF200+ axons are also visible within the lesion core.

[0636] Figure 5 B shows a modest number of MAP1b-stained sprouting axons visible around the lesion in vehicle-treated animals, with few, if any, MAP1b axons present within the core of the lesion. In contrast, MAP1b staining revealed flower-like axon sprouting around the lesion in rBoNT / A(0)-treated animals, and also branching throughout the core of the lesion.

[0637] The rapid onset of improved performance in functional tests suggests that rBoNT / A(0) induces axonal sprouting, establishing useful functional synapses beneath the lesion. Qualitative immunohistochemistry provided evidence for localized sprouting of BoNT-induced flower-like axons through the SDC lesion site.

[0638] These in vivo data are clear evidence for the role of rBoNT / A(0) in the treatment of neurological disorders.

[0639] Example 5

[0640] Effects of full-length catalytically inactive recombinant BoNT, BoNT fragments & variants on the number of neurites per cell

[0641] A number of full-length catalytically inactive recombinant BoNT serotypes as well as BoNT fragments and variants were tested for their modulation of neurite outgrowth in vitro.

[0642] Materials and methods

[0643] Cells exposed to the polypeptides were compared to those exposed to a positive control (1 ng / ml BDNF).Mouse motor neuron-like heterozygous (NSC34) cells were differentiated and exposed to 3 different doses (0.1 nM, 1 nM and 10 nM) of different polypeptides for 4 days in vitro (DIV).

[0644] NSC34 cells were generated by fusing embryonic mouse spinal cord cells rich in motor neurons and mouse neuroblastoma (Cashman et al., Dev Dyn. 1992 July; 194(3): 209-21, which is incorporated herein by reference). The cells mimic many properties of motor neurons, including choline acetyltransferase, acetylcholine synthesis, storage and release, and neurofilament triad proteins. In addition, NSC34 spinal motor neurons express glutamate receptor proteins and generate action potentials. NSC34 neurons have been widely used to study the mechanisms of neuronal signaling and neuronal degeneration.

[0645] The following experimental protocol was used: Screening on a neuronal cell line (NSC34):

[0646]

[0647] NSC34 cells were cultured on poly-D-lysine coated glass coverslips in DMEM plus 10% FCS.

[0648] After coating, cells were differentiated into motor neurons by exposure to retinoic acid and low serum levels for 4 days. Cells were cultured in the presence / absence of polypeptides at specific time points (i.e., 4 DIV). Test data were compared with the effects observed in positive (BDNF) and negative (BSA) control data.

[0649] After 4 days in vitro (DIV), cells were fixed in 4% paraformaldehyde, stained with specific neuronal markers (β-tubulin), and neurite outgrowth (neurite extension, axon elongation, branching) was quantified. Images were acquired using an Operetta CLS HCS microscope (PerkinElmer) through a 20× objective. Six (6) fields of view were acquired for each well. Neurite outgrowth analysis was performed and the average neurite per cell was assessed.

[0650] result

[0651] Figure 6-10 The mean values ​​of the number of neurites counted per cell evaluated in three independent experimental sessions are shown. The data were normalized to untreated control cells. The polypeptides statistically significantly increased the number of neurites per cell when compared to BSA.

[0652] For BoNT / A, LH N / A fragment (light chain plus translocation domain) and cell binding domain (H C domain) fragments have improved activity compared to (see Figure 6 ).

[0653] For BoNT / FA and BoNT / F, C Compared with the domain fragments, LH N and LC (light chain only) fragments showed improved activity (see Figure 7 and 8 ).

[0654] Finally, variant H C The domain fragments all showed high efficacy ( Fig. 9 and 10 ), where the cation H C / A domain (SEQ ID NO:42- Fig. 9 ) showed excellent activity, which was improved relative to BDNF at 2 of the 3 concentrations. C The high activity of the α / β-domain is also evident in the full-length polypeptide comprising the domain (whether catalytically inactive or catalytically active).

