Use of clostridial neurotoxin variant for treating neurological disorders

Abstract

To provide safer methods for treating neurological disorders.SOLUTION: The present invention is directed to a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, where 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). Additional polypeptides for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient are also provided, as are corresponding methods and uses.SELECTED DRAWING: Figure 9

Description

[Technical field]

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

[0002] Neurological disorders include neuronal injury, neurodegenerative diseases, sensory disorders, and autonomic disorders.

[0003] Neuronal injuries, such as spinal cord injury (SCI), induce degeneration of damaged axons, impairing normal sensory, motor, and autonomic functions. Recovery can proceed through intrinsic mechanisms, such as regeneration of damaged axons and collateral sprouting of undamaged axons, resulting in regeneration of the paralyzed target area. However, the regenerative capacity of damaged neurons, especially in the spinal cord, is limited in adult mammals, and patients can suffer from a variety of physical disabilities that significantly affect their quality of life.

[0004] Conventional treatments for neuronal injury include interleukin-6 (IL-6) and stem cell transplantation, but few are in development for use in the outpatient clinic. Thus, there remains a need for therapies for neuronal injury that can promote neuronal growth or repair.

[0005] Bacteria of the genus Clostridia produce highly potent and unique protein toxins that are capable of poisoning neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and those produced by C. botulinum serotypes A to G and X (BoNT) (see WO 2018 / 009903 A2), as well as those produced by C. baratii and C. butyricum.

[0006] Clostridial neurotoxins are some of the most potent toxins known. For example, botulinum neurotoxins have median lethal doses (LD50s) for mice ranging from 0.5 to 5 ng / kg depending on the serotype. 50) value. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Botulinum toxins act at the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, while tetanus toxin acts within the central nervous system.

[0007] Originally, clostridial neurotoxins are synthesized as single-chain polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often called the activation site, located between cysteine ​​residues that result in the interchain disulfide bond. It is this two-chain form that is the active form of the toxin. The two chains are designated heavy chains (H chains), with a molecular weight of approximately 100 kDa, and light chains (L chains), with a molecular weight of approximately 50 kDa. The H chain is connected to an N-terminal translocation component (H N domain) and the C-terminal targeting component (H C The cleavage site is located between the L chain and the translocation domain components. C Following binding of the domain to its target neuron and uptake of the bound toxin into the cell via endosomes, H N The domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0008] Non-cytotoxic proteases act by proteolytically cleaving intercellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin). The acronym SNARE is derived from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intercellular vesicle fusion and therefore secretion of molecules from cells via vesicular transport. Protease function is a zinc-dependent endopeptidase activity that shows high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cells, non-cytotoxic proteases are capable of inhibiting cellular secretion from the target cells. L-chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

[0009] In view of the ubiquitous nature of SNARE proteins, clostridial neurotoxins, such as botulinum toxins, have been successfully employed in a wide range of therapeutic treatments.

[0010] WO 2016 / 170,501 A1 discloses a method for the treatment of paralysis due to spinal cord injury (L chain and H chain). N and H CWO 2016 / 170501 A1 describes the use of catalytically active full-length BoNT / A (including the heavy chain containing the cytotoxic domain). WO 2016 / 170501 A1 teaches that each of the functional domains of BoNT / A is essential for the observed therapeutic effect, including the binding and translocation capabilities of the heavy chain and the non-cytotoxic proteolytic activity of the light chain. As noted above, full-length clostridial neurotoxins are extremely potent, necessitating the adoption of specific safety procedures when working with the toxins. Furthermore, the spread of the toxin away from the target tissue is believed to be the cause of unwanted side effects that may be life-threatening in extreme cases. This may be a particular concern when using high doses, high concentrations, and high injection volumes of clostridial neurotoxin therapies (such as BoNT therapies). Reported adverse effects associated with this problem for public BoNT / A therapies include asthenia, generalized muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and dyspnea. Difficulties in swallowing and breathing can be life-threatening, and deaths have been reported related to the spread of the toxin's effects.Therefore, safer therapies to promote neuronal growth or repair are needed.

[0011] Given their size, the use of full-length Clostridial neurotoxins (~150 kDa) or their complete H chains (~100 kDa) entails an increased risk of eliciting an immune response in patients being treated with said polypeptides. Moreover, the use of the entire H chain (and in particular the H C The presence of the .alpha.-domain) results in the polypeptide binding to the target receptor of the clostridial neurotoxin, which may be accompanied by unwanted off-target effects in the patient to whom the polypeptide is administered. Summary of the Invention

[0012] The present invention overcomes one or more of the problems set forth above.

[0013] The present inventors have discovered that fragments of the Clostridial neurotoxin L chain and / or the Clostridial neurotoxin H chain (e.g., the translocation domain (H N) or receptor binding domain (H C We have surprisingly found that polypeptides comprising the heavy chain (H-chain) and the full-length clostridial neurotoxin (L-C) promote neuronal growth or repair, and therefore find utility in the treatment of neurological disorders. Advantageously, this allows the use of non-toxic (or substantially non-toxic) fragments of clostridial neurotoxins, which, given their smaller size (compared to full-length heavy chains or full-length clostridial neurotoxins), are less likely to cause an immune response in patients administered said fragments. Furthermore, non-toxic (or substantially non-toxic) fragments are less expensive and / or less complex to manufacture than full-length clostridial neurotoxins. In addition, non-toxic (or substantially non-toxic) fragments constitute more clearly defined therapeutic approaches than full-length clostridial toxins, and, given the shorter length of the polypeptide, for example, cysteine ​​shuffling between domains is less likely.

[0014] Thus, in one aspect, the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the 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).

[0015] In a related embodiment, a method is provided for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the method comprising administering to the patient a polypeptide, the polypeptide comprising a Clostridial neurotoxin L chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin H chain.

[0016] In another embodiment, there is provided a use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the polypeptide comprising a Clostridial neurotoxin L chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin H chain.

[0017] In one aspect, the invention provides a polypeptide for use in treating a neurological disorder in a patient, the 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).

[0018] In a related embodiment, a method for treating a neurological disorder in a patient is provided, the method comprising administering to the patient a polypeptide, the polypeptide comprising a Clostridial neurotoxin L chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin H chain.

[0019] In another embodiment, there is provided a use of a polypeptide in the manufacture of a medicament for treating a neurological disorder in a patient, said polypeptide comprising a Clostridial neurotoxin L chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin H chain.

[0020] In one embodiment, a polypeptide of the invention comprises a Clostridial neurotoxin L chain. Preferably, the L chain is catalytically inactive.

[0021] Thus, in one aspect, the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain.

[0022] In a related aspect, the invention provides a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the method comprising administering to the patient a polypeptide, the polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain.

[0023] In another related aspect, the invention provides the use of a polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient.

[0024] In one aspect, the invention provides a polypeptide for use in treating a neurological disorder in a patient, the polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain.

[0025] In a related embodiment, the invention provides a method for treating a neurological disorder in a patient, the method comprising administering to the patient a polypeptide, the polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain.

[0026] In another related aspect, the invention provides the use of a polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain in the manufacture of a medicament for the treatment of a neurological disorder in a patient.

[0027] The present inventors have shown for the first time that the catalytic activity of a Clostridial neurotoxin L-chain is not required to promote neuronal growth or neuronal repair, thus making it possible to provide safer (less toxic) therapeutic approaches.

[0028] The active clostridial neurotoxin L chain has non-cytotoxic protease activity. In particular, the active clostridial neurotoxin L chain has endopeptidase activity and is capable of cleaving a protein of the exocytic fusion apparatus in a target cell. The protein of the exocytic fusion apparatus is preferably a SNARE protein, such as SNAP-25, synaptobrevin / VAMP, or syntaxin.

[0029] The term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L chain means that the L chain does not exhibit substantially any non-cytotoxic protease activity, and preferably, the term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L chain means that the L chain does not exhibit any non-cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin L chain does not cleave proteins of the exocytic 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, for example, less than 2%, less than 1%, or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain. Non-cytotoxic protease activity can be determined in vitro by incubating a test Clostridial neurotoxin L chain with a SNARE protein and comparing the amount of SNARE protein cleaved by a test Clostridial neurotoxin L chain when compared to the amount of SNARE protein cleaved by a catalytically active Clostridial neurotoxin L chain under the same conditions. Routine techniques such as SDS-PAGE and Western blotting can also be used to quantify the amount of cleaved SNARE protein. Suitable in vitro tests are described in WO 2019 / 145,577 A1, which is incorporated herein by reference.

[0030] 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 translocation domain has non-cytotoxic protease activity. Assays such as the digit abduction score (DAS), dorsal root ganglion (DRG) assay, spinal cord neuron (SCN) assay, and mouse phrenic nerve hemidiaphragm (PNHD) assay are routine in the art. Suitable assays for determining non-cytotoxic protease activity may be those described in Pharmacol Res Perspect, e00446 1-14 (2018) by Donald et al., incorporated herein by reference.

[0031] A catalytically inactive L chain may have one or more mutations that inactivate the catalytic activity. For example, a catalytically inactive BoNT / AL chain may include mutations of active site residues such as His223, Glu224, His227, Glu262, and / or Tyr366. The numbering of the positions corresponds to the amino acid positions of SEQ ID NO:62 and can be determined by aligning the polypeptide to SEQ ID NO:62. Since the presence of a methionine residue at position 1 of SEQ ID NO:62 is optional, those skilled in the art will take into account the presence / absence of the methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO:62 contains a methionine, the numbering of the positions will be as defined above (e.g., His223 becomes His223 in SEQ ID NO:62). Alternatively, if a methionine is absent in SEQ ID NO:62, the numbering of the amino acid residues should be amended by -1 (e.g., His223 becomes His222 in SEQ ID NO:62). Similar considerations apply to the presence / absence of a 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.

[0032] In a particularly preferred embodiment, the polypeptide of the present invention is a modified BoNT / A or a fragment thereof (preferably, a BoNT / AH or a fragment thereof). CThe modified BoNT / A or fragment thereof may include a modification at one or more amino acid residues selected from ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277. This modified BoNT / A or fragment thereof can demonstrate reduced or absent side effects compared to the use of known BoNT / As. The enhanced tissue retention properties of the modified BoNT / As of the present invention can also provide increased efficacy and / or duration of activity (or increased dosage without any additional adverse effects) compared to known clostridial toxin therapies, thus providing an additional advantage.

[0033] The modifications may be modifications relative to the unmodified BoNT / A shown as SEQ ID NO:62, and the numbering of the amino acid residues is determined by alignment with SEQ ID NO:62. Because the presence of a methionine residue at position 1 of SEQ ID NO:62 (as well as each SEQ ID NO corresponding to a modified BoNT / A polypeptide or fragment thereof described herein) is optional, one of skill in the art would take into consideration the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:62 contains a methionine, the numbering of the positions would be as defined above. Alternatively, if the methionine is absent in SEQ ID NO:2, the numbering of the amino acid residues would be corrected by -1 (e.g., ASN886 would become ASN885 in SEQ ID NO:62). Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and one of skill in the art would readily determine the correct amino acid residue numbering using techniques routine in the art.

[0034] The amino acid residues indicated for modification above are surface-exposed amino acid residues.

[0035] A modified BoNT / A or fragment thereof may include a modification at one or more amino acid residues selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277.

[0036] The term "one or more amino acid residues" when used in the context of a modified BoNT / A or a fragment thereof preferably means at least 2, 3, 4, 5, 6, or 7 of the indicated amino acid residues. Thus, a modified BoNT / A may contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications at the indicated amino acid residues. A modified BoNT / A or a fragment thereof may contain 1-30, 3-20, or 5-10 amino acid modifications. More preferably, the term "one or more amino acid residues" when used in the context of a modified BoNT / A or a fragment thereof means all of the indicated amino acid residues.

[0037] Preferably, beyond one or more amino acid modifications at the indicated amino acid residues, the modified BoNT / A or fragment thereof does not contain any additional amino acid modifications when compared to SEQ ID NO:62.

[0038] The modification is i. Substitution of acidic surface-exposed amino acid residues with basic amino acid residues; ii. replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; iii. Substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue, and v. Deletion of acidic surface-exposed amino acid residues.

[0039] The modifications set forth above result in a modified BoNT / A or fragment thereof having an increased positive surface charge and an increased isoelectric point when compared to the corresponding unmodified BoNT / A or fragment thereof.

[0040] Isoelectric point (pl) is an intrinsic property of a particular protein. As is well known in the art, proteins are made up of unique sequences of amino acids (also called amino acid residues when in a protein). Each amino acid in the standard set of 20 has a different side chain (or R group), meaning that each amino acid residue in a protein exhibits different chemical properties such as charge and hydrophobicity. These properties can be influenced by the surrounding chemical environment such as temperature and pH. The overall chemical characteristics of a protein depend on the sum of these various factors.

[0041] Certain amino acid residues (described in more detail below) have ionizable side chains that may exhibit a charge depending on the pH of their surroundings. Whether or not this side chain is charged at a particular pH depends on the pKa of the associated ionizable moiety, which is the negative logarithm of the acid dissociation constant (Ka) of a particular proton from its conjugate base.

[0042] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values ​​of approximately 4.1 (the exact pKa value may depend on temperature, ionic strength, and the microenvironment of the ionizable group). Thus, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains lose their charge by adding a proton.

[0043] Conversely, basic residues such as lysine and arginine have nitrogen-containing side groups with pKa values ​​of approximately 10 to 12. Thus, these side chains are positively charged at a pH of 7.4. At higher pH values, these side chains lose a proton and lose their charge.

[0044] Thus, the total (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), as well as on the surrounding pH. Changing the surrounding pH changes the total charge of the protein. Thus, for each protein, there is a specific pH at which the number of positive and negative charges is equal and the protein exhibits no overall net charge. This point is known as the isoelectric point (pl). The isoelectric point is a standard concept in protein biochemistry with which those skilled in the art are familiar.

[0045] Thus, the isoelectric point (pl) is defined as the pH value at which a protein exhibits a zero net charge. An increase in pl means that a higher pH value is required for the protein to exhibit a zero net charge. Thus, an increase in pl represents an increase in the net positive charge of the protein at a particular pH. Conversely, a decrease in pl means that a lower pH value is required for the protein to exhibit a zero net charge. Thus, a decrease in pl represents a decrease in the net positive charge of the protein at a particular pH.

[0046] Methods for determining the pl of a protein are known in the art and will be familiar to those skilled in the art. As an example, the pl of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pl"). This calculation can be performed using computer programs known in the art, such as the Compute pl / MW Tool from ExPASy (https: / / web.expasy.org / compute_pl / ), which is a preferred method for calculating pl according to the present invention. Comparison of pl values ​​between different molecules should be performed using the same calculation techniques / programs.

[0047] Where appropriate, the calculated pl of a protein can be experimentally confirmed using the technique of isoelectric focusing ("observed pl"). This technique uses electrophoresis to separate proteins by their pl. Isoelectric focusing is typically performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH at which it has zero net charge, which becomes the pl of the protein. The results provided by isoelectric focusing are typically relatively low resolution in nature, and therefore the inventors believe that the results provided by calculated pl (as described above) are more appropriate to use.

[0048] Throughout this specification, unless otherwise stated, "pl" means "calculated pl."

[0049] The pl 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 pl can be brought about by decreasing the number of acidic residues or by increasing the number of basic residues.

[0050] A modified BoNT / A or fragment thereof of the present invention can have a pl value that is at least 0.2, 0.4, 0.5, or 1 pl unit higher than that of an unmodified BoNT / A (e.g., SEQ ID NO: 62) or fragment thereof. Preferably, the modified BoNT / A or fragment thereof can have a pl of at least 6.6, e.g., at least 6.8.

[0051] The properties of the 20 common amino acids are shown in the table below. [Table 1]

[0052] The following amino acids are considered to be charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0053] At pH 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.

[0054] The following amino acids are considered to be uncharged and polar (meaning that they can participate in hydrogen bonding): asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0055] The following amino acids are considered to be uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0056] In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into a BoNT / A polypeptide sequence or fragment thereof, thus increasing the total number of amino acid residues in said sequence. In an amino acid deletion, an amino acid residue is removed from a Clostridial toxin amino acid sequence, thus decreasing the total number of amino acid residues in said sequence.

