Cationic neurotoxins

Abstract

To provide clostridial toxins increased in tissue retention at an administration site compared to known clostridial toxins.SOLUTION: The present invention provides an engineered clostridial toxin comprising at least one amino acid modification, where the at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is higher than the pI of a clostridial toxin that is identical except for lacking the at least one amino acid modification, by at least 0.2 pI units. Also provided are corresponding uses of the engineered clostridial toxin in therapy.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to modified Clostridium toxins comprising at least one amino acid modification, and the pharmaceutical and therapeutic use of such modified Clostridium toxins. [Background technology]

[0002] Clostridium bacteria produce highly potent and specific protein toxins capable of poisoning neurons and other cells upon delivery. Examples of these Clostridium toxins include neurotoxins produced by C. tetani (TeNT) and C. botulinum (BoNT) serotype AG, as well as those produced by C. baratii and C. butyricum.

[0003] Some Clostridium toxins are known to be among the most potent. For example, botulinum neurotoxin has a median lethal dose (LD50) in mice. 50 The serotype ranges from 0.5 to 5 ng / kg. Both tetanus and botulinum toxin act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction, inhibiting cholinergic transmission in the peripheral nervous system, while tetanus toxin acts in the central nervous system.

[0004] Originally, Clostridium toxin is synthesized as a single-chain polypeptide, and after translation, it is modified by a protein cleavage event, forming two polypeptide chains linked together by disulfide bonds. The cleavage occurs at specific cleavage sites located between cysteine ​​residues that result in interchain disulfide bonds. This double-chain form is the active form of the toxin. These double chains are called the heavy chain (H chain) with a molecular weight of approximately 100 kDa and the light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain has an N-terminal transposition component (H N Domain) and C-terminal targeting component (H CIt includes the domain. The cleavage site is located between the L chain and the transposition domain component. C Following the binding of the domain to target neurons and the internal translocation of the binding toxin into the cell via endosomes, H N The domain translocates the light chain (L) across the endosomal membrane into the cytosol, where it provides protease function (also known as a non-cytotoxic protease).

[0005] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin). See Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE stands for Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and therefore essential for the secretion of molecules via vesicle transport from cells. The protease function is zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Therefore, when delivered to the desired target cells, non-cytotoxic proteases can inhibit cellular secretion from those target cells. Clostridium toxin light chain proteases are non-cytotoxic proteases that cleave SNARE proteins.

[0006] Because SNARE proteins are ubiquitous, the use of Clostridium toxins, such as botulinum toxin, has been successful in a wide range of therapeutic applications.