[0655] All publications mentioned in the above specification are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the method and system of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the modes for implementing the present invention that are apparent to those skilled in the art of biochemistry and biotechnology or related fields are intended to fall within the scope of the appended claims.

[0656] Terms

[0657] 1. A polypeptide for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises:

[0658] Clostridial neurotoxin light chain (L-chain) or fragments thereof; and / or

[0659] Fragments of the heavy chain (H-chain) of the Clostridial neurotoxin.

[0660] 2. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises:

[0661] Clostridial neurotoxin L-chain or fragments thereof; and / or

[0662] Fragments of the H-chain of Clostridial neurotoxins.

[0663] 3. Use of a polypeptide in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises:

[0664] Clostridial neurotoxin L-chain or fragments thereof; and / or

[0665] Fragments of the H-chain of Clostridial neurotoxins.

[0666] 4. The polypeptide for use according to item 1, the method according to item 2 or the use according to item 3, wherein the L-chain is catalytically inactive.

[0667] 5. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide consists essentially of a Clostridial neurotoxin light chain (L-chain) or a fragment thereof; and / or a fragment of a Clostridial neurotoxin heavy chain (H-chain).

[0668] 6. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide consists of a Clostridial neurotoxin light chain (L-chain) or a fragment thereof; and / or a fragment of a Clostridial neurotoxin heavy chain (H-chain).

[0669] 7. A polypeptide for use, a method or a use according to any preceding clause, wherein the fragment of the clostridial neurotoxin H-chain comprises: a translocation domain (H N ) or a fragment thereof; or a Clostridial neurotoxin receptor binding domain (H C ) or a fragment thereof.

[0670] 8. A polypeptide for use, a method or a use according to any preceding clause, wherein the fragment of the H-chain of the clostridial neurotoxin comprises H N domain or fragments thereof.

[0671] 9. A polypeptide for use, a method or a use according to any preceding clause, wherein the fragment of the H-chain of the clostridial neurotoxin consists of H N domain or its fragments.

[0672] 10. A polypeptide for use, a method or a use according to any preceding clause, wherein the fragment of the H-chain of the clostridial neurotoxin comprises H C domain or fragments thereof.

[0673] 11. A polypeptide for use, a method or a use according to any preceding clause, wherein the fragment of the H-chain of the clostridial neurotoxin consists of C domain or its fragments.

[0674] 12. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide lacks a Clostridial neurotoxin receptor binding domain (H CC )'s C-terminal portion.

[0675] 13. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide does not comprise Clostridial neurotoxin H N Domain and H C Domains both.

[0676] 14. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide does not further comprise a non-Clostridial catalytic domain.

[0677] 15. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide comprises: a Clostridial neurotoxin L-chain or a fragment thereof, and H N domain or fragments thereof.

[0678] 16. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide consists of: a Clostridial neurotoxin L-chain or a fragment thereof, and H N domain or fragments thereof.

[0679] 17. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide consists of: a Clostridial neurotoxin L-chain and an H N Structural domain.

[0680] 18. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide:

[0681] a. encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 or 49; or

[0682] b. comprising (preferably consisting of) a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48 or 50.

[0683] 19. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide:

[0684] a. encoded by a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 or 49; or

[0685] b. comprising (preferably consisting of) a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NO: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48 or 50.

[0686] 20. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide:

[0687] a. encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 or 49; or

[0688] b. comprising (preferably consisting of) a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NO: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48 or 50.

[0689] 21. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide:

[0690] a. encoded by a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 or 49; or

[0691] b. comprising (preferably consisting of) a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NO: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48 or 50.

[0692] 22. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide:

[0693] a. encoded by a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NO: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 or 49; or

[0694] b. comprising (preferably consisting of) a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NO: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48 or 50.

[0695] 23. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide:

[0696] a. encoded by a nucleotide sequence having at least 99.9% sequence identity to any one of SEQ ID NO: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 or 49; or

[0697] b. comprising (preferably consisting of) a polypeptide sequence having at least 99.9% sequence identity to any one of SEQ ID NO: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48 or 50.