[0057] 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 forming part of the BoNT / A polypeptide sequence or fragment thereof is replaced with a different amino acid residue. The replacement amino acid residue may be one of the 20 standard amino acids listed above. Alternatively, the replacement amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the standard set of 20). By way of example, the replacement amino acid may 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.

[0058] In one embodiment, the substitutions are selected from substitutions of acidic amino acid residues with basic amino acid residues, substitutions of acidic amino acid residues with uncharged amino acid residues, and substitutions of uncharged amino acid residues with basic amino acid residues. In one embodiment, the substitutions are substitutions of acidic amino acid residues with uncharged amino acid residues, where the acidic amino acid residues are replaced with their corresponding uncharged amide amino acid residues (i.e., aspartic acid is replaced with asparagine, glutamic acid is replaced with glutamine).

[0059] Preferably, the basic amino acid residue is a lysine or arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0060] Preferably, the modified BoNT / A or fragment thereof for use in the present invention is Clostridial toxin H CNThe modified BoNT / A or fragment thereof preferably has a pl of at least 6.6. The modified BoNT / A or fragment thereof preferably has at least four amino acid modifications selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, and ASN1052, the modifications comprising amino acid substitutions with lysine or arginine residues. For example, the modified BoNT / A or fragment thereof may have at least five amino acid modifications selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, and GLN1229, the modifications comprising amino acid substitutions with lysine or arginine residues.

[0061] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. By way of example, amino acid modifications can be introduced by modification of the DNA sequence encoding the polypeptide (e.g., encoding the unmodified BoNT / A or a fragment thereof). This can be achieved using standard molecular cloning techniques, such as by site-directed mutagenesis, where a short stretch of DNA (oligonucleotide) coding for the desired amino acid is used to replace the original coding sequence using a polymerase enzyme, or by insertion / deletion of parts of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized.

[0062] In one aspect, the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0063] In a related embodiment, a method is provided for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the method comprising administering to the patient a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0064] In a further related aspect, there is provided the use of a polypeptide in the manufacture of a medicament for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0065] In one aspect, the invention provides a polypeptide for use in treating a neurological disorder in a patient, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0066] In a related embodiment, a method for treating a neurological disorder in a patient is provided, the method comprising administering to the patient a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42, and / or the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0067] In a further related aspect, there is provided a use of a polypeptide in the manufacture of a medicament for treating a neurological disorder in a patient, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41.

[0068] In one embodiment, a polypeptide for use according to the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 42. Preferably, a polypeptide for use according to the invention comprises the polypeptide sequence shown in SEQ ID NO: 42.

[0069] In one embodiment, a polypeptide for use according to the invention comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 41. Preferably, a polypeptide for use according to the invention comprises a polypeptide sequence encoded by the nucleotide sequence shown in SEQ ID NO: 41.

[0070] In one embodiment, a polypeptide for use according to the 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 to SEQ ID NO: 61 or 65. Thus, in one embodiment, a polypeptide for use according to the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 61 or 65. Preferably, a polypeptide for use according to the invention may comprise (and 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).

[0071] In one embodiment, a polypeptide for use according to the invention (e.g. comprising SEQ ID NO: 42 or encoded by SEQ ID NO: 41) may be encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 60. Thus, in one embodiment, a polypeptide for use according to the invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 60. Preferably, a polypeptide for use according to the invention may be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 60. In one embodiment, the polypeptide comprises a catalytically inactive light chain.

[0072] SEQ ID NO: 42 is an example of a modified BoNT / A fragment, and SEQ ID NO: 61 and 65 are examples of catalytically active and inactive modified BoNT / A polypeptides, respectively. These modified BoNT / A polypeptides and fragments are particularly preferred for use according to the present invention. The polypeptides shown as SEQ ID NO: 42, 61, and 62 have a number of amino acid modifications (e.g., substitutions) compared to wild-type BoNT / A that increase the isoelectric point of the polypeptide. Without being 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, thus increasing retention and duration of action at the administration site. Thus, it is expected that the neuronal growth and / or repair properties of SEQ ID NO: 42, 61, and 65 are improved compared to comparable polypeptides without the modifications.

[0073] For the catalytically active repaired BoNT / A polypeptide (e.g., SEQ ID NO: 61), one way in which these advantageous properties (representing an increased therapeutic index) can be defined is in terms of the safety factor of the modified BoNT / A. In this regard, the unwanted effects of the clostridial neurotoxins (caused by diffusion of the toxin away from the administration site) can be experimentally evaluated by measuring the rate of weight loss in relevant animal models (e.g., mice, this weight loss was detected within 7 days of administration). Conversely, the desired on-target effects of the clostridial neurotoxins can be experimentally evaluated by the digit abduction score (DAS) test, which is a measurement of muscle paralysis. The DAS test can be performed by injection of 20 μl of the clostridial neurotoxin formulated in gelatin phosphate buffer into the gastrocnemius / soleus muscle complex of mice, followed by evaluation of the digit abduction score using the method of Aoki (Aoki KR, Toxicon ("Toxicology") 39: 1815-1820; 2001). In the DAS assay, mice are briefly suspended by the tail to elicit a specific startle response in which the mice extend their hind limbs and abduct their hind digits. Following injection of a clostridial neurotoxin, various degrees of digit abduction are scored on a 5-point scale (0 = normal to 4 = maximal reduction in digit abduction and limb extension).

[0074] The safety factor of a clostridial neurotoxin can be expressed as the ratio between the amount of toxin required for a 10% reduction in body weight (measured at peak effect within the first 7 days after administration to mice) and the amount of toxin required for a DAS score of 2. Thus, a high safety factor score is desirable, which indicates a toxin that can effectively paralyze the target muscle with few unwanted off-target effects. The catalytically active modified BoNT / A of the present invention can have a safety factor higher than that of the equivalent unmodified (native) botulinum toxin (e.g., SEQ ID NO: 62).

[0075] Thus, in one embodiment, a catalytically active modified BoNT / A of the invention has a safety factor of at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), which is the DAS ED of toxin required for ∼10% body weight change (pg / mouse). 50 (pg / mouse) [ED 50 = dosage required to produce a DAS score of 2].

[0076] In one embodiment, a catalytically active modified BoNT / A of the invention has a safety factor of at least 10. In one embodiment, a modified BoNT / A or fragment thereof of the invention has a safety factor of at least 15.

[0077] Polypeptides comprising at least 70% sequence identity to SEQ ID NO:61 are described in WO 2015 / 004,461 A1, the entirety of which is incorporated by reference herein.

[0078] 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 encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 or 60 comprises substitutions at one or more (preferably 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more, more preferably all) of positions 930, 955, 991, 1026, 1052, 1229, and 886. The numbering of the positions corresponds to the positions of SEQ ID NO: 62 and can be determined by aligning the polypeptide sequence to 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, one skilled in the art would take into account the presence / absence of a methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO:62 contains a methionine, the numbering of the positions will be as defined above (e.g., position 886 becomes ASN 885 of SEQ ID NO:62). Alternatively, if methionine is absent in SEQ ID NO:62, the numbering of the amino acid residues should be corrected by -1 (e.g., position 886 becomes ASN 885 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 be readily able to determine the correct amino acid residue numbering using techniques routine in the art.

[0079] Preferably, 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 lysine or arginine (more preferably, a lysine) at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, the polypeptide comprises a lysine or arginine (more preferably, a lysine) at at least two, three, four, five, six, or all of positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the polypeptide comprises a lysine or arginine (more preferably, a lysine) at all of positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0080] The polypeptides of the present invention promote neuronal growth and / or neuronal repair. Thus, the polypeptides find utility in treating neurological disorders. As used herein, the term "neurological disorder" refers to a disorder treatable by promoting the growth and / or repair of neurons in a patient.

[0081] Thus, in one embodiment, the invention provides a method for promoting neuronal growth and / or neuronal repair, the method comprising administering to a patient a polypeptide, the 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 embodiment, the invention provides a method for promoting neuronal growth and / or neuronal repair, the method comprising administering to a patient a polypeptide, the polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain. In another embodiment, a method for promoting neuronal growth or neuronal repair is provided, the method comprising administering to a patient a polypeptide, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, and / or the polypeptide comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41. In another embodiment, a method for promoting neuronal growth or neuronal repair is provided, the method comprising administering to a patient a polypeptide, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63.

[0082] The term "promoting neuronal growth and / or neuronal repair" can mean that the polypeptide of the present invention initiates neuronal growth and / or neuronal repair, for example, when neuronal growth and / or neuronal repair does not occur. In another embodiment, the term "promoting neuronal growth and / or neuronal repair" can mean that the polypeptide of the present invention increases the rate of neuronal growth and / or neuronal repair. The increase can be 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 neural circuits, thereby restoring activity and / or neuronal transmission in a network or population of neurons. Thus, the term "neuronal repair" as used herein can include the repair of specific neurons as well as the repair of neural circuits.

[0083] The term "neuron growth and / or neuronal repair" may also include neuroplasticity. Thus, in one embodiment, the polypeptide of the present invention promotes neuroplasticity. As used herein, the term "neuroplasticity" includes axonal sprouting, dendritic sprouting, neurogenesis (e.g., the generation of new neurons), maturation, differentiation, and / or synaptic plasticity (e.g., including changes to synaptic strength, activity, structure, and / or connection). In one embodiment, the polypeptide of the present invention promotes the establishment of functional synapses (e.g., at or near the site of injury).

[0084] Neuronal growth and / or repair may be increased by at least 10%, 20%, 30%, 40, 50%, 60%, or 70% (preferably at least 80%) in the presence of a polypeptide of the invention when compared to neuronal growth and / or repair in the absence of a polypeptide of the invention or in the presence of an alternative polypeptide. In some embodiments, neuronal growth and / or repair may be increased by at least 100%, 150%, or 200% in the presence of a polypeptide of the invention when compared to neuronal growth and / or repair in the absence of a polypeptide of the invention or in the presence of an alternative polypeptide.

[0085] 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 may increase the length of neurites, the number of neurites (e.g., the number of neurites per cell), and / or increase the length and / or number of processes from the cell body or cell membrane of a neuron. Preferably, the polypeptide of the present invention promotes axonal growth of a neuron, e.g., a neuron of a patient. In other words, preferably, the polypeptide of the present invention may increase axonal growth, e.g., axonal sprouting. The axonal growth may promote connections and / or chemical communication between neurons.

[0086] The neurological disorder treated by the polypeptides of the present invention may be a neuronal injury, a neurodegenerative disorder, a sensory disorder, or an autonomic disorder.

[0087] The neurological disorder may be neuronal injury. In one embodiment, the neuronal injury may be neurotrauma, neuropathy (e.g., peripheral neuropathy), spinal cord injury, nerve section, brain injury (e.g., traumatic brain injury), non-traumatic injury (e.g., stroke or spinal cord infarction), or injury to the brachial plexus, such as Erb's palsy or Klumpke's palsy.

[0088] In one embodiment, the nerve injury may be caused by a wound and / or a fracture. In such cases of nerve injury, nerve terminals are damaged. The polypeptide of the present invention advantageously allows the repair of said nerve terminals or distal nerve terminals, allowing the treatment of nerve injury.

[0089] The neuronal injury may be paralysis, such as paralysis caused by spinal cord injury (eg, by compression, constriction, and / or stretching). In one embodiment, the spinal cord injury is paraplegia or quadriplegia.

[0090] The neuropathy may be a sensory disorder. In one embodiment, the sensory disorder is sensory neuropathy, sensorimotor polyneuropathy, diabetic neuropathy, pain, Brown-Séquard syndrome, Charcot-Marie-Tooth disease, or Devic's syndrome. Preferably, the sensory disorder described herein is not pain. In other words, preferably, the neuropathy described herein is not pain.

[0091] The neuropathy may be an autonomic neuropathy. In one embodiment, the autonomic neuropathy is an autonomic neuropathy, multiple system atrophy, acute idiopathic polyneuropathy, autonomic failure, familial autonomic failure, diabetic autonomic failure, pure autonomic failure, thermoregulation disorder, hyperhidrosis, neuromediated syncope (vasovagal, cough, swallowing, and other situational types), erectile dysfunction, orthostatic hypertension, orthostatic tachycardia syndrome (PoTS), or Guillain-Barre syndrome.

[0092] The neurological disorder may be a neurodegenerative disorder. In one embodiment, the neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, Parkinson's disease-related disorders, motor neuron diseases, peripheral neuropathies, motor neuron neuropathy, prion diseases, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, monolimbic muscular atrophy, Friedreich's ataxia, Hallervorden-Spatz disease, or frontotemporal lobe mutations. Preferably, the neurodegenerative disorder is Parkinson's disease or a motor neuron disease. Advantageously, it is believed that the polypeptides of the invention find utility in the treatment of neurodegenerative disorders by virtue of their ability to promote neuronal growth and / or neuronal repair (including, for example, neuronal plasticity), and further by virtue of their ability to reconstruct damaged neuronal circuits, thereby restoring activity and / or neuronal communication within a network or population of neurons.

[0093] The polypeptide of the present invention may be considered to be a neurotrophic polypeptide in view of its ability to promote neuronal growth and / or neuronal repair. The neuron described herein may be one or more selected from motor neurons (including autonomic neurons), sensory neurons, spinal interneurons, and brain interneurons. Thus, in one embodiment, the polypeptide of the present invention promotes the growth and / or repair of motor neurons, sensory neurons, and / or interneurons. Preferably, the polypeptide of the present invention promotes the growth and / or repair of motor neurons.

[0094] As used herein, a "patient" may be a mammal, such as a human or other mammal. Preferably, "patient" refers to a human patient.

[0095] As used herein, the term "disorder" also encompasses "disease." In one embodiment, the disorder is a disease.

[0096] As used herein, the term "treat" or "treatment" encompasses prophylactic treatment (e.g., to prevent the onset of a disorder) as well as corrective treatment (treatment of a patient already suffering from a disorder). Preferably, as used herein, "treat" or "treatment" refers to corrective treatment.

[0097] As used herein, the term "treat" or "treatment" is directed to a disorder and / or its symptoms.

[0098] Thus, the polypeptides of the invention may be administered to a patient in a therapeutically effective amount or in a prophylactically effective amount. Preferably, the polypeptides of the invention are administered to a patient in a therapeutically effective amount.

[0099] A "therapeutically effective amount" is an amount of any of the polypeptides that, when administered alone or in combination to a patient for treating said disorder (or a symptom thereof), is sufficient to effect treatment of the disorder or a symptom thereof.

[0100] A "prophylactically effective amount" is an amount of any of the polypeptides that, when administered to a patient alone or in combination, inhibits or delays the onset or recurrence of a disorder (or symptoms thereof). In some embodiments, a prophylactically effective amount prevents the onset or recurrence of a disorder altogether. By "inhibiting" onset, it is meant either a reduction in the likelihood of onset of a disorder (or symptoms thereof) or prevention of onset altogether.

[0101] The polypeptides of the invention may be formulated in any suitable manner for administration to a patient, for example as part of a pharmaceutical composition. Thus, in one aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the invention and a pharma- ceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt. In some embodiments, the polypeptides of the invention may be in single-chain form, while in other embodiments, the polypeptides may be in two-chain form, for example, where the two chains are linked by a disulfide bridge. Preferably, the polypeptides are in two-chain form.

[0102] The polypeptides of the invention may be formulated for oral, parenteral, continuous infusion, inhalation, or topical application. Compositions suitable for injection may be in the form of a solution, suspension, or emulsion, or a dry powder that is dissolved or suspended in a suitable excipient prior to use.

[0103] In the case of locally delivered polypeptides, the polypeptides may be formulated as a cream (eg, for topical use) or for subcutaneous injection.

[0104] Localized delivery means may include an aerosol or other spray (e.g., a nebulizer). In this regard, an aerosol formulation of the polypeptide allows for delivery to the lungs and / or other nasal and / or bronchial or airway passages.

[0105] The polypeptides of the invention may be administered to the patient by intrathecal or epidural injection into the spinal column at the level of the spinal segment involved in the innervation of the affected organ.

[0106] The route of administration may be via laparoscopy or local injection. In one embodiment, the polypeptide of the present invention is administered at or near the site of injury, preferably at the site of injury. For example, if the injury is a spinal cord injury, the polypeptide may be administered intrathecally or intraspinally (preferably intrathecally). In one embodiment, the route of administration of the polypeptide of the present invention may be peripheral, intraneural, intraspinal, and / or intrathecal.