[0007] For example, William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004) describes the use of Clostridium toxins, such as botulinum neurotoxin (BoNT), BoNT / A, BoNT / B, BoNT / C1, BoNT / D, BoNT / E, BoNT / F, and BoNT / G, which inhibit neuronal signaling, as well as tetanus neurotoxin (TeNT), in various therapeutic and cosmetic applications. TMIt is currently approved as a treatment for the following conditions: achalasia, adult spasticity, anal fissure, back pain, blepharospasm, bruxism, cervical dystonia, essential tremor, frown lines or hyperactive facial wrinkles, headache, hemifacial spasm, hyperkinesia, hyperhidrosis, childhood cerebral palsy, multiple sclerosis, myoclonus disorders, nasolabial folds, spasmodic dysphonia, strabismus, and VII neuropathy. In addition, Clostridium toxin therapy is also described for the treatment of: neuromuscular disorders (see US6,872,397), uterine disorders (see US2004 / 0175399), ulcers and gastroesophageal reflux disease (see US2004 / 0086531), dystonia (see US6,319,505), eye disorders (see US2004 / 0234532), blepharospasm (see US2004 / 0151740), strabismus (see US2004 / 0126396), pain (see US6,869,610, US6,641,820, US6,464,986, and US6,113,915), and fibromyalgia (see US6,623,742, US (See 2004 / 0062776), treatment of lower back pain (see US2004 / 0037852), treatment of muscle injuries (see US6,423,319), treatment of sinusitis headaches (see US6,838,434), treatment of tension headaches (see US6,776,992), treatment of headaches (see US6,458,365), reduction of migraines (see US5,714,469), treatment of cardiovascular diseases (see US6,767,544), treatment of neurological disorders such as Parkinson's disease (see US6,620,415, US6,306,403), treatment of neuropsychiatric disorders (see US2004 / 0180061, US (See 2003 / 0211121), treatment of endocrine disorders (see US6,827,931), treatment of thyroid disorders (see US6,740,321), treatment of cholinergic-affected sweat gland disorders (see US6,683,049), treatment of diabetes (see US6,337,075, US 6,416,765), treatment of pancreatic disorders (see US6,261,572, US6,143,306), treatment of cancer such as bone tumors (see US6,565,870, US6,368,605, US6,139,845, US2005 / 0031648), treatment of ear disorders (see US6,358,926, US6,265,379), treatment of autonomic nerve disorders such as gastrointestinal muscle disorders and other smooth muscle dysfunction (see US5,(See US 437, 291), treatment of skin lesions with impaired skin cell proliferation (see US 5,670, 484), management of neurogenic inflammatory disorders (see US 6,063, 768), reduction of hair loss and stimulation of hair growth (see US 6,299, 893), treatment of downward-turning corners of the mouth (see US 6,358, 917), reduction of appetite (see US 2004 / 40253274), dental treatments and procedures (see US 2004 / 0115139). ), treatment of neuromuscular disorders and conditions (see US2002 / 0010138), treatment of various disorders and conditions and associated pain (see US2004 / 0013692), treatment of conditions caused by excessive mucus secretion such as asthma and COPD (see WO00 / 10598), and treatment of non-neuronal conditions such as inflammation, endocrine conditions, exocrine conditions, immunological conditions, cardiovascular conditions, and bone conditions (see WO01 / 21213). All of the above-mentioned published documents shall be considered part of this specification in their entirety by clearly indicating the source.

[0008] The use of non-cytotoxic proteases such as Clostridium toxins (e.g., BoNT and TeNT) in therapeutic and cosmetic procedures in humans and other mammals is expected to expand, along with the range of diseases and conditions for which the properties of these toxins may be beneficial.

[0009] To avoid systemic neurological effects, many Clostridium toxin therapies utilize direct administration of Clostridium toxin therapeutic agents to designated target sites (target tissues, etc.). A problem with administering Clostridium toxin-based therapeutic agents in this manner is the spread of the toxin from the administration site into surrounding tissues or the systemic circulation. Toxin diffusion from target tissues is thought to cause undesirable side effects, potentially life-threatening in extreme cases. This is a particular concern when using Clostridium toxin therapeutic agents (BoNT therapeutic agents) at high doses, concentrations, and infusion volumes. Side effects associated with these problems reported for commercially available BoNT / A therapeutic agents include asthenia, generalized muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and dyspnea. Dysphagia and dyspnea can be life-threatening, and deaths associated with the spread of toxic effects have been reported. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] Therefore, in this field, there is a need for a Clostridium toxin that exhibits increased tissue retention at the administration site and thus reduced diffusion from the administration site compared to known Clostridium toxins. [Means for solving the problem]

[0012] This invention solves the above-mentioned problems by providing a modified Clostridium toxin as specified in the claims. [Modes for carrying out the invention]

[0013] In one aspect, the present invention provides a modified Clostridium toxin comprising at least one amino acid modification, wherein the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of the same Clostridium toxin except for lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the modified Clostridium toxin to a value that is at least 0.4 pI units higher than the pI of the same Clostridium toxin except for lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the modified Clostridium toxin to a value that is at least 0.5 pI units higher than the same Clostridium toxin except for lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the modified Clostridium toxin to a value that is at least 0.6 pI units higher than the pI of the same Clostridium toxin except for lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the modified Clostridium toxin to a value that is at least 0.8 pI units higher than the pI of the same Clostridium toxin except for lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the modified Clostridium toxin to a value that is at least 1 pI unit higher than the pI of the same Clostridium toxin except for lacking the at least one amino acid modification.