[0698] 24. A polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a catalytically inactive Clostridial neurotoxin L-chain.

[0699] 25. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a catalytically inactive Clostridial neurotoxin L-chain.

[0700] 26. Use of a polypeptide comprising a catalytically inactive Clostridial neurotoxin L-chain in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject.

[0701] 27. A polypeptide for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:41.

[0702] 28. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:41.

[0703] 29. Use of a polypeptide in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:42 and / or wherein the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:41.

[0704] 30. A polypeptide for use, method or use according to any one of clauses 27 to 29, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 42 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 41.

[0705] 31. A polypeptide for use, method or use according to any one of clauses 27 to 30, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 42 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 41.

[0706] 32. A polypeptide for use, method or use according to any one of clauses 27 to 31, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 42 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 41.

[0707] 33. A polypeptide for use, method or use according to any one of clauses 27 to 32, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 42 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 41.

[0708] 34. A polypeptide for use, method or use according to any one of clauses 27 to 33, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 99.9% sequence identity to SEQ ID NO: 42 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 99.9% sequence identity to SEQ ID NO: 41.

[0709] 35. A polypeptide for use, method or use according to any one of clauses 27 to 34, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 61 or 65 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 60.

[0710] 36. A polypeptide for use, method or use according to any one of clauses 27 to 35, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 61 or 65 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60.

[0711] 37. A polypeptide for use, method or use according to any one of clauses 27 to 36, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 61 or 65 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 60.

[0712] 38. A polypeptide for use, method or use according to any one of clauses 27 to 37, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 61 or 65 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 60.

[0713] 39. A polypeptide for use, method or use according to any one of clauses 27 to 38, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 61 or 65 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 60.

[0714] 40. A polypeptide for use, method or use according to any one of clauses 27 to 39, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 99.9% sequence identity to SEQ ID NO: 61 or 65 and / or wherein the polypeptide is encoded by a nucleotide sequence having at least 99.9% sequence identity to SEQ ID NO: 60.

[0715] 41. A polypeptide for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0716] 42. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering to the subject a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0717] 43. Use of a polypeptide in the preparation of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 63 or 64.

[0718] 44. A polypeptide for use, method or use according to any one of clauses 41 to 43, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 63 or 64.

[0719] 45. A polypeptide for use, method or use according to any one of clauses 41 to 44, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 63 or 64.

[0720] 46. ​​A polypeptide for use, method or use according to any one of clauses 41 to 45, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 63 or 64.

[0721] 47. A polypeptide for use, method or use according to any one of clauses 41 to 46, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 63 or 64.

[0722] 48. A polypeptide for use, method or use according to any one of clauses 41 to 47, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence having at least 99.9% sequence identity to SEQ ID NO: 63 or 64.

[0723] 49. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide does not comprise a native Clostridial neurotoxin H-chain.

[0724] 50. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide is neurotrophic.

[0725] 51. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide promotes neuronal growth and / or neuronal repair.

[0726] 52. A polypeptide for use, method or use according to any preceding clause, wherein the neurological disorder is a disorder treatable by promoting neuronal growth and / or repair.

[0727] 53. A polypeptide for use, method or use according to any preceding clause, wherein the neurological disorder is neuronal damage, a neurodegenerative disorder, a sensory disorder or an autonomic disorder.

[0728] 54. A polypeptide for use, method or use according to any preceding clause, wherein the neurological disorder is neuronal damage selected from the group consisting of neurotrauma (e.g. caused by scarring and / or by bone fractures), neuropathy (e.g. peripheral neuropathy), spinal cord injury (e.g. including paralysis), nerve severance, brain injury (e.g. traumatic brain injury), non-traumatic injury (e.g. stroke or spinal cord infarction) and brachial plexus injury, e.g. Erb's palsy or Klumpke's palsy.