[0107] Dosage ranges for administration of the polypeptides of the present invention are those ranges to produce the desired therapeutic and / or prophylactic effect. It will be understood that the required dosage range will depend on the particular nature of the Clostridial neurotoxin or composition, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, any contraindications, and the judgment of the attending physician. Various dosage levels can be adjusted using standard empirical routines for optimization.

[0108] In one embodiment, the dosage of the polypeptide is a flat dose. The flat dose may range from 50 pg to 250 μg, preferably 100 pg to 100 μg. In one embodiment, the flat dose may be at least 50 pg, 100 pg, 500 pg, 1 ng, 50 ng, 100 ng, 500 ng, 1 μg, or 50 μg. The dosage may be a single flat dose.

[0109] Fluid dosage forms are typically prepared using the polypeptide and a sterile pyrogen-free extensor. The Clostridial neurotoxin can be either dissolved or suspended in the extensor, depending on the extensor and concentration used. In preparing the solution, the polypeptide can be dissolved in the extensor, the solution is made isotonic by adding sodium chloride if necessary, and sterilized by filtration through a sterile filter using an aseptic method before being filled into suitable sterile vials or ampoules and sealed. Alternatively, if the stability of the solution is appropriate, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics can also be dissolved in the extensor.

[0110] Dry powders, dissolved or suspended in a suitable excipient prior to use, may be prepared by filling the pre-sterilized ingredients in a sterile container using aseptic techniques in a sterile area. Alternatively, the ingredients may be dissolved in a suitable container using aseptic techniques in a sterile area. The product is then freeze-dried and the container is aseptically sealed.

[0111] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile ingredients are suspended in a sterile excipient instead of being dissolved, and sterilization cannot be achieved by filtration. The ingredients may be isolated in a sterile state or, alternatively, may be sterilized after isolation, for example by gamma irradiation.

[0112] Advantageously, a suspending agent, such as polyvinylpyrrolidone, is included in the composition to promote uniform distribution of the components.

[0113] Administration according to the present invention may utilize a variety of delivery technologies including microparticle, encapsulation, or high pressure aerosol injection.

[0114] The polypeptide of the present invention may be a Clostridial neurotoxin or a fragment thereof, preferably a fragment thereof.

[0115] In one embodiment, a polypeptide of the invention may be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 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, a polypeptide of the invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 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 polypeptides of the invention may be encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 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.

[0116] 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 NOs: 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 one of SEQ ID NOs: 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, a polypeptide of the invention may comprise the polypeptide sequence of any one of SEQ ID NOs: 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.

[0117] In one embodiment, the invention encompasses 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.

[0118] The term "Clostridial neurotoxin" includes C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified Clostridial neurotoxins or derivatives derived from any of the foregoing.

[0119] Botulinum neurotoxins (BoNTs) are produced by C. botulinum in the form of large protein complexes, consisting of BoNT itself complexed to many accessory proteins. Currently, there are eight different classifications of botulinum neurotoxins, namely botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X, all of which share a similar structure and mode of action. The different BoNT serotypes are distinguishable based on inactivation by certain neutralizing antisera, with such classification by serotype correlating with the percentage sequence identity at the amino acid level. The BoNT proteins of certain serotypes have been further divided into different subtypes based on the percentage amino acid sequence identity.

[0120] Each BoNT is absorbed in the gastrointestinal tract and enters the systemic circulation, where it binds to the presynaptic membrane of cholinergic nerve terminals and inhibits 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 syntaxin. BoNT / X has been shown to cleave SNAP-25, VANP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

[0121] Tetanus toxin is produced in a single serotype by C. tetani; C. butyricum produces BoNT / E, whereas C. baratii produces BoNT / F.

[0122] The term "Clostridial neurotoxin" is intended to encompass modified Clostridial neurotoxins and derivatives thereof, including but not limited to those described below. Modified Clostridial neurotoxins or derivatives may contain one or more amino acids that are modified compared to the native (unmodified) form of Clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of Clostridial neurotoxin. For example, modified Clostridial neurotoxins may have modified amino acid sequences in one or more domains compared to the sequence of native (unmodified) Clostridial neurotoxin. This modification may modify the functional aspects of the toxin, such as biological activity or persistence. Thus, in one embodiment, the Clostridial neurotoxin of the present invention is a modified Clostridial neurotoxin, or a modified Clostridial neurotoxin derivative, or a Clostridial neurotoxin derivative.

[0123] Modified Clostridial neurotoxins (modified H C The neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., domain), which heavy chain binds to target neurons with higher or lower affinity than native (unmodified) Clostridial neurotoxin. C This modification in the domain is H C This can include modifications of residues in the ganglioside binding site of the domain, or in the protein (SV2 or synaptotagmin) binding site and / or protein receptor of the target neuronal cell that alter binding to the ganglioside receptor. Examples of such modified Clostridial neurotoxins are described in WO 2006 / 027,207 and WO 2006 / 114,308, both of which are incorporated herein by reference in their entireties.

[0124] 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 that can 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 U.S. Patent Application Publication No. 2011 / 0,318,385, both of which are incorporated herein by reference in their entireties.

[0125] The modified clostridial neurotoxin may contain 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 may contain a leucine-based or tyrosine-based motif that increases or decreases the biological activity and / or biological persistence of the modified clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxIL, and xExxxLM, where x is any amino acid. Suitable tyrosine-based motifs include Yxx-Hy, where Hy is a hydrophobic amino acid. Examples of modified clostridial neurotoxins that contain leucine-based and tyrosine-based motifs are described in WO 2002 / 08268, which is incorporated herein by reference in its entirety.

[0126] As described above, a modified Clostridial neurotoxin (or a Clostridial neurotoxin fragment) may include one or more modifications that increase the isoelectric point of the Clostridial neurotoxin compared to an equivalent unmodified Clostridial neurotoxin lacking said one or more modifications. Suitable modified Clostridial neurotoxins are described above and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 61 and 42 described herein.

[0127] The term "clostridial neurotoxin" is intended to encompass hybrid and chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype, 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 comprise the entire light chain from one clostridial neurotoxin subtype and a heavy chain from another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin may comprise a portion of a heavy chain (e.g., a binding domain) from one clostridial neurotoxin subtype, along with another portion of a heavy chain from another clostridial neurotoxin subtype. Also, or alternatively, a therapeutic element may comprise light chain portions from different clostridial neurotoxins. The hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of the clostridial neurotoxins to patients who have immune resistance to a particular clostridial neurotoxin subtype, who may have a lower than average concentration of receptors for a particular clostridial neurotoxin heavy chain binding domain, or who may have antiprotease mutants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in U.S. Patent No. 8,071,110, the entirety of which is incorporated herein by reference. Thus, in one embodiment, the clostridial neurotoxins (or fragments thereof) of the present invention are hybrid clostridial neurotoxins or chimeric clostridial neurotoxins.

[0128] In a particularly preferred embodiment, the polypeptide of the present invention comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H CThe chimeric Clostridial neurotoxin may be a chimeric Clostridial neurotoxin comprising (preferably consisting of) the C-terminal domain (or a portion thereof). Suitable chimeric and / or hybrid Clostridial neurotoxins may be those taught in WO 2017 / 191315, which is incorporated herein by reference. The preferred sequences include SEQ ID NOs: 44, 63, and 64.

[0129] BoNT / A LH N The domain is BoNT / BH C The chimeric BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0130] LH N The C-terminal amino acid residue of the domain is the LH of BoNT / A. N Domain and H C Splitting the Domain 3 10 may correspond to the first amino acid residue of the helix, and H C The N-terminal amino acid residue of the domain is the LH of BoNT / B. N Domain and H C Splitting the Domain 3 10 It may correspond to the second amino acid residue of the helix.

[0131] As used herein, the term "BoNT / A LH N Domain and H C Splitting the Domain 3 10 The reference to "the first amino acid residue of the helix" is N Domain and H C Splitting the Domain 3 10 It refers to the N-terminal residue of the helix.

[0132] As used herein, the term "BoNT / B LH N Domain and H C Splitting the Domain 3 10 The reference to "the second amino acid residue of the helix" is N Domain and H C Splitting the Domain 3 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0133] "3 10 A "helix" is a type of secondary structure found in proteins and polypeptides, along with alpha helices, beta sheets, and reverse helices. 3 10 The amino acids in the helix are arranged in a right-handed helical conformation, where one full turn is completed by three residues and ten atoms that break intramolecular hydrogen bonds between them. Each amino acid has ten atoms in a ring formed by hydrogen bonding, corresponding to a turn of 120 degrees in the helix (i.e., the helix has three residues per turn) and a translation of 2.0 Å (=0.2 nm) along the axis of the helix. Most importantly, the NH group of the amino acid forms hydrogen bonds earlier with the C=O groups of three residues of the amino acid, and this repeated i+3→i hydrogen bond is 3 10 Define a spiral. 3 10 A helix is ​​a standard concept in structural biology that is well known to those skilled in the art.

[0134] These three 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of these four residues. N Domain and H C Splitting the Domain 3 10 The "helix" consists of these six residues.

[0135] Through performing structural analysis and sequence alignment, LH N Domain and H C Splitting the Domain 3 10 The helix was identified. 10 The helix is ​​located at its N-terminus (i.e., LH N in the C-terminal portion of the domain) and an α-helix in its C-terminus (i.e. C It is surrounded by β-strands (at the N-terminal part of the domain). 10 The first (N-terminal) residue of the helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0136] LH N Domain and H C Splitting the Domain 3 10 The helices can be determined, for example, from the publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0137] Publicly available computational modeling and alignment tools, such as the homology modeling servers LOOPP (Learning, Observing, and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ), and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and the Internet Resources for Molecular and Cell Many other tools / services, such as those listed in the Internet Resources for Molecular and Cellular Biologists (http: / / molbiol-tools.ca / ), also support the use of LH in other neurotoxins. N Domain and H C Splitting the Domain 3 10It can be used to determine the location of the helix. N / H CN The region surrounding the junction is highly structurally conserved, making it an ideal region for superimposing different serotypes.

[0138] For example, the following method is 10 Can be used to determine the sequence of the helix: 1. The structural homology modeling tool LOOP (http: / / loopp.org) was used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb); 2. The structure (pdb) file obtained in this way is CN The N-terminal end of the domain and (H N The protein was then edited to include only the previous 80 residues (part of the H domain), which is structurally highly conserved. N / H CN " held territory; 3. The protein superposition server SuperPose ( http: / / wishart.biology.ualberta.ca / superpose / ) was used to superimpose each serotype onto the 3BTA.pdb structure; 4. The overlapping pdb files are the H of BoNT / A1. C 3 at the start of the domain 10 were examined to find the location of the helix and subsequently identify the corresponding residues in other serotypes; 5. Other BoNT serotype sequences were aligned with Clustal Omega to ensure that corresponding residues were correct.

[0139] LH determined by this method N , H C , and 3 10 Examples of each domain of the helix are shown below. [Table 2]

[0140] Using structural analysis and sequence alignment, LH N Domain and H C Splitting the Domain 3 10 The helix followed by a β-strand is a conserved structure in all botulinum and tetanus neurotoxins, and LH N Domain and H C Splitting the Domain 3 10 It has been found to begin at the eighth residue (eg, residue 879 for BoNT / A1) when starting from the first residue of the helix.

[0141] BoNT / AB chimera is a chimera derived from BoNT / B C LH from BoNT / A covalently linked to the domain N may include a domain, ·LH N The C-terminal amino acid residue of the domain is H C corresponds to the eighth amino acid residue N-terminal to the β-strand at the start (N-terminus) of the domain, and H C The N-terminal amino acid residue of the domain is H of BoNT / B. C It corresponds to the seventh amino acid residue N-terminally following the β-strand at the start (N-terminus) of the domain.

[0142] BoNT / AB chimera is a chimera derived from BoNT / B C LH from BoNT / A covalently linked to the domain N may include a domain, ·LH N The C-terminal amino acid residue of the domain is the LH of BoNT / A. N corresponds to the C-terminal amino acid residue of an alpha helix located at the end (C-terminus) of the domain, and H C The N-terminal amino acid residue of the domain is the LH of BoNT / B. N It corresponds to the amino acid residue adjacent to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain.

[0143] The rationale for the design process of the BoNT / AB chimera was to ensure that the secondary structure was unknown, thereby minimizing any changes to the tertiary structure and function of each domain. 10 It is hypothesized that not disrupting the four central amino acid residues of the helix ensures an optimal conformation for the chimeric neurotoxin, thereby allowing the chimeric neurotoxin to function to its full potential.

[0144] 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 domain from BoNT / A N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:62.

[0145] 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. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:52.

[0146] Preferably, the BoNT / AB chimera is BoNT / A LH N Domains and BoNT / BH C More preferably, the LH domain is N The domain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 62), CThe domain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO:52).

[0147] Preferably, BoNT / BH C Domain has the effect of increasing the binding affinity of BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence CC The BoNT / BH subdomain further comprises at least one amino acid residue substitution, addition, or deletion. CC Suitable amino acid residue substitutions, additions, or deletions in the subdomains are disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated by reference herein).

[0148] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the subdomains include substitution mutations selected from the group consisting of 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.

[0149] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomain further include a set of two substitution mutations selected from the group consisting of 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 W178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0150] BoNT / BH CC Suitable amino acid residue substitutions, additions or deletions in the subdomains also include the set of three substitution mutations: E1191M, S1199W, and W1178Q.

[0151] Preferably, BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the subdomains include a set of two substitution mutations, E1191M and S1199Y.

[0152] The modifications may be modifications relative to unmodified BoNT / B shown as SEQ ID NO:52, and the numbering of the amino acid residues 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, one of skill in the art would take into account the presence / absence of the methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO:52 contains a methionine, the numbering of the positions would be as defined above (e.g., E1191 would become E1191 in SEQ ID NO:52). Alternatively, if a methionine is absent in SEQ ID NO:52, the numbering of the amino acid residues would be modified by -1 (e.g., E1191 would become E1190 in SEQ ID NO:52). Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and one of skill in the art would readily determine the correct amino acid residue number using techniques routine in the art.

[0153] Thus, in one aspect, the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0154] In a related embodiment, a method is provided for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the method comprising administering to the patient a polypeptide, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:63 or 64.

[0155] In a further related embodiment, there is provided a use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.

[0156] In one aspect, the invention provides a polypeptide for use in treating a neurological disorder in a patient, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:63 or 64.

[0157] In a related embodiment, a method for treating a neurological disorder in a patient is provided, the method comprising administering to the patient a polypeptide, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:63 or 64.

[0158] In a further related embodiment, there is provided a use of a polypeptide in the manufacture of a medicament for treating a neurological disorder in a patient, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:63 or 64.

[0159] In one embodiment, a polypeptide for use according to the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 63 or 64. Preferably, a polypeptide for use according to the invention comprises (more preferably consists of) the polypeptide sequence shown in SEQ ID NO: 63 or 64.

[0160] Preferably, the polypeptide sequence having at least 70% sequence identity to SEQ ID NO:63 includes a catalytically inactive L chain, such as SEQ ID NO:64.

[0161] Chimeric and / or hybrid Clostridial neurotoxins for use in the present invention can include a portion of a BoNT / A polypeptide and a portion of a BoNT / B polypeptide, examples of which include the polypeptide set forth herein as SEQ ID NO:44.

[0162] A suitable chimeric Clostridial neurotoxin may comprise BoNT / FA. Indeed, in a particularly preferred mode of implementation, the polypeptide of the invention may comprise BoNT / FA or a fragment thereof. Catalytically inactive forms of BoNT / FA are set forth herein as SEQ ID NOs: 26 and 34. Suitable fragments of BoNT / FA are also set forth herein as SEQ ID NOs: 28, 30, and 32.

[0163] The term "clostridial neurotoxin" may also encompass members of the newly discovered botulinum neurotoxin protein family expressed by non-clostridial microorganisms, such as the Enterococcus-encoded toxin with closest sequence identity to BoNT / X, the Weisselia oryzae-encoded toxin called BoNT / Wo (NCBI Ref.Seq:WP_027699549.1) that cleaves VAMP2 in W89-W90, the Enterococcus faecium-encoded toxin (GenBank:OTO22244.1) that cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero-encoded toxin (NCBI Ref.Seq:WP_034687872.1).