[0014] In one aspect, the present invention provides a modified Clostridium toxin comprising at least one amino acid modification, wherein the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin to a value that is at least 1 pI unit higher than the pI of the same Clostridium toxin except for lacking the at least one amino acid modification.

[0015] In certain embodiments, the modified Clostridium toxin comprises at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 amino acid modifications.

[0016] In certain embodiments, the at least one amino acid modification increases the pI of the modified Clostridium toxin to a value that is at least 2, 3, 4, or 5 pI units higher than the pI of the same Clostridium toxin except lacking the at least one amino acid modification.

[0017] The inventors have found that by increasing the pI of a Clostridium toxin, for example, by at least 0.2 pI units, or 0.5 pI units, or 1 pI unit (by introduction of at least one amino acid modification into the Clostridium toxin protein), the resulting modified Clostridium toxin has the advantage of increased tissue retention and decreased diffusion from the site of administration, while maintaining the ability to bind to, translocate into, and cleave target SNARE protein(s). Thus, the spread of the Clostridium toxin from the site of administration is significantly reduced compared to the same Clostridium toxin except lacking the at least one amino acid modification.

[0018] The modified Clostridium toxin of the present invention is suitable for use in any of the above-described therapies and has the advantage that it may exhibit a reduction or absence of side effects compared to the use of known Clostridium toxin therapeutics. The increased tissue retention of the modified Clostridium toxin of the present invention further results in an increase in efficacy and / or duration of action, making it further advantageous as it allows for a reduction in the dose to be used (or an increase in the dose without adding any additional side effects) compared to known Clostridium toxin therapeutics.

[0019] As will be described in more detail below, the increase in pI resulting from at least one amino acid modification means that the modified Clostridium toxin of the present invention has a higher positive net charge at a given pH than the net charge of the same Clostridium toxin except for the lack of the at least one amino acid modification. While we do not wish to be bound by any one theory, we believe that this increased positive charge enables the modified Clostridium toxin of the present invention to exhibit a longer tissue retention time at the administration site due to good electrostatic interactions between the modified Clostridium toxin and anionic extracellular components (such as cell membranes and heparan sulfate proteoglycans) at the administration site. This improved electrostatic interaction plays a role in reducing the diffusion of the modified Clostridium toxin from the administration site and thus improves tissue retention.

[0020] For example, the improved tissue retention properties of the modified Clostridium toxin of the present invention enable (i) high doses to be administered to individual muscles such as the sternocleidomastoid muscle without spreading to muscles near the neck and causing dysphagia, and (ii) high total doses (to all muscles) in a single treatment without spreading into the circulation and causing systemic effects such as respiratory distress. Benefits for patients include increased efficacy in treating large muscles such as the sternocleidomastoid muscle, increased opportunities to inject into several different muscles during each treatment, and a longer duration of effective treatment due to the high dose (longer time before retreatment is needed).

[0021] In one embodiment, the modified Clostridium toxin of the present invention has a positive net charge during use (for example, when the modified Clostridium toxin is located at the desired administration site in the tissue during use).

[0022] The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are formed by specific sequences of amino acids (also called amino acid residues in proteins). Each of the 20 standard sets of amino acids 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 are determined by the sum of these various factors.

[0023] Certain amino acid residues (detailed below) have ionizable side chains that can exhibit a charge depending on the surrounding pH. Whether or not these side chains become charged at a given pH depends on the pKa of the relevant ionizable moiety, where pKa is the negative logarithm of the acid dissociation constant (Ka) for a particular proton from the conjugate base.

[0024] For example, acidic residues such as aspartic acid and glutamic acid have side-chain carboxylic