[0729] 55. A polypeptide for use, method or use according to any preceding clause, wherein the neurological disorder is a neurodegenerative disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, Parkinson's disease-related disorders, motor neurone disease, peripheral neuropathy, motor neuropathy, prion disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, monomelic muscular atrophy, Friedreich's ataxia, Hallervorden-Spatz disease or frontotemporal lobar degeneration.

[0730] 56. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide promotes the growth or repair of motor neurons.

[0731] 57. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide is a modified Clostridial neurotoxin, such as a chimeric Clostridial neurotoxin or a hybrid Clostridial neurotoxin.

[0732] 58. A polypeptide for use, method or use according to any one of clauses 24 to 34 or 49 to 57, wherein the polypeptide is catalytically inactive and:

[0733] a. encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 60; or

[0734] b. comprising (preferably consisting of) a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0735] 59. A polypeptide for use, method or use according to any one of clauses 24 to 34 or 49 to 58, wherein the polypeptide is catalytically inactive and:

[0736] a. encoded by a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 60; or

[0737] b. comprising (preferably consisting of) a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0738] 60. A polypeptide for use, method or use according to any one of clauses 24 to 34 or 49 to 59, wherein the polypeptide is catalytically inactive and:

[0739] a. encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 60; or

[0740] b. comprising (preferably consisting of) a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0741] 61. A polypeptide for use, method or use according to any one of clauses 24-34 or 49-60, wherein the polypeptide is catalytically inactive and:

[0742] a. encoded by a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 60; or

[0743] b. comprising (preferably consisting of) a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0744] 62. A polypeptide for use, method or use according to any one of clauses 24 to 34 or 49 to 61, wherein the polypeptide is catalytically inactive and:

[0745] a. encoded by a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 60; or

[0746] b. comprising (preferably consisting of) a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0747] 63. A polypeptide for use, method or use according to any one of clauses 24 to 34 or 49 to 58, wherein the polypeptide is catalytically inactive and:

[0748] a. encoded by a nucleotide sequence having at least 99.9% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 60; or

[0749] b. comprising (preferably consisting of) a polypeptide sequence having at least 99.9% sequence identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.

[0750] 64. A polypeptide for use, method or use according to any one of clauses 24-26 or 49-63, wherein the polypeptide:

[0751] a. encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25 or 33; or

[0752] b. comprising (preferably consisting of) a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65.

[0753] 65. A polypeptide for use, method or use according to any one of clauses 24-26 or 49-64, wherein the polypeptide:

[0754] a. Encoded by a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25 or 33; or

[0755] b. comprising (preferably consisting of) a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65.

[0756] 66. A polypeptide for use, method or use according to any one of clauses 24-26 or 49-65, wherein the polypeptide:

[0757] a. encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25 or 33; or

[0758] b. comprising (preferably consisting of) a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65.

[0759] 67. A polypeptide for use, method or use according to any one of clauses 24-26 or 49-66, wherein the polypeptide:

[0760] a. Encoded by a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25 or 33; or

[0761] b. comprising (preferably consisting of) a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65.

[0762] 68. A polypeptide for use, method or use according to any one of clauses 24-26 or 49-67, wherein the polypeptide:

[0763] a. Encoded by a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25 or 33; or

[0764] b. comprising (preferably consisting of) a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65.

[0765] 69. A polypeptide for use, method or use according to any one of clauses 24-26 or 49-68, wherein the polypeptide:

[0766] a. Encoded by a nucleotide sequence having at least 99.9% sequence identity to any one of SEQ ID NO: 1, 9, 11, 13, 15, 17, 25 or 33; or

[0767] b. comprising (preferably consisting of) a polypeptide sequence having at least 99.9% sequence identity to any one of SEQ ID NO: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65.

[0768] 70. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide is administered at or near the site of injury, preferably wherein the polypeptide is administered intrathecally.

[0769] 71. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide does not further comprise a domain that binds to a cellular receptor.