[0164] The polypeptides of the present invention are functional H of Clostridial neurotoxins. C It may lack a domain, and may also lack a functionally equivalent exogenous ligand targeting moiety (TM).

[0165] Thus, in a particularly preferred form of implementation, the Clostridial neurotoxin of the present invention is not a retargeted Clostridial neurotoxin. In a retargeted Clostridial neurotoxin, the Clostridial neurotoxin has been modified to include an exogenous ligand known as a targeting moiety (TM). The TM is selected to provide binding specificity for the desired target cell, and as part of the retargeting process, a unique binding moiety of the Clostridial neurotoxin (e.g., H C Domain or H CC Retargeting techniques are described, for example, in EP 0689459, WO 1994 / 021300, EP 0939818, U.S. Pat. No. 6,461,617, U.S. Pat. No. 7,192,596, WO 1998 / 007864, EP 0826051, U.S. Pat. No. 5,989,545, U.S. Pat. No. 6,395,513, U.S. Pat. No. 6,962,703, WO 1996 / 033273, EP 0996468, U.S. Pat. No. 7,052,702, WO 1999 / 017806 ... No. 1107794, U.S. Pat. No. 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.

[0166] As discussed above, (full-length) clostridial neurotoxins are formed from two polypeptide chains, a heavy chain (H chain) having a molecular weight of about 100 kDa and a light chain (L chain) having a molecular weight of about 50 kDa. The H chain contains a C-terminal targeting component (the receptor binding domain or H C domain) and N-terminal translational component (H N domain).

[0167] The Clostridial neurotoxin may be selected from 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 BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X.

[0168] In one embodiment, the Clostridial neurotoxin may be BoNT / A. The reference BoNT / A sequence is set forth as SEQ ID NO: 51. In another embodiment, the Clostridial neurotoxin may be BoNT / B. The reference BoNT / B sequence is set forth as SEQ ID NO: 52. In another embodiment, the Clostridial neurotoxin may be BoNT / C. The reference BoNT / C sequence is set forth as SEQ ID NO: 53. In another embodiment, the Clostridial neurotoxin may be BoNT / D. The reference BoNT / D sequence is set forth as SEQ ID NO: 54. In another embodiment, the Clostridial neurotoxin may be BoNT / E. The reference BoNT / E sequence is set forth as SEQ ID NO: 55. In another embodiment, the Clostridial neurotoxin may be BoNT / F. The reference BoNT / F sequence is set forth as SEQ ID NO: 56. In another embodiment, the Clostridial neurotoxin may be BoNT / G. The reference BoNT / G sequence is set forth as SEQ ID NO: 57. In another embodiment, the Clostridial neurotoxin may be TeNT. A reference TeNT sequence is provided as SEQ ID NO: 58. In another embodiment, the Clostridial neurotoxin may be BoNT / X. A reference BoNT / X sequence is provided as SEQ ID NO: 59.

[0169] 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.

[0170] In embodiments, when the polypeptides described herein comprise a tag for purification (eg, a His tag) and / or a linker, said tag and / or linker are optional.

[0171] Suitable full-length Clostridial neurotoxins are described herein.

[0172] 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 NOs: 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. 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 NOs: 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, a polypeptide of the invention may comprise a polypeptide sequence comprising any one of SEQ ID NOs: 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.

[0173] In one embodiment, the polypeptide of the invention may be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. In one embodiment, the polypeptide of the invention is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. Preferably, the polypeptide of the invention is encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60, with the proviso that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive.

[0174] In one embodiment, the polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65, provided that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. In one embodiment, the polypeptide of the invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65, provided that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive. Preferably, the polypeptide of the invention comprises any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65, provided that the Clostridial neurotoxin L chain of the polypeptide is catalytically inactive.

[0175] In one embodiment, the polypeptide of the invention is a full-length Clostridial neurotoxin selected from BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT.

[0176] In one embodiment, the polypeptide of the present invention may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 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 SEQ ID NO: 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 SEQ ID NO: 52-59, 61, or 63. Preferably, the polypeptide of the present invention may comprise (and more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NO: 52-59, 61, or 63.

[0177] In a particularly preferred mode of practice, the polypeptides of the invention are not full-length catalytically active Clostridial neurotoxins, eg, are not full-length catalytically active BoNT / A.

[0178] A polypeptide of the invention may comprise (or consist of) a fragment of a Clostridial neurotoxin, for example, a fragment of any full-length Clostridial neurotoxin described herein.

[0179] 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 NOs: 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%, 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, or 65. Preferably, a polypeptide of the invention may comprise a fragment of a polypeptide sequence including any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, or 65.

[0180] In one embodiment, the polypeptide of the invention comprises (or consists of) a Clostridial neurotoxin L chain or a fragment thereof. The Clostridial neurotoxin L chain fragment may have ≦400, ≦350, ≦300, ≦250, ≦200, ≦150, ≦100, or ≦50 amino acid residues of the Clostridial neurotoxin L chain. In one embodiment, the Clostridial neurotoxin L chain fragment has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the Clostridial neurotoxin L chain. For example, the Clostridial neurotoxin L chain fragment may have 20-400, 50-300, or 100-200 amino acid residues of the Clostridial neurotoxin L chain.

[0181] Examples of light chain reference sequences include the following: Botulinum type A neurotoxin: amino acid residues 1-448 Clostridium botulinum type B neurotoxin: amino acid residues 1-440 Clostridium botulinum type C1 neurotoxin: amino acid residues 1-441 Clostridium botulinum type D neurotoxin: amino acid residues 1-445 Clostridium botulinum type E neurotoxin: amino acid residues 1-422 Clostridium botulinum type F neurotoxin: amino acid residues 1-439 Clostridium botulinum type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457

[0182] For the recently identified BoNT / X, the light chain has been reported as corresponding to amino acid residues 1-439 thereof, with the light chain boundary potentially varying by approximately 25 amino acids (eg, 1-414 or 1-464).

[0183] The reference sequences identified above should be considered as a guideline, as slight variations may occur depending on the subserotype. By way of example, U.S. Patent Application No. 2007 / 0,166,332 (hereby incorporated by reference in its entirety) refers to the following slightly different Clostridial sequences: Clostridium botulinum type A neurotoxin: Amino acid residues M1 to K448 Clostridium botulinum type B neurotoxin: Amino acid residues M1-K441 Clostridium botulinum type C1 neurotoxin: Amino acid residues M1 to K449 Clostridium botulinum type D neurotoxin: Amino acid residues M1-R445 Clostridium botulinum type E neurotoxin: Amino acid residues M1-R422 Clostridium botulinum type F neurotoxin: Amino acid residues M1 to K439 Clostridium botulinum type G neurotoxin: Amino acid residues M1 to K446 Tetanus neurotoxin: amino acid residues M1-A457

[0184] Suitable Clostridial neurotoxin L chains are described herein.

[0185] A Clostridial neurotoxin L chain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 6, 24, 32, or 40, or a fragment thereof. In one embodiment, a Clostridial neurotoxin L chain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 6, 24, 32, or 40, or a fragment thereof. Preferably, a Clostridial neurotoxin L chain comprises (and more preferably consists of) a polypeptide sequence comprising SEQ ID NOs: 6, 24, 32, or 40, or a fragment thereof.

[0186] The Clostridial neurotoxin L chain may be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 5, 23, 31, or 39, or a fragment thereof. In one embodiment, the Clostridial neurotoxin L chain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 5, 23, 31, or 39, or a fragment thereof. Preferably, the Clostridial neurotoxin L chain is encoded by a nucleotide sequence comprising SEQ ID NOs: 5, 23, 31, or 39, or a fragment thereof.

[0187] In one embodiment, the polypeptide of the invention comprises (or consists of) a fragment of a Clostridial neurotoxin H chain. The fragment of a 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 a 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, a fragment of a Clostridial neurotoxin H chain may have 20 to 800, 30 to 600, 40 to 400, 50 to 300, or 100 to 200 amino acid residues of the Clostridial neurotoxin H chain.

[0188] Clostridial neurotoxin H chains contain two structural / functional domains, the translocation domain (H N ) and the receptor binding domain (H C ).

[0189] In one embodiment, the polypeptide of the invention comprises (or consists of) a Clostridial neurotoxin translocation domain or a fragment thereof. A fragment of a Clostridial neurotoxin translocation domain may have ≦400, ≦350, ≦300, ≦250, 200, ≦150, ≦100, or ≦50 amino acid residues of the 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 the 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 the Clostridial neurotoxin translocation domain.

[0190] The translocation domain is a fragment of the H chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H chain, or a domain corresponding to that fragment in an intact H chain. C The function is to modify (either at the DNA synthesis level or at the post-synthetic level by treatment with nucleases or proteases) H C It may be removed by deletion of the amino acid sequence. C The function may be inactivated by chemical or biological treatment, and thus, in some embodiments, the H chain may be incapable of binding to the binding site on the target cell to which the native Clostridial neurotoxin (i.e., the holotoxin) binds.

[0191] Examples of suitable (reference) translocation domains include: Botulinum type A neurotoxin - amino acid residues (449-871) Botulinum type B neurotoxin - amino acid residues (441-858) Botulinum type C neurotoxin - amino acid residues (442-866) Botulinum type D neurotoxin - amino acid residues (446-862) Botulinum type E neurotoxin - amino acid residues (423-845) Botulinum type F neurotoxin - amino acid residues (440-864) Botulinum type G neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879)

[0192] The reference sequences set forth above should be considered as a guideline, as slight variations may occur depending on the subserotype. By way of example, U.S. Patent Application No. 2007 / 0,166,332 (hereby incorporated by reference) refers to the following slightly different Clostridial sequences:

[0193] Botulinum type A neurotoxin - amino acid residues (A449-K871) Botulinum type B neurotoxin - amino acid residues (A442-S858) Clostridium botulinum type C1 neurotoxin - amino acid residues (T450~N866) Botulinum type D neurotoxin - amino acid residues (D446 to N862) Botulinum type E neurotoxin - Amino acid residues (K423-K845) Botulinum type F neurotoxin - amino acid residues (A440~K864) Botulinum type G neurotoxin - amino acid residues (S447-S863) Tetanus neurotoxin - amino acid residues (S458~V879)

[0194] In accordance with the present invention, various Clostridial neurotoxins H that contain a translocation domain are N These active fragments can be useful in each aspect of the 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 execution of the entire cellular machinery, whereby clostridial neurotoxins proteolytically cleave substrates. H from clostridial neurotoxin heavy chains can be used to cleave substrates. N The region is approximately 410-430 amino acids in length and contains the translocation domain. N It has been shown that the overall length of the region is not necessary for the translocation activity of the Translocation Domain, and therefore aspects of this embodiment include, for example, Clostridial neurotoxin H that includes a Translocation Domain having a length of at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. NOther aspects of this embodiment include, for example, Clostridial neurotoxin H that includes a translocation domain having a length of at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, and at most 425 amino acids. N It may contain regions.

[0195] For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see The Clostridia: Molecular Biology and Pathogenesis by Henderson et al., Academic Press, (1997).

[0196] Term H N is a naturally occurring neurotoxin H N and modified H having non-naturally occurring amino acid sequences and / or synthetic amino acid residues. N In one embodiment, the modified H N Part still exhibits the translocation function described above.

[0197] In a preferred embodiment, the polypeptide of the present invention comprises a Clostridial neurotoxin receptor binding domain (H C ) or a fragment thereof. C ) fragment of the Clostridial neurotoxin receptor binding domain (H C In one embodiment, the clostridial neurotoxin receptor binding domain (H) may have ≦350, ≦300, ≦250, ≦200, ≦150, ≦100, or ≦50 amino acid residues. C A fragment of a Clostridial neurotoxin receptor binding domain (H) has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues. C) fragment of the Clostridial neurotoxin receptor binding domain (H C ) may have 20 to 350, 50 to 300, or 100 to 200 amino acid residues.

[0198] Clostridial neurotoxin receptor binding domain (H C ) reference sequences include: BoNT / A - N872~L1296 BoNT / B - E859~E1291 BoNT / C1 - N867~E1291 BoNT / D - S863~E1276 BoNT / E - R846~K1252 BoNT / F - K865~E1274 BoNT / G - N864~E1297 TeNT-I880~D1315

[0199] For the recently identified BoNT / X, C The domains correspond to amino acids 893-1306, with the domain boundaries reportedly potentially varying by approximately 25 amino acids (eg, 868-1306 or 918-1306).

[0200] The clostridial neurotoxin H chain may further comprise a translocation facilitating domain, which facilitates delivery of the L chain into the cytosol of a target cell, as described, for example, in WO 08 / 008,803 and WO 08 / 008,805, each of which is incorporated herein by reference.

[0201] For example, the translocation-facilitating domain is Clostridial neurotoxin H CN More particularly, the clostridial neurotoxin H CNThe translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. CN The translocation facilitating domain preferably has a length of at most 200 amino acids, more preferably at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific examples include: Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111) Botulinum type D neurotoxin - amino acid residues (863-1098) Botulinum type E neurotoxin - amino acid residues (846-1085) Botulinum type F neurotoxin - amino acid residues (865-1105) Botulinum type G neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127)

[0202] The above sequence positions may vary slightly depending on the serotype / subtype and may be modified by the appropriate (see reference) Clostridial neurotoxin H CN Further examples of domains include: Botulinum type A neurotoxin - amino acid residues (874-1110) Botulinum type B neurotoxin - amino acid residues (861-1097) Botulinum type C neurotoxin - amino acid residues (869-1111) Botulinum type D neurotoxin - amino acid residues (865-1098) Botulinum type E neurotoxin - amino acid residues (848-1085) Botulinum type F neurotoxin - amino acid residues (867-1105) Botulinum type G neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127)

[0203] Suitable Clostridial neurotoxins H C Domains are described herein.

[0204] 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. C The domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50, or a fragment thereof. C The domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50, or a fragment thereof.

[0205] Clostridial neurotoxin H C The domain may be 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. C The domain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49, or a fragment thereof. C The domain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49, or a fragment thereof.

[0206] In one embodiment, the Clostridial neurotoxin H for use in the present invention is C The domain is mutant BoNT / AH C The mutant BoNT / AH C The domain may include modifications of one or more amino acid residues selected from Y1117, F1252, H1253, and L1278. For example, the mutant BoNT / AH C The domain may include one or more (preferably two or more) of the following modifications: Y1117V, F1252Y, H1253K, and L1278F or L1278H.

[0207] In one embodiment, the mutant BoNT / AH C The domain contains the following modifications: Y1117V and H1253K, or Y1117V, F1252Y, H1253K, and L1278F, or Y1117V, F1252Y, H1253K, and L1278H.

[0208] Preferably, the mutant BoNT / AH C The domain contains the following modifications: Y1117V and H1253K, or Y1117V, F1252Y, H1253K, and L1278H.

[0209] The modifications may be modifications in comparison with the unmodified BoNT / A shown as SEQ ID NO:62, and the numbering of the amino acid residues is determined by aligning 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 numbering of the amino acid residues. For example, if SEQ ID NO:62 contains a methionine, the numbering of the positions will be as defined above (e.g., Y1117 aligns with Y1117 of SEQ ID NO:62). Alternatively, if a methionine is absent in SEQ ID NO:62, the numbering of the amino acid residues should be corrected by -1 (e.g., Y1117 aligns with Y1116 of SEQ ID NO:52). Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and the skilled artisan will readily determine the correct numbering of the amino acid residues using routine techniques in the art.

[0210] Mutant BoNT / AH C The domain is a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46, 48, or 50, or a fragment thereof, and is designated as a mutant BoNT / AH polypeptide. C In one embodiment, the mutant BoNT / AH domain may include the above-mentioned modifications. C The domain is 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, as a mutant BoNT / AH polypeptide. C provided that the domain contains the above modifications. C The domain is 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, and is used as a mutant BoNT / AH polypeptide. C Provided that the domain contains the above modifications. Preferably, the mutant BoNT / AH CThe domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NO: 46, 48, or 50, or a fragment thereof.

[0211] Mutant BoNT / AH C The domain is a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NO: 46 or 50, or a fragment thereof, and is used as a mutant BoNT / AH polypeptide. C In one embodiment, the mutant BoNT / AH domain may include the above-mentioned modifications. C The domain is a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 46 or 50, or a fragment thereof, as a mutant BoNT / AH polypeptide. C provided that the domain contains the above modifications. C The domain is 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, and is used as a mutant BoNT / AH polypeptide. C Provided that the domain contains the above modifications. Preferably, the mutant BoNT / AH C The domain comprises (more preferably consists of) a polypeptide sequence comprising SEQ ID NO: 46, or 50, or any one of a fragment thereof.

[0212] Mutant BoNT / AH C The domain is a mutant BoNT / AH C The domain may be 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 domain contains the modifications described above. C The domain is a mutant BoNT / AH CIn one embodiment, the mutant BoNT / AH domain is 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 domain contains the modifications described above. C The domain is a mutant BoNT / AH C Preferably, the mutant BoNT / AH domain is 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 domain contains the modifications described above. C The domain is encoded by any one of SEQ ID NO: 45, 47, or 49, or a fragment thereof.

[0213] Mutant BoNT / AH C The domain is a mutant BoNT / AH C The domain may be encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 45 or 49, or any one of its fragments, provided that the domain contains the modifications described above. C The domain is a mutant BoNT / AH C In one embodiment, the mutant BoNT / AH domain is 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 domain contains the modifications described above. C The domain is a mutant BoNT / AH C Preferably, the mutant BoNT / AH domain is encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to SEQ ID NO: 45 or 49, or any one of its fragments, provided that the domain contains the above-mentioned modifications. C The domain is encoded by SEQ ID NO: 45 or 49, or any one of a fragment thereof.

[0214] Any of the above facilitating domains can be combined with any of the above translocation domain peptides suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain can be combined with a non-clostridial translocation domain peptide or 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: Botulinum type A neurotoxin - amino acid residues (449-1110) Botulinum type B neurotoxin - amino acid residues (442-1097) Botulinum type C neurotoxin - amino acid residues (450-1111) Botulinum type D neurotoxin - amino acid residues (446-1098) Botulinum type E neurotoxin - amino acid residues (423-1085) Botulinum type F neurotoxin - amino acid residues (440-1105) Botulinum type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127)

[0215] In some embodiments, the clostridial neurotoxins of the present invention are functional H of the clostridial neurotoxin. CIn one embodiment, the Clostridial neurotoxin preferably lacks the last 50 C-terminal amino acids of a 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 a Clostridial neurotoxin holotoxin. Alternatively, the Clostridial neurotoxin may lack the last 50 C-terminal amino acid residues of a Clostridial neurotoxin holotoxin. C Binding activity may be abolished / reduced by mutagenesis; for convenience, referring to BoNT / A, modification of one or two amino acid residue mutations (W1266 to L and Y1267 to F) in the ganglioside binding pocket results in H C Similar mutations can be made to non-serogroup A clostridial peptide components, such as constructs based on C. botulinum B with the mutations W1262 to L and Y1263 to F, or C. botulinum E (W1224 to L and Y1225 to F). Other mutations to the active site include H C The same truncation of receptor binding activity, e.g., Y1267S in botulinum type A toxin and the corresponding highly conserved residues in other clostridial neurotoxins, is achieved. Details of this and other mutations are described in Rummel et al. (Molecular Microbiol. 51:631-634) (2004), which is incorporated herein by reference.

[0216] Natural Clostridial Neurotoxin H C The peptide contains approximately 400-440 amino acid residues and is divided into two functionally distinct domains, each of approximately 25 kDa, i.e., CN The N-terminal region (called a peptide or domain) and the CCIt consists of a C-terminal region called a peptide or domain. This fact has been confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC, Nat. Struct. Biol. 4: 788-792 (1997); Herreros J, Biochem. J 347: 199-204 (2000); Halpern J, J. Biol. Chem. 268, 15, pp. 11188-11192 (1993); Rummel A, PNAS 104: 359-364 (2007); Lacey DB, Nat. Struct. Biol. 5: 898-902 (1998); Knapp, Am. Cryst. Assoc. Abstract 1999, 14: 1117-1118 (2007); Papers (Proceedings of the American Crystallographic Society) 25: 90 (1998), Swaminathan and Eswaramoorthy, Nat. Struct. Biol. 7: 1751-1759 (2000), and Rummel A, Mol. Microbiol. 51(3), 631-643 (2004). In addition, the C-terminal region (H) consisting of 160 to 200 amino acid residues at the C-terminus CC It is well documented that the H β -terminal domain of the clostridial neurotoxin is responsible for the binding of the clostridial neurotoxin to its native cellular receptor, i.e., to the nerve endings at the neuromuscular junction, and this fact is also confirmed by the above publication. Thus, it is necessary to have a functional heavy chain H β -terminal domain that prevents the heavy chain from binding to the cell surface receptor to which the native clostridial neurotoxin binds. C References throughout this specification to a Clostridial heavy chain lacking a peptide (or domain) are intended to indicate that the Clostridial heavy chain is merely a functional H CC This means that the peptide is missing. CCThe peptide region may be deleted, either partially or entirely, or otherwise modified (e.g., via conventional chemical or proteolytic treatment) to reduce its natural binding ability to the nerve endings at the neuromuscular junction.

[0217] Thus, in one embodiment, the Clostridial neurotoxin H of the present invention N The peptides were derived from the C-terminal peptide portion (H CC ) and therefore lacks the H of the native clostridial neurotoxin. C By way of 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 Clostridial neurotoxin heavy chain. N The peptides were derived from the C-terminal peptide portion (H CC ) and therefore lacks the H of the native clostridial neurotoxin. C By way of 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 Clostridial neurotoxin heavy chain. N The peptide is selected from the group consisting of: CC Lacking a reference sequence: Botulinum type A neurotoxin - amino acid residues (Y1111 to L1296) Botulinum type B neurotoxin - amino acid residues (Y1098 to E1291) Botulinum type C neurotoxin - amino acid residues (Y1112 to E1291) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086 to K1252) Botulinum type F neurotoxin - amino acid residues (Y1106 to E1274) Botulinum type G neurotoxin - amino acid residues (Y1106 to E1297) Tetanus neurotoxin - amino acid residues (Y1128~D1315)

[0218] The reference sequences identified above should be considered as guidelines since slight variations may occur among subserotypes.

[0219] In a preferred embodiment, the polypeptide of the invention comprises (or consists of) a Clostridial neurotoxin L chain or a fragment thereof and a Clostridial neurotoxin H chain fragment. For example, the polypeptide comprises a Clostridial neurotoxin L chain or a fragment thereof and a Clostridial neurotoxin translocation domain (H N Preferably, the polypeptide comprises (or consists of) a Clostridial neurotoxin receptor binding domain (H C ) or at least the C-terminal portion of the Clostridial neurotoxin receptor binding domain (H CC ) is not further included. Thus, in one embodiment, the polypeptide of the invention does not further include the C-terminal portion of the Clostridial neurotoxin receptor binding domain (H CC Advantageously, the polypeptide lacks endogenous Clostridial neurotoxin receptor binding capacity and therefore exhibits fewer off-target effects in patients receiving said polypeptide.

[0220] 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. The term "consists essentially of" as used in this context means that the polypeptide does not further comprise one or more amino acid residues that provide additional functionality to the polypeptide, for example, when administered to a patient. In other words, a polypeptide that essentially consists 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 (to the amino acid residues of the Clostridial neurotoxin L chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin H chain), but said one or more additional amino acid residues do not provide additional functionality to the polypeptide, for example, when administered to a patient. The additional functionality may include enzymatic activity, binding activity, and / or any physiological activity whatsoever.

[0221] In one embodiment, the polypeptide may include a non-clostridial neurotoxin sequence in addition to any clostridial neurotoxin sequence. The non-clostridial neurotoxin sequence preferably does not interfere with the ability of the polypeptide of the invention to promote neuronal growth or neuronal repair. Preferably, the non-clostridial neurotoxin sequence does not have catalytic activity, e.g., enzymatic activity. Preferably, the non-clostridial sequence does not bind to a cellular receptor. In other words, most preferably, the non-clostridial sequence is not a ligand for a cellular receptor. The cellular receptor may be a proteinaceous cellular receptor, such as an integral membrane protein. Examples of cellular receptors can be found in the IUPHAR Guide to Pharmacology Database, version 2019.4, available at https: / / www.guidetopharmacology.org / download.jsp#db_reports. The non-clostridial neurotoxin sequence may include a tag to aid in purification, such as a His tag. Preferably, any Clostridial neurotoxin sequence contained in the polypeptide comprises a Clostridial neurotoxin light chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin heavy chain. In one embodiment, the Clostridial neurotoxin sequence contained in the polypeptide may comprise a Clostridial neurotoxin light chain. In one embodiment, the Clostridial neurotoxin sequence contained in the polypeptide may comprise a Clostridial neurotoxin translocation domain. In one embodiment, the Clostridial neurotoxin sequence contained in the polypeptide may comprise a Clostridial neurotoxin receptor binding domain. In one embodiment, the Clostridial neurotoxin sequence contained in the polypeptide may comprise a Clostridial neurotoxin light chain and a Clostridial neurotoxin translocation domain.

[0222] Suitable polypeptides comprising (or consisting of) a Clostridial neurotoxin L chain and translocation domain are described herein.

[0223] A 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, a 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, a Clostridial neurotoxin comprising (or consisting of) a Clostridial neurotoxin L chain and a Translocation Domain comprises (more preferably consists of) a polypeptide sequence comprising SEQ ID NOs: 4, 20, 28, or 36, or a fragment thereof.

[0224] The Clostridial neurotoxin comprising (or consisting of) a Clostridial neurotoxin L chain and a Translocation Domain may be 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 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 encoded by a nucleotide sequence comprising SEQ ID NOs: 3, 19, 27, or 35, or a fragment thereof.

[0225] The polypeptides of the invention may be free of each complex protein present in a naturally occurring Clostridial neurotoxin complex.

[0226] The polypeptides of the invention can be produced using recombinant nucleic acid techniques. Thus, in one embodiment, the polypeptide (as described above) is a recombinant polypeptide.

[0227] In one embodiment, a nucleic acid (e.g., DNA) is provided that comprises a nucleic acid sequence that encodes a polypeptide. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector that includes a promoter and a terminator.

[0228] In a preferred embodiment, the vector has a promoter selected from: Promoter / Inducer / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-Iac operator / IPTG / 0.2mM (0.05~2.0mM)

[0229] In another preferred embodiment, the vector has a promoter selected from: Promoter / Inducer / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-Iac operator / IPTG / 0.2mM (0.05~2.0mM) T5-Iac operator / IPTG / 0.2mM (0.05~2.0mM)

[0230] The nucleic acid molecule may be prepared using any suitable process known in the art. Thus, the nucleic acid molecule may be prepared using chemical synthesis techniques. Alternatively, the nucleic acid molecule of the present invention may be prepared using molecular biology techniques.

[0231] The DNA constructs of the present invention are preferably designed on a computer and then synthesized by conventional DNA synthesis techniques.

[0232] The above nucleic acid sequence information is optionally modified for codon bias due to the final host cell (eg, E. coli) expression system to be employed.

[0233] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0234] The polypeptides of the invention (and in particular any Clostridial neurotoxin portions thereof) may exist as single chains or as two chains.

[0235] The invention provides a method for producing a single-chain polypeptide having a light chain and a heavy chain, comprising expressing a nucleic acid described herein in an expression host, lysing the host cell to provide a host cell homogenate comprising the single-chain polypeptide, and isolating the single-chain polypeptide. In one aspect, the invention provides a method for activating a polypeptide described herein, comprising contacting the polypeptide with a proteolytic enzyme that hydrolyzes a peptide bond in the activation loop of the polypeptide, thereby converting the (single-chain) polypeptide into the corresponding two-chain polypeptide (e.g., with the light and heavy chains linked together by a disulfide bond).

[0236] Thus, the present invention provides a double-chain polypeptide obtainable by the method of the present invention.

[0237] It is intended that the various therapeutic use related embodiments of the present invention apply equally to methods of treatment, the polypeptides of the present invention, and vice versa.

[0238] sequence homology

[0239] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, individual methods, and hybrid methods such as, for example, segmental approaches. Defined procedures for determining percent identity are routine procedures within the skill of the art. Global methods align the sequence from beginning to end of the molecule and determine the best alignment by combining the scores of individual residue pairs and imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W ("Clustal Double"), see, e.g., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice by Julie D. Thompson et al., 22(22) Nucleic Acids Research 4673-4680 (1994), and iterative refinement, see, e.g., 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). Individual methods align sequences by identifying one or more conserved motifs that are shared by all of the input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences by Eric Depiereux and Ernest Feytmans, 8(5) CABIOS 501 -509 (1992); Gibbs sampling (see, e.g., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment by C.E. Lawrence et al., 262(5131 ) Science 208-214 (1993); Align-M (see, e.g., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent by Ivo Van WaIIe et al. Sequences ("AlignEm - a new algorithm for multiple alignment of highly divergent sequences"), 20(9) Bioinformatics:1428-1435 (2004), including:

[0240] Thus, the percentage sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio., 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap open penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.), shown below (amino acids are shown by standard one-letter code):

[0241] The "percentage sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by each sequence. Thus, % identity may 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 may also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The comparison of sequences between two or more sequences and the determination of percentage identity can be performed using certain mathematical algorithms such as BLAST ("blast"), which are well known to those skilled in the art.

number

[0242] Therefore, the percentage identity is calculated as follows:

number

[0243] Each substantially homologous polypeptide is characterized by having one or more amino acid substitutions, deletions, or additions, which are preferably minor, such as substitutions of conserved amino acid residues (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, as well as small deletions, typically of about 30 amino acids, and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, small linker peptides of up to about 20-25 residues, or affinity tags.

[0244] Conservative amino acid substitutions

[0245] Basicity: Arginine lysine Histidine Acidic: Glutamic acid Aspartic acid Polar: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Ballin Aromatic: Phenylalanine Tryptophan Tyrosine Small things: glycine Alanine Serine Threonine Methionine

[0246] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conserved amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the invention can also include non-naturally occurring amino acid residues.

[0247] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, in vitro systems can be employed 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 carried out in cell-free systems including E. coli S30 extracts and commercially available enzymes, as well as other translation systems. Proteins are 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 the second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996).In a 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-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring 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 species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0248] A limited number of non-conserved amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0249] Essential amino acids in the polypeptides of the invention can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, involving mutation of putative contact site amino acids. See, e.g., 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 identity of essential amino acids can also be inferred from analysis of homologies with related components of the polypeptides of the invention (eg, translocation or proteolytic enzyme components).

[0250] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions within a polypeptide, selecting functional polypeptides, and subsequently sequencing the mutagenized polypeptides to determine the spectrum of permissible 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, WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0251] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY by Singleton et al., 20th ED., John Wiley and Sons, New York, (1994) and THE HARPER COLLINS DICTIONARY OF BIOLOGY by Hale & Marham, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0252] The 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 embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range, and unless otherwise indicated, all nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0253] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0254] Amino acids are referred to herein using their amino acid name, three-letter abbreviation, or one-letter abbreviation. 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 instances, the term "amino acid sequence" is synonymous with the term "peptide." In some instances, 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 one-letter and three-letter codes for amino acid residues can be used. The three-letter code for amino acids as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It will be understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0255] Other definitions of terms may appear throughout the specification. Before each of the exemplary embodiments is described in more detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure is defined only by the appended claims.

[0256] Where a range of values ​​is provided, it will be understood that each intermediate value between the upper and lower limits of that range is also specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated value or intermediate value in a stated range and any other stated value or intermediate value within that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range is also encompassed within the disclosure when either or both of the upper and lower limits are included in the smaller range, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0257] It should 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, reference to "the Clostridial neurotoxin" includes reference to one or more Clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0258] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims which follow herefrom.