[0770] 72. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide lacks the functional H of a clostridial neurotoxin C domain, and also lacks any functionally equivalent exogenous ligand targeting moiety (TM).

[0771] 73. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide is not expressed in cells of the subject.

[0772] 74. A polypeptide for use, method or use according to any preceding clause, wherein the clostridial sequence of the polypeptide consists of a clostridial neurotoxin light chain (L-chain) or a fragment thereof; and / or a fragment of a clostridial neurotoxin heavy chain (H-chain).

[0773] 75. A polypeptide for use, method or use according to any preceding clause, wherein the polypeptide further comprises one or more non-Clostridial neurotoxin sequences.

[0774] 76. A polypeptide for use, method or use according to clause 75, wherein the one or more non-Clostridial neurotoxin sequences do not bind to a cellular receptor.

[0775] 77. A polypeptide for use, method or use according to clause 75 or 76, wherein the one or more non-Clostridial neurotoxin sequences do not comprise a ligand for a cellular receptor.

[0776] 78. A polypeptide for use, method or use according to any of clauses 1-40 or 49-77, wherein the polypeptide is a modified BoNT / A or fragment thereof comprising a modification at one or more amino acid residues selected from the group consisting of ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY1215, ASN 1216, GLN1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from:

[0777] i. replacing surface exposed acidic amino acid residues with basic amino acid residues;

[0778] ii. replacing surface exposed acidic amino acid residues with uncharged amino acid residues;

[0779] iii. replacing uncharged surface-exposed amino acid residues with basic amino acid residues;

[0780] iv. inserting a basic amino acid residue; and

[0781] v. Deletion of surface exposed acidic amino acid residues.

[0782] 79. A polypeptide for use, method or use according to any one of clauses 1-26 or 41-77, wherein the polypeptide is a polypeptide comprising a BoNT / A light chain and a translocation domain and a BoNT / B receptor binding domain (H C domain).

Claims

1. Use of a polypeptide in the preparation of a medicament for treating a neurological disorder in a subject, wherein the neurological disorder is neuronal damage, and wherein the polypeptide consists of the following components: (a) a botulinum neurotoxin serotype A (BoNT / A), a botulinum neurotoxin serotype B (BoNT / B), a botulinum neurotoxin serotype C (BoNT / C), a botulinum neurotoxin serotype E (BoNT / E), a botulinum neurotoxin serotype F (BoNT / F), or a botulinum neurotoxin serotype FA (BoNT / FA) light chain (L-chain), wherein the L-chain is catalytically inactive; or (b) a BoNT / A, BoNT / B, BoNT / C, BoNT / E, BoNT / F or BoNT / FA L-chain, wherein the L-chain is catalytically inactive, and a BoNT / A, BoNT / B, BoNT / C, BoNT / E, BoNT / F or BoNT / FA translocation domain (HN domain).

2. The use according to claim 1, wherein the polypeptide consists of a botulinum neurotoxin L-chain, wherein the botulinum neurotoxin L-chain is catalytically inactive.

3. The use according to claim 1, wherein the polypeptide consists of: Botulinum neurotoxin L-chain, and H N domain, wherein the botulinum neurotoxin L-chain is catalytically inactive.

4. The use according to claim 1, wherein the polypeptide is neurotrophic.

5. The method of claim 1, wherein the neuronal injury is selected from the group consisting of neurotrauma, neuropathy, spinal cord injury, nerve transection, brain injury, non-traumatic injury, and brachial plexus injury. The use according to claim 1 , wherein the polypeptide promotes the growth or repair of motor neurons.

7. The use according to claim 1, wherein the polypeptide is a modified botulinum neurotoxin.

8. The use according to claim 7, wherein the modified botulinum neurotoxin is a chimeric botulinum neurotoxin or a hybrid botulinum neurotoxin.

9. The use according to claim 1, wherein the polypeptide is administered at or near the site of injury.

10. The use according to claim 1, wherein the polypeptide is administered intrathecally.

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