[0259] Embodiments of the invention will now be described, by way of example only, with reference to the following drawings and embodiments. [Brief description of the drawings]

[0260] [Figure 1] Neurotrophic effect of recombinantly expressed catalytically inactive different BoNT / serotypes compared to the positive control brain-derived neurotrophic factor (BDNF) in the motor neuron-like cell line NSC34. *p<0.05 vs. untreated control, one-way ANOVA followed by Dunnett's multiple comparison test. Data are the mean ± SEM of three independent experiments, each experiment performed in six replicate wells. [Diagram 2] Neurotrophic effect of botulinum neurotoxin serotype A fragments in the motor neuron-like cell line NSC34 and the effect of recombinantly expressed catalytically inactive BoNT / A. BDNF was used as a positive control. *p<0.05 vs. untreated control, one-way ANOVA followed by Dunnett's multiple comparison test. Data are means ± SEM of three independent experiments, each experiment performed in six replicate wells. [Diagram 3] Neurotrophic effects of negative control and recombinantly expressed catalytically inactive BoNT / A (BoNT / A(0)) in the motor neuron-like cell line NSC34. BDNF was used as a positive control. *p<0.05 vs. untreated control, one-way ANOVA followed by Dunnett's multiple comparison test. Data are means ± SEM of three independent experiments, each experiment performed in six replicate wells. [Figure 4] FIG. 1 shows the results of a horizontal bridge test on mice administered either an expander control (PBS) or rBoNT / A(0) at 100 pg, 100 ng, or 50 μg. [Diagram 5](A) Immunohistochemistry using an antibody binding to neurofilament 200 (NF200) 4 weeks after administration of stretching agent (PBS) (left panel) or 100 ng of rBoNT / A(0) (right panel) and (B) immunohistochemistry using an antibody binding to MAP1b 4 weeks after administration of stretching agent (PBS) (left panel) or 100 ng of rBoNT / A(0) (right panel). Lesion sites are indicated by * (and for FIG. 5B, white arrows). [Figure 6] Figure 1 shows the effect of (A) catalytically inactive BoNT / A (0), (B) BoNT / A light chain plus translocation domain fragment (LHN / A), (C) BoNT / A light chain (LC / A, i.e., L / A), and (D) BoNT / A receptor binding domain (HC / A) 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 vs. BSA control, one-way ANOVA followed by Dunnett's post-hoc test. Data are means ± SEM. [Figure 7] Figure 1 shows the effect of (A) catalytically inactive BoNT / FA (0), (B) BoNT / FA light chain plus translocation domain fragment (LHN / FA), (C) BoNT / FA light chain (LC / FA, i.e., L / FA), and (D) BoNT / FA receptor binding domain (HC / FA) 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 vs. BSA control, one-way ANOVA followed by Dunnett's post-hoc test. Data are means ± SEM. [Figure 8]Figure 1 shows the effect of (A) BoNT / F light chain plus translocation domain fragment (LHN / F), (B) BoNT / F light chain (LC / F, i.e., L / F), and (C) BoNT / F receptor binding domain (HC / F) 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 vs. BSA control, one-way ANOVA followed by Dunnett's post-hoc test. Data are mean ± SEM. [Figure 9] FIG. 1 shows the effect of cationic rHC / A (i.e., mrHC / A) on the number of neurites per cell. Cationic 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 vs. BSA control, one-way ANOVA followed by Dunnett's post-hoc test. Data are means ± SEM. [Figure 10] Figure 1 shows the effect of (A) toxHC / A YH (i.e., rHC / A Variant Y1117V H1253K) and (B) toxHC / A YFHL(L to H) (i.e., rHC / A Variant Y1117V F1252Y H1253K L1278H) on the number of neurites per cell. Mutant 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 vs. BSA control, one-way ANOVA followed by Dunnett's post-hoc test. Data are means ± SEM.

[0261] Sequence Listing

[0262] Where the first Met amino acid residue or the corresponding first codon is shown in any of the following SEQ ID NOs, said residue / codon is optional.

[0263] SEQ ID NO:1 - Nucleotide sequence, catalytically inactive recombinant BoNT / A (rBoNT / A(0)) SEQ ID NO:2 - Polypeptide sequence, rBoNT / A(0) SEQ ID NO:3 - nucleotide sequence, rLH N / A (light chain and translocation domain only). SEQ ID NO:4 - Polypeptide sequence, rLH N / A SEQ ID NO:5 - nucleotide sequence, rL / A (light chain only) SEQ ID NO:6 - Polypeptide sequence, rL / A SEQ ID NO:7 - nucleotide sequence, rH C / A SEQ ID NO:8 - Polypeptide sequence, rH C / A SEQ ID NO:9 - nucleotide sequence, rBoNT / B(0) SEQ ID NO:10 - Polypeptide sequence, rBoNT / B(0) SEQ ID NO:11 - nucleotide sequence, rBoNT / C(0) SEQ ID NO:12 - Polypeptide sequence, rBoNT / C(0) SEQ ID NO:13 - nucleotide sequence, rBoNT / E(0) SEQ ID NO:14 - Polypeptide sequence, rBoNT / E(0) SEQ ID NO:15 - nucleotide sequence, rBoNT / F(0) SEQ ID NO:16 - Polypeptide sequence, rBoNT / F(0) SEQ ID NO:17 - Nucleotide sequence, rBoNT / A(0) (His tagged) SEQ ID NO:18 - Polypeptide sequence, rBoNT / A(0) (His tagged) SEQ ID NO:19 - nucleotide sequence, rLH N / A (His tagged) SEQ ID NO:20 - Polypeptide sequence, rLH N / A (His tagged) SEQ ID NO:21 - nucleotide sequence, rH C / A (His tagged) SEQ ID NO:22 - Polypeptide sequence, rH C / A (His tagged) SEQ ID NO:23 - Nucleotide sequence, rLC / A (His tagged) SEQ ID NO:24 - Polypeptide sequence, rLC / A (His tagged) SEQ ID NO:25 - Nucleotide sequence, rBoNT / FA(0) (His tagged) SEQ ID NO:26 - Polypeptide sequence, rBoNT / FA(0) (His tagged) SEQ ID NO:27 - nucleotide sequence, rLH N / FA (His tagged) SEQ ID NO:28 - Polypeptide sequence, rLH N / FA (His tagged) SEQ ID NO:29 - nucleotide sequence, rH C / FA (His tagged) SEQ ID NO:30 - Polypeptide sequence, rH C / FA (His tagged) SEQ ID NO:31 - Nucleotide sequence, rLC / FA (His tagged) SEQ ID NO:32 - Polypeptide sequence, rLC / FA (His tagged) SEQ ID NO:33 - Nucleotide sequence, rBoNT / F(0) (His tagged) SEQ ID NO:34 - Polypeptide sequence, rBoNT / F(0) (His tagged) SEQ ID NO:35 - nucleotide sequence, rL H N / F (His tagged) SEQ ID NO:36 - Polypeptide sequence, rL H N / F (His tagged) SEQ ID NO:37 - nucleotide sequence, rH C / F (His tagged) SEQ ID NO:38 - Polypeptide sequence, rH C / F (His tagged) SEQ ID NO:39 - Nucleotide sequence, rLC / F (His tagged) SEQ ID NO:40 - Polypeptide sequence, rLC / F (His tagged) SEQ ID NO:41 - nucleotide sequence, cationic rH C / A (His tagged) SEQ ID NO:42 - Polypeptide sequence, cationic rH C / A (His tagged) SEQ ID NO:43 - nucleotide sequence, rH C / AB (His tagged) SEQ ID NO:44 - Polypeptide sequence, rH C / AB (His tagged) SEQ ID NO:45 - nucleotide sequence, rH C / A Mutant Y1117V H1253K (His tagged) SEQ ID NO:46 - Polypeptide sequence, rH C / A Mutant Y1117V H1253K (His tagged) SEQ ID NO:47 - nucleotide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278F (His tagged) SEQ ID NO:48 - Polypeptide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278F (His tagged) SEQ ID NO:49 - nucleotide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278H (His tagged) SEQ ID NO:50 - Polypeptide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278H (His tagged) SEQ ID NO:51 - Polypeptide sequence, BoNT / A - UniProt P10845 SEQ ID NO:52 - Polypeptide sequence, BoNT / B - UniProt P10844 SEQ ID NO:53 - Polypeptide sequence, BoNT / C - UniProt P18640 SEQ ID NO:54 - Polypeptide sequence, BoNT / D - UniProt P19321 SEQ ID NO:55 - Polypeptide sequence, BoNT / E - UniProt Q00496 SEQ ID NO:56 - Polypeptide sequence, BoNT / F - UniProt A7GBG3 SEQ ID NO:57 - Polypeptide sequence, BoNT / G - UniProt Q60393 SEQ ID NO:58 - Polypeptide sequence, TeNT - UniProt P04958 SEQ ID NO:59 - Polypeptide sequence, BoNT / X SEQ ID NO:60 - Nucleotide sequence, mrBoNT / A SEQ ID NO:61 - Polypeptide sequence, mrBoNT / A SEQ ID NO:62 - Polypeptide sequence, unmodified BoNT / A1 SEQ ID NO:63 - Polypeptide sequence, mrBoNT / AB SEQ ID NO:64 - Polypeptide sequence, mrBoNT / AB(0) SEQ ID NO:65 - Polypeptide sequence, mrBoNT / A(0)

[0264] SEQ ID NO:1 - nucleotide sequence, rBoNT / A(0) SEQ ID NO:2 - Polypeptide sequence, rBoNT / A(0) SEQ ID NO:3 - nucleotide sequence, rLH N / A SEQ ID NO:4 - Polypeptide sequence, rLH N / A MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAV TLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSG KFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKT KSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKY TMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIF SGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDD LSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEAHHHHHHHHHH SEQ ID NO:5 - nucleotide sequence, rL / A SEQ ID NO:6 - Polypeptide sequence, rL / A MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTL AHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLGLEAHHHHHHHHHH SEQ ID NO:7 - nucleotide sequence, rH C / A SEQ ID NO:8 - Polypeptide sequence, rH C / A MHHHHHHKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENN SGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVK NKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO:9 - nucleotide sequence, rBoNT / B(0) SEQ ID NO:10 - Polypeptide sequence, rBoNT / B(0) SEQ ID NO:11 - nucleotide sequence, rBoNT / C(0) SEQ ID NO:12 - Polypeptide sequence, rBoNT / C(0) SEQ ID NO:13 - nucleotide sequence, rBoNT / E(0) SEQ ID NO:14 - Polypeptide sequence, rBoNT / E(0) SEQ ID NO:15 - nucleotide sequence, rBoNT / F(0) SEQ ID NO:16 - Polypeptide sequence, rBoNT / F(0) SEQ ID NO:17 - Nucleotide sequence, rBoNT / A(0) (His tagged) SEQ ID NO:18 - Polypeptide sequence, rBoNT / A(0) (His tagged) SEQ ID NO:19 - nucleotide sequence, rLH N / A (His tagged) SEQ ID NO:20 - Polypeptide sequence, rLH N / A (His tagged) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVT LAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGK FSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKS LDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMF HYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGA VILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSS KLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTENLYFQGASHHHHHHHH SEQ ID NO:21 - nucleotide sequence, rH C / A (His tagged) SEQ ID NO:22 - Polypeptide sequence, rH C / A (His tagged) MHHHHHHENLYFQGKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIIN CMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFD KELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVV KNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO:23 - Nucleotide sequence, rLC / A (His tagged) SEQ ID NO:24 - Polypeptide sequence, rLC / A (His tagged) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVT LAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGK FSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEENLYFQGASHHHHHHHH SEQ ID NO:25 - Nucleotide sequence, rBoNT / FA(0) (His tagged) SEQ ID NO:26 - Polypeptide sequence, rBoNT / FA(0) (His tagged) SEQ ID NO:27 - nucleotide sequence, rLH N / FA (His tagged) SEQ ID NO:28 - Polypeptide sequence, rLH N / FA (His tagged) MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLE EIKNAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLTTNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEYATAFNDISIASHSPSLFIKD PALILMHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAKDS NGVYSVDLNKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNIGALSINYQGQNIGSDINSIKKLQGQGVVSRVVRLCSNSNTKNSLC ITVNNRDLFFIASQESYGENTINTYKEIDDTTTLDPSFEDILDKVILNFNEQVIPQMPNRNVSTDIQKDNYIPKYDYNRTDIIDSYEVGRNYNTFFYLNAQKFSPNESN ITLTSSFDTGLLEGSKVYTFFSSDFINNINKPVQALLFIEWVKQVIRDFTTEATKTSTVDKLKDISLVVPYIGLALNIGDEIYKQHFAEAVELVGAGLLLEFSPEFLIPT LLIFTIKGYLTGSIRDKDKIIKTLDNALNVRDQKWKELYRWVVSKWLTTINTQFNKRKEQMYKALKNQATAIKKIIENKYNNYTTDEKSKIDSSYNINEIERTLNEKINL AMKNIEQFITESSIAYLINIINNETIQKLKSYDDLVRRYLLGYIRNHSSILGNSVEELNSKVNNHLDNGIPFELSSYTNDSLLIRYFNKNYGEENLYFQGASHHHHHHHH SEQ ID NO:29 - nucleotide sequence, rH C / FA (His tagged) SEQ ID NO:30 - Polypeptide sequence, rH C / FA (His tagged) MLKYNCILNIKYEMDRDKLVDSSGYRSRINIGTGVKFSEIDKNQVQLSNLESSKIEVILNNGVIYNSMYENFSTSFWIRIPKYFRNINNEYKIISCMQNNSGWEVSLNFSN MNSKIIWTLQDTEGIKKTVVFQYTQNINISDYINRWIFVTITNNRLSNSKIYINGRLINEESISDLGNIHASNNIMFKLDGCRDPHRYIWIKYFNLFDKELNKKEIKDLYDN QSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYLDVNNVGIRGYMYLKGPRGRIVTTNIYLNSTLYMGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSAVEIPDVGNLSQVVVMKSENDQGIRNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIGKASRTFGCSWEFIPVDDGWGESSLENLYFQGASHHHHHHHH SEQ ID NO:31 - Nucleotide sequence, rLC / FA (His tagged) SEQ ID NO:32 - Polypeptide sequence, rLC / FA (His tagged) MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLEEIK NAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLTTNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEYATAFNDISIASHSPSLFIKDPALIL MHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAKDSNGVYSVDL NKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNIGALSINYQGQNIGSDINSIKKLQGQGVVSRVVRLCSNSENLYFQGASHHHHHHHH SEQ ID NO:33 - Nucleotide sequence, rBoNT / F(0) (His tagged) SEQ ID NO:34 - Polypeptide sequence, rBoNT / F(0) (His tagged) SEQ ID NO:35 - nucleotide sequence, rL H N / F (His tagged) SEQ ID NO:36 - Polypeptide sequence, rL H N / F (His tagged) MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQE ISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIAD PAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNA DGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAP PRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLNNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGE TNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELLI PTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVS LAMENIERFITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKENLYFQGASHHHHHHHH SEQ ID NO:37 - nucleotide sequence, rH C / F (His tagged) SEQ ID NO:38 - Polypeptide sequence, rH C / F (His tagged) MIKDNSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDCIRNNNNSGWKIS LNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNSRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIET LYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYA DISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQENENLYFQGASHHHHHHHH SEQ ID NO:39 - Nucleotide sequence, rLC / F (His tagged) SEQ ID NO:40 - Polypeptide sequence, rLC / F (His tagged) MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEIS YAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAIS LAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTV NENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSENLYFQGASHHHHHHHH SEQ ID NO:41 - nucleotide sequence, cationic rH C / A (His tagged) SEQ ID NO:42 - Polypeptide sequence, cationic rH C / A (His tagged) MIINTSILNLRYESKHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFNKISLNNEYTIINCMENNSGWKVSLNY GEIIWTLQDTKEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDN QSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQ AGVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLKLAAALEHHHHHH SEQ ID NO:43 - nucleotide sequence, rH C / AB (His tagged) SEQ ID NO:44 - Polypeptide sequence, rH C / AB (His tagged) MILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGN RIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYS EYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMK LFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEKLAAALEHHHHHH SEQ ID NO:45 - nucleotide sequence, rH C / A Mutant Y1117V H1253K (His tagged) SEQ ID NO:46 - Polypeptide sequence, rH C / A Mutant Y1117V H1253K (His tagged) MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLN YGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLY DNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNA SQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFKQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLHHHHHHHHHH SEQ ID NO:47 - nucleotide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278F (His tagged) SEQ ID NO:48 - Polypeptide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278F (His tagged) MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLN YGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLY DNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNA SQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNNGDIGFIGYKQFNNIAKLVASNWYNRQIERSSRTFGCSWEFIPVDDGWGERPLHHHHHHHHHH SEQ ID NO:49 - nucleotide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278H (His tagged) SEQ ID NO:50 - Polypeptide sequence, rH C / A Mutant Y1117V F1252Y H1253K L1278H (His tagged) MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSL NYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKD LYDNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLA TNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNNGDIGFIGYKQFNNIAKLVASNWYNRQIERSSRTHGCSWEFIPVDDGWGERPLHHHHHH SEQ ID NO:51 - Polypeptide sequence, BoNT / A - UniProt P10845 MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELN EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAK LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO:52 - Polypeptide sequence, BoNT / B - UniProt P10844 SEQ ID NO:53 - Polypeptide sequence, BoNT / C - UniProt P18640 SEQ ID NO:54 - Polypeptide sequence, BoNT / D - UniProt P19321 SEQ ID NO:55 - Polypeptide sequence, BoNT / E - UniProt Q00496 SEQ ID NO:56 - Polypeptide sequence, BoNT / F - UniProt A7GBG3 SEQ ID NO:57 - Polypeptide sequence, BoNT / G - UniProt Q60393 SEQ ID NO:58 - Polypeptide sequence, TeNT - UniProt P04958 SEQ ID NO:59 - Polypeptide sequence, BoNT / X MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIP LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEG TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNNALDKRD QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAK IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNL GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWS SFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMG ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED SEQ ID NO:60 - Nucleotide sequence, mrBoNT / A SEQ ID NO:61 - Polypeptide sequence, mrBoNT / A SEQ ID NO:62 - Polypeptide sequence, unmodified BoNT / A1 SEQ ID NO:63 - Polypeptide sequence, mrBoNT / AB SEQ ID NO:64 - Polypeptide sequence, mrBoNT / AB(0) SEQ ID NO:65 - Polypeptide sequence, mrBoNT / A(0) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHQLIYAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESKHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIP KYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHAS NKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYL NSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFI GFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL EXAMPLES

[0265] Example 1

[0266] Catalytically inactive BoNT serotypes increase total neurite length compared to untreated control cells.

[0267] material and method

[0268] Five catalytically inactive (i.e., endopeptidase-inactive) botulinum neurotoxin (BoNT) serotypes were recombinantly expressed in E. coli, corresponding to serotypes A, B, C, E, and F, designated rBoNT / A(0), rBoNT / B(0), rBoNT / C(0), rBoNT / E(0), and rBoNT / F(0). As a result of being catalytically inactive, these molecules were unable to cleave their respective (SNARE) proteinaceous substrates.

[0269] Motor neuron-like hybrid cell line (NSC34 cells) (Tebu-Bio, Cedariane laboratories, France) was cultured at 5000 cells / well on black multiwell plates coated with D-lysine only and cultured in DMEM with additional 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, followed by treatment with rBoNT / A(0), rBoNT / B(0), rBoNT / C(0), rBoNT / E(0), and rBoNT / F(0) at three different concentrations, 0.1, 1, and 10 nM, 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 neurite outgrowth. Cells were fixed with paraformaldehyde 4%-sucrose 4% and subsequently stained with appropriate antibodies. In particular, anti-βIII tubulin mAb (Promega G7121) was diluted (1:1,000) in 1× PBS+2% BSA+0.3% TritonX-10 and plates were kept at 37°C for 3 h. Then, Alexa Fluor 488 goat anti-house mouse lgG(H+L) secondary antibody (Life Tech cat. A-11001) was administered (1:2,000 in 1× PBS+2% BSA+0.3% TritonX-100) for 1 h at 37°C. Nuclei were stained with DAPI. Image analysis: Six images per well were taken with a 10x objective using an ArrayScan XTI HCA reader (Thermo Fisher Scientific). All analyses were performed using Image J software (open source software from NIH, MD, USA). Three independent experiments were performed, each containing six replicates.

[0270] result

[0271] Cells were exposed to different catalytically inactive BoNT serotypes for 4 days (Figure 1). Figure 1 shows the average neurite outgrowth of NSC34 cells exposed to three different concentrations. The graph presents the average of three independent experimental runs. The average neurite outgrowth data confirms that rBoNT / A(0) increases neurite length per NSC34 cell when compared to untreated controls as well as the positive control BDNF. rBoNT / B(0), rBoNT / C(0), rBoNT / E(0), and rBoNT / F(0) were also found to increase neurite length per NSC34 cell.

[0272] Thus, these data support that the neurotrophic properties of BoNT / A are extrapolable to other BoNT serotypes.

[0273] Example 2

[0274] BoNT L chain and LH N Increases the total length of neurites relative to controls

[0275] material and method

[0276] The catalytically inactive botulinum toxin rBoNT / A(0) was recombinantly expressed in E. coli. Fragments of BoNT / A were also expressed in E. coli. These consist of the light chain (L / A), the light chain and the translocation domain (LH N / A), and a cell-binding domain fragment of the heavy chain (H C NSC34 cells were exposed to BoNT / A fragments as per Example 1 as well as full-length rBoNT / A(0).

[0277] result

[0278] Figure 2 shows the results of three different concentrations of rBoNT / A(0), rL / A, and rLH. N / A and rH C 1 shows the average neurite outgrowth of NSC34 cells exposed to 100 mM NaCl / A. The graph represents the average of three independent experiments performed.

[0279] rH C / A as well as rL / A and rLH N Both rL / A and rLH were found to increase neurite length per NSC34 cell at each concentration when compared to untreated controls as well as the positive control BDNF. N It was not anticipated that the / A fragment would be neurotrophic, since both (rH C This is because it lacks the clostridial toxin receptor binding domain (which is present in / A).

[0280] Example 3

[0281] Other proteins administered at similar concentrations as BoNT / A(0) or its fragments did not increase neurite outgrowth

[0282] material and method

[0283] NSC34 cells were differentiated and subsequently cultured for 4 days under the following experimental conditions: (1) Untreated cell control: cells were subjected to the same number of manipulations, i.e. washing / feeding, as compound-treated cells, but untreated control cells were exposed to growth medium only, (2) BDNF-positive assay control, 1 ng / ml; (3) BoNT / A(0) at three doses (0.1, 1, and 10 nM); (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 expressed lysate without botulinum neurotoxin or fragments thereof. All negative control proteins were tested at a final concentration of 1.5 μg / ml, which corresponds to 10 nM of BoNT / A(0). Protein solutions were in PBS, except for Annexin 4, which was in 20 mM Tris-HCl buffer (pH 8.0) containing 20% ​​glycerol and 0.2 M NaCl. All protein solutions were at 1 mg / ml. Cells were stained using anti-beta III-tubulin dilution 1:1,000 in 1x PBS-4% BSA-0.3% TritonX100 and secondary antibody anti-Mus musculus Alexa Fluor488, with DAPI as nuclear stain. All original images of beta 3-tubulin signal were processed using NeurophologyJ (Image J Macro, NIH, MD, USA).

[0284] result

[0285] Cells were exposed to different experimental conditions. Figure 3 shows the average neurite length in NSC34 cells. The graph presents the average of three independent experimental runs. The data for average neurite outgrowth confirm that rBoNT / A(0) increases the length of neurites per NSC34 cell compared to the untreated control, while the positive control BDNF does as well. In contrast, none of the other "negative control" conditions increased neurite length. This is consistent with the results of rL / A and rLH. N / A (as well as various BoNT serotypes and rH CThis study confirms the neurotrophic effect observed upon exposure to botulinum toxin (A) and demonstrates that the effect does not simply result from exposure of NSC34 cells to the protein or to putative residual E. coli components present in the botulinum toxin preparation.

[0286] Example 4

[0287] Treating neuronal damage in vivo

[0288] A study was designed to investigate the efficacy of catalytically inactive botulinum toxin rBoNT / A(0) in enhancing functional recovery and nerve regeneration using an in vivo mouse spinal column lesion model. This model is useful for analyzing the efficacy of molecules that induce local sprouting and / or regeneration of long axons. As is well recognized, crush injury is a common feature of spinal cord injury, and therefore this model mimics most of the pathological changes that occur in the spinal cord after trauma (for details on this model and the injury response see Lagord et al., Molecular and Cellular Neuroscience 20:69 (2002); Esmaelli et al., Neural Regeneration Research 9:1653 (2014); Surey et al., Neuroscience 275C:62 (2014); Almutiri et al., Scientific Reports 8:10707 (2018)).

[0289] material and method

[0290] Mouse model of spinal cord injury

[0291] Before surgery, C57 / BL mice were injected with buprenorphine subcutaneously and 1.8 ml / l O 2Anesthesia was administered using 5% isoflurane in the blood, and body temperature and heart rate were monitored throughout the procedure.After a partial laminectomy at thoracic level 8 (T8), ascending sensory axons, descending motor axons, and segmental proprioceptive axons (SPA) of the spinal column (SDC) were crushed bilaterally using calibrated watchmaker's forceps 1 mm deep × 1 mm wide.

[0292] Drug administration

[0293] Administration of rBoNT / A(0) was by a single 10 μl intrathecal injection (into the CSF of the spinal canal) of one of three doses (100 pg, 100 ng, and 50 μg / mouse) at the time of surgery. Treatment groups for each of the three doses were as follows: 1. Distensor (phosphate-buffered saline [PBS]), ie, SDC injury plus immediate single 10 μl intrathecal injection of distensor, n = 6 mice. 2. Mice treated with BoNT, i.e., SDC lesion 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.

[0294] Intrathecal injections of BoNT were performed as follows: mice were placed in a prone position and injections were made between the L5 and S1 vertebrae. The spinous process was incised and reflected rostrally to expose the ligamentum flavum, and a blunt 25G needle was inserted horizontally at 60° through the ligamentum flavum, and access to the intrathecal space was confirmed by the presence of cerebrospinal fluid (CSF) reflux and a "tail flick." 10 μl of injection solution was then slowly injected over 1 min, and the tail was gently lifted to encourage CSF expression.

[0295] Measured Endpoints 1. Locomotor function was measured using the horizontal ladder walking test at baseline (pre-injury) and again 2 days, 1 week, 2 weeks, 3 weeks, and 4 weeks after SDC injury. 2. Qualitative histological assessment of sprouting and regeneration from motor and sensory neurons / axons at 4 weeks, i.e., axonal growth over short (<1mm) and long (~5mm) distances. Tissue sections stained for Neurofilament200 (NF200) detect mature axons. Phosphorylated MAP1b is present within growing axons and growth cones, where it maintains a dynamic balance between cytoskeletal components and regulates the stability and interactions of cellular microtubules and actin, promoting axonal growth, neural connections, and regeneration in the central nervous system. Staining for MAP1b reveals areas of active axonal sprouting.

[0296] Horizontal ladder test

[0297] This tests locomotor function and is performed on a 0.6 m long horizontal ladder, 8 cm wide with randomly aligned rungs with variable spacing of 1-2 cm. Mice were assessed for ladder traversal before injury and again 2 days, 1 week, 2 weeks, 3 weeks, and 4 weeks after SDC injury, and the number of left and right hindlimb slips was recorded along with the total number of steps taken by careless individuals in the treatment group. To calculate the mean error rate, the number of slips was divided by the total number of steps.

[0298] Tissue preparation and cryosectioning

[0299] Four weeks after SDC injury, mice were perfused intracardially with 4% formaldehyde (Raymond A Lamb, Peterborough, UK) and transected sections of T8 spinal cord containing the DC injury site (lesion site + 5 mm on either side) along with the tibialis capitis muscle were postfixed for 2 h at room temperature, cryosectioned in a graded sucrose series, trapped in optimal cutting temperature medium (OCT, Raymond A Lamb) and sectioned at 15 μm thickness using a Bright cryostat.

[0300] Immunocytochemical status

[0301] Sections were thawed for 30 min at room temperature before being washed twice in 0.1 M phosphate-buffered saline, pH 7.4 (PBS, Raymond A Lamb). Sections were then permeabilized for 10 min in 0.1% TritonX100 in PBS (Sigma) and trapped in PBS containing 0.5% bovine serum albumin (BSA) and 0.1% Triton-X100 (all from Sigma) for 30 min at room temperature. Sections were then incubated with the appropriate primary antibody diluted in antibody dilution buffer (ADB, PBS containing 0.5% BSA and 0.05% Tween-20 (all from Sigma)) and incubated overnight at 4°C in a humidified chamber. Sections were then washed in PBS and incubated with the appropriate fluorescently labeled secondary antibody diluted in ADB. Sections were then washed in PBS and coverslip mounted using Vectashield with DAPI (Vector Laboratories, Peterborough, UK). Negative controls were included in each run involving omission of the primary antibody, and these were used to set background threshold levels for image capture. Sections were viewed and images were captured using an Axioplan2 epifluorescence microscope equipped with an AxiocamHRc running Axiovision software.

[0302] The primary antibodies used were as follows: Rabbit anti-NF200, Sigma, Poole, UK (1:300 dilution) Rabbit MAP1b, Abcam, Cambridge, UK (1:400 dilution)

[0303] The secondary antibodies used were as follows: Alexa488 anti-rabbit lgG Invitrogen, Paisley, UK (1:400 dilution) Alexa594 anti-rabbit lgG Invitrogen, Paisley, UK (1:400 dilution)

[0304] statistics

[0305] Statistical analysis of functional data was performed using SPSS20 (IBM, USA). A normal distribution test was performed to determine the most appropriate statistical analysis for comparing each treatment. Statistical significance was determined at p<0.05.

[0306] result

[0307] Figure 4 shows that administration of rBoNT / A(0) reduced the magnitude of spinal cord injury-induced locomotor deficits at 2 days compared to extensor controls for doses of 100 pg and 100 ng. Administration of rBoNT / A(0) significantly reduced spinal cord injury-induced locomotor deficits and recovery rates at 4 weeks compared to extensor controls at all doses tested. Furthermore, the effect was more pronounced when rBoNT / A(0) was administered intrathecally than intraspinally (data not shown).

[0308] Immunohistochemical evaluation employed the use of antibodies to Neurofilament200 (NF200) and MAP1b. Neurofilament200 (NF200) is expressed in mature axons, and pMAP1b antibody reveals neurofilaments at the terminals of actively sprouting axons, representing axons still actively sprouting around and within the lesion site.

[0309] Figure 5A shows that many NF200 stained axons are visible surrounding the lesion in stretch-treated animals, with very few, if any, NF200+ axons present within the core of the lesion in untreated animals, whereas many NF200 stained axons are visible surrounding the lesion in rBoNT / A(0)-treated animals, with many more NF200+ axons also visible within the core of the lesion.

[0310] Figure 5B shows that a few MAP1b-stained sprouting axons are visible surrounding the lesion in stretch-treated animals, with very few, if any, MAP1b axons present within the lesion core, whereas MAP1b staining reveals bright red axonal sprouting surrounding the lesion.

[0311] The rapid onset of improved performance on functional tests indicates that rBoNT / A(0) caused axonal sprouting with the establishment of useful functional synapses below the lesion. Qualitative immunohistochemistry provided evidence of localized BoNT-induced bright red axonal sprouting through the SDC lesion site.

[0312] These in vivo data provide clear evidence validating a role for rBoNT / A(0) in the treatment of neurological disorders.

[0313] Example 5

[0314] Effect of catalytically inactive full-length recombinant BoNT, BoNT fragments, and mutants on the number of neurites per cell

[0315] Several catalytically inactive full-length recombinant BoNT serotypes as well as BoNT fragments and mutants were tested for their modulatory effects on neurite outgrowth in vitro.

[0316] material and method

[0317] Cells exposed to the polypeptides were compared to those exposed to a positive control (1 ng / ml BDNF). Mouse motor neuron-like hybrid (NSC34) cells were differentiated and exposed to different polypeptides at three different doses (0.1 nM, 1 nM, and 10 nM) for 4 days ex vivo (DIV).

[0318] NSC34 cells were generated by fusion of mouse neuroblastoma with embryonic mouse spinal cord cells enriched for motor neurons (Cashman et al., Dev Dyn. ("Developmental Dynamics") Jul;194(3):209-21, (1992), incorporated herein by reference). The cells mimic many properties of motor neurons, including choline acetyltransferase, acetylcholine synthesis, storage, release, and neurofilament triple protein. In addition, NSC34 spinal motor neurons express glutamate receptor proteins and generate action potentials. NSC34 neurons have been used extensively to study mechanisms of neuronal signaling and neuronal degeneration.

[0319] The following experimental scheme was adopted: Selection against a neuronal cell line (NSC34).

number

[0320] NSC34 cells were cultured on poly-D-lysine-coated glass coverslips in DMEM supplemented with 10% FCS.

[0321] After plating, cells were differentiated into motor neurons by exposure to retinoic acid and low serum levels for 4 days. Cells were cultured in either the presence or absence of polypeptides at specific time points (i.e., 4 DIV). Test data were compared to the effects seen in positive (BDNF) and negative (BSA) controls.

[0322] After 4 days in vitro (DIV), cells were fixed in 4% paraformaldehyde, stained with a specific neuronal marker (beta-tubulin), and quantitatively assayed for neurite outgrowth (neurite spreading, axonal extension, and dendritic branching). Images were acquired using an Operetta CLS HCS microscope (PerkinElmer) with a 20x objective. Six (6) fields were acquired per well. Analysis of neurite outgrowth was performed to assess the average neurites per cell.

[0323] result

[0324] Figures 6-10 represent the average number of neurites counted on each cell assessed in three independent experimental runs. Data were normalized to untreated control cells. Compared to BSA, the polypeptides statistically significantly increased the number of neurites per cell.

[0325] For BoNT / A, the cell-binding domain (H C domain) fragments, compared to LH N The / A fragment (light chain plus the translocation domain) improved activity (see FIG. 6).

[0326] For both BoNT / FA and BoNT / F, C Compared to the domain fragment, LH N and the LC (light chain only) fragment showed improved activity (see Figures 7 and 8).

[0327] Finally, the mutant H C The domain fragments all showed high efficacy (Figures 9 and 10), and cationic H C The / A domain (SEQ ID NO: 42 - FIG. 9) displayed exceptional activity, which was improved against BDNF at two of three concentrations. C The high activity of the / A domain is expected to be evident in full-length polypeptides containing said domain (whether catalytically inactive or active).

[0328] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred forms of implementation, it should be understood that the invention as claimed should not be unduly limited to those specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and bioengineering or related fields are intended to be within the scope of the following claims.

[0329] item 1. A polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, comprising a Clostridial neurotoxin light chain (L chain) or a fragment thereof, and / or a fragment of a Clostridial neurotoxin heavy chain (H chain). 2. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, comprising administering to the patient a polypeptide, said polypeptide comprising: a Clostridial neurotoxin L chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin H chain. 3. Use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, said polypeptide comprising: a Clostridial neurotoxin L chain or a fragment thereof, and / or a fragment of a Clostridial neurotoxin H chain. 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. 5. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide essentially 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). 6. A polypeptide for use, method or use according to any one of the preceding claims, 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). 7. 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. 8. The fragment of the Clostridial neurotoxin H chain is H N 8. A polypeptide for use, method or use according to any one of the preceding items comprising the domain or a fragment thereof. 9. The fragment of the Clostridial neurotoxin H chain is H N 8. A polypeptide for use, method or use according to any one of the preceding items, consisting of a domain or a fragment thereof. 10. The fragment of the Clostridial neurotoxin H chain is H C 8. A polypeptide for use, method or use according to any one of the preceding items comprising the domain or a fragment thereof. 11. The fragment of the Clostridial neurotoxin H chain is H C 8. A polypeptide for use, method or use according to any one of the preceding items, consisting of a domain or a fragment thereof. 12. The polypeptide is a C-terminal portion of the Clostridial neurotoxin receptor binding domain (H CC 5. A polypeptide for use, method or use according to any one of the preceding claims, lacking: 13. The polypeptide is Clostridial neurotoxin H N Domain and H C 8. A polypeptide for use, method or use according to any one of the preceding items, which does not comprise both of the domains. 14. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide does not further comprise a non-clostridial catalytic domain. 15. The polypeptide comprises a Clostridial neurotoxin L chain or a fragment thereof, and N 8. A polypeptide for use, method or use according to any one of the preceding items comprising the domain or a fragment thereof. 16. The polypeptide comprises a Clostridial neurotoxin L chain or a fragment thereof, and N 8. A polypeptide for use, method or use according to any one of the preceding items, consisting of a domain or a fragment thereof. 17. The polypeptide comprises a Clostridial neurotoxin L chain and an H chain. N 8. A polypeptide for use, method or use according to any one of the preceding items, comprising the domain. 18. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, or 49; or b. Comprising (preferably consisting of) a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, or 50. 19. A polypeptide for use, a method or a use according to any one of the preceding items, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, or 49; or b. Comprising (preferably consisting of) a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, or 50. 20. A polypeptide for use, a method or a use according to any one of the preceding items, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, or 49; or b. Comprising (preferably consisting of) a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, or 50. 21. A polypeptide for use, a method or a use according to any one of the preceding items, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, or 49; or b. Comprising (preferably consisting of) a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, or 50. 22. A polypeptide for use, a method or a use according to any one of the preceding items, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, or 49; or b. Comprising (preferably consisting of) a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, or 50. 23. A polypeptide for use, a method or a use according to any one of the preceding items, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 99.9% sequence identity to any one of SEQ ID NOs: 3, 5, 7, 19, 21, 23, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, or 49; or b. Comprising (preferably consisting of) a polypeptide sequence having at least 99.9% sequence identity to any one of SEQ ID NOs: 4, 6, 8, 20, 22, 24, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, or 50. 24. A polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain. 25. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, comprising administering to the patient a polypeptide, said polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain. 26. Use of a polypeptide comprising a catalytically inactive Clostridial neurotoxin L chain in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient. 27. A polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:42 and / or a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:41. 28. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, comprising administering to the patient a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and / or the polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41. 29. Use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, wherein said polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and / or said polypeptide comprises a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41. 30. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 41. 31. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 41. 32. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 41. 33. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 41. 34. The polypeptide for use, method or use according to any one of items 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. 35. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 60. 36. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60. 37. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 60. 38. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 60. 39. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 60. 40. The polypeptide for use, method or use according to any one of items 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 the polypeptide is encoded by a nucleotide sequence having at least 99.9% sequence identity to SEQ ID NO: 60. 41. A polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64. 42. A method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, comprising administering to the patient a polypeptide, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64. 43. Use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a patient, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64. 44. A polypeptide for use, a method or a use according to any one of items 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. 45. A polypeptide for use, a method or a use according to any one of items 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. 46. ​​The polypeptide for use, method or use according to any one of items 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. 47. A polypeptide for use, a method or a use according to any one of items 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. 48. A polypeptide for use, a method or a use according to any one of items 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. 49. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide does not comprise a native Clostridial neurotoxin heavy chain. 50. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is neurotrophic. 51. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide promotes neuronal growth and / or neuronal repair. 52. A polypeptide for use, method or use according to any one of the preceding items, wherein said neurological disorder is a disorder treatable by promoting neuronal growth and / or repair. 53. A polypeptide for use, method or use according to any one of the preceding items, wherein the neurological disorder is neuronal damage, a neurodegenerative disorder, a sensory disorder or an autonomic neuropathy. 54. The polypeptide for use, method or use according to any one of the preceding items, wherein the neurological disorder is neuronal damage selected from nerve trauma (e.g. resulting from injury and / or fracture), neuropathy (e.g. peripheral neuropathy), spinal cord injury (including, e.g., paralysis), nerve transection, brain injury (e.g. traumatic brain injury), non-traumatic injury (e.g. stroke or spinal cord infarction), and injury to the brachial plexus, e.g. Erb's palsy or Klumpke's palsy. 55. The polypeptide for use, method or use according to any one of the preceding items, wherein the neurological disorder is a neurodegenerative disorder selected from Alzheimer's disease, Parkinson's disease, Parkinson's related disorders, motor neuron diseases, peripheral neuropathies, motor neuron neuropathy, prion diseases, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, monolimbic muscular atrophy, Friedreich's ataxia, Hallervorden-Spatz disease, and frontotemporal lobe dysplasia. 56. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide promotes growth or repair of motor neurons. 57. The polypeptide for use, method, or use according to any one of the preceding items, wherein the polypeptide is a modified Clostridial neurotoxin, such as a chimeric Clostridial neurotoxin or a hybrid Clostridial neurotoxin. 58. A polypeptide for use, a method or a use according to any one of items 24 to 34 or 49 to 57, wherein the polypeptide is catalytically inactive and: a. encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 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 b. Comprising (preferably consisting of) a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 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. 59. A polypeptide for use, a method or a use according to any one of items 24 to 34 or 49 to 58, wherein the polypeptide is catalytically inactive and: a. encoded by a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 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 b. Comprising (preferably consisting of) a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 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. 60. A polypeptide for use, a method or a use according to any one of items 24 to 34 or 49 to 59, wherein the polypeptide is catalytically inactive and: a. encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 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 b. Comprising (preferably consisting of) a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 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. 61. A polypeptide for use, a method or a use according to any one of items 24 to 34 or 49 to 60, wherein the polypeptide is catalytically inactive and: a. encoded by a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 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 b. Comprising (preferably consisting of) a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 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. 62. A polypeptide for use, a method or a use according to any one of items 24 to 34 or 49 to 61, wherein the polypeptide is catalytically inactive and: a. encoded by a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 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 b. comprising (preferably consisting of) a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 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. 63. A polypeptide for use, a method or a use according to any one of items 24 to 34 or 49 to 62, wherein the polypeptide is catalytically inactive and: a. encoded by a nucleotide sequence having at least 99.9% sequence identity to any one of SEQ ID NOs: 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 b. comprises (preferably consists of) a polypeptide sequence having at least 99.9% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 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. 64. A polypeptide for use, a method or a use according to any one of items 24 to 26 or 49 to 63, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, or 33; or b. comprising (preferably consisting of) a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65. 65. A polypeptide for use, a method or a use according to any one of items 24 to 26 or 49 to 64, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, or 33; or b. comprising (preferably consisting of) a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65. 66. A polypeptide for use, a method or a use according to any one of items 24 to 26 or 49 to 65, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, or 33; or b. comprising (preferably consisting of) a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65. 67. A polypeptide for use, a method or a use according to any one of items 24 to 26 or 49 to 66, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, or 33; or b. comprising (preferably consisting of) a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65. 68. A polypeptide for use, a method or a use according to any one of items 24 to 26 or 49 to 67, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, or 33; or b. comprising (preferably consisting of) a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65. 69. A polypeptide for use, a method or a use according to any one of items 24 to 26 or 49 to 68, wherein the polypeptide is: a. encoded by a nucleotide sequence having at least 99.9% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, or 33; or b. comprising (preferably consisting of) a polypeptide sequence having at least 99.9% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64, or 65. 70. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is administered at or near the site of injury, preferably the polypeptide is administered intrathecally. 71. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide does not further comprise a domain that binds to a cellular receptor. 72. The polypeptide is a functional H of a Clostridial neurotoxin. C 8. A polypeptide for use, method or use according to any one of the preceding items, lacking the domain and also lacking any functionally equivalent exogenous ligand targeting moiety (TM). 73. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is not expressed in cells of the patient. 74. A polypeptide for use, method or use according to any one of the preceding items, 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). 75. The polypeptide for use, method, or use of any one of the preceding items, wherein the polypeptide further comprises one or more non-clostridial neurotoxin sequences. 76. The polypeptide for use, method or use according to item 75, wherein the one or more non-clostridial neurotoxin sequences do not bind to a cellular receptor. 77. A polypeptide for use, method or use according to item 75 or 76, wherein the one or more non-clostridial neurotoxin sequences do not comprise a ligand for a cellular receptor. 78. A polypeptide, method or use according to items 1 to 40 or 49 to 77, wherein the polypeptide is a modified BoNT / A or a fragment thereof comprising a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, wherein the modification is selected from: i. replacement of acidic surface-exposed amino acid residues with basic amino acid residues; ii. replacement of acidic surface exposed amino acid residues with uncharged amino acid residues; iii. replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue; and v. Deletion of acidic surface exposed amino acid residues. 79. The polypeptide comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C8. The polypeptide, method or use according to items 1 to 26 or 41 to 77, wherein the BoNT is a chimeric BoNT comprising a β-actin domain. [Explanation of symbols]

[0330] [Figures 1, 2, 3] Neurite Length per Cell: Neurite length per cell Untreated control: Untreated control [Figure 3] Negative controls: Annexin IV: Annexin IV Ubiquitin: E. coli lysate: Escherichia coli lysate [Figure 4] Mean ratio (slips / total steps): Vehicle control: Baseline: Baseline value 2d: 2 days week: week [Figure 5] Vehicle Control: Vehicle control Animal: Animal lesion: damage [Figure 6] Number of neurites / cell: Number of neurites / cell SBA Control

Claims

1. A pharmaceutical composition for treating a neurological disorder in a patient, comprising a polypeptide: (i) comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 63, 61, 44, or 42; (ii) comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43, 60, or 41; and / or (iii) a two-chain polypeptide in which a light chain (L chain) of the polypeptide is linked to a heavy chain (H chain) of the polypeptide by a disulfide bond, the two-chain polypeptide being obtainable by a process comprising contacting a single-chain polypeptide comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:63 or SEQ ID NO:61 with a protease that hydrolyzes a peptide bond in the activation loop of the single-chain polypeptide, thereby converting the single-chain polypeptide into the corresponding two-chain polypeptide; and The amount of the polypeptide administered to the patient is at least 50 ng.

2. The pharmaceutical composition of claim 1, wherein the amount of the polypeptide administered to a patient is 500 ng or less.

3. The pharmaceutical composition of claim 1 or 2, wherein the neurological disorder is a sensory disorder, neuronal injury, or a neurodegenerative disorder.

4. The pharmaceutical composition described in claim 3, wherein the sensory disorder is selected from pain, sensory neuropathy, sensorimotor polyneuropathy, diabetic neuropathy, Brown-Séquard syndrome, Charcot-Marie-Tooth disease, and Devic's syndrome.

5. A pharmaceutical composition for treating a neurological disorder in a patient, comprising a polypeptide, wherein the polypeptide is (i) comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 63, 61, 44, or 42; (ii) comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43, 60, or 41; and / or (iii) a two-chain polypeptide in which a light chain (L chain) of the polypeptide is linked to a heavy chain (H chain) of the polypeptide by a disulfide bond, the two-chain polypeptide being obtainable by a process comprising contacting a single-chain polypeptide comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:63, or SEQ ID NO:61, with a protease that hydrolyzes a peptide bond in the activation loop of the single-chain polypeptide, thereby converting the single-chain polypeptide into the corresponding two-chain polypeptide; the neurological disorder is a sensory disorder, neuronal injury, or a neurodegenerative disorder; and The sensory disturbance is not pain.

6. The pharmaceutical composition of claim 5, wherein the amount of said polypeptide administered to a patient is 100 pg to 500 ng.

7. The pharmaceutical composition described in claim 5, wherein the sensory disorder is selected from sensory neuropathy, sensorimotor polyneuropathy, diabetic neuropathy, Brown-Séquard syndrome, Charcot-Marie-Tooth disease, and Devic's syndrome.

8. The pharmaceutical composition of claim 3 or 5, wherein (i) the neuronal injury is selected from neurotrauma, neuropathy, spinal cord injury, nerve transection, brain injury, non-traumatic injury, and injury to the brachial plexus; or (ii) the neurodegenerative disorder is selected from Alzheimer's disease, motor neuron disease, peripheral neuropathies, motor neuron neuropathies, prion diseases, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, monolimbic muscular atrophy, Friedreich's ataxia, Hallervorden-Spatz disease, and frontotemporal lobe dysplasia.

9. The pharmaceutical composition of claim 8, wherein the neurological trauma results from injury and / or fracture; the neuropathy is a peripheral neuropathy; the spinal cord injury includes paralysis; the brain injury is a traumatic brain injury; the non-traumatic injury is a stroke or spinal cord infarction; or the injury to the brachial plexus is Erb's palsy or Klumpke's palsy.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the polypeptide is neurotrophic.

11. A pharmaceutical composition described in any of claims 1 to 10, wherein the polypeptide promotes neuronal growth and / or neuronal repair to treat the neurological disorder.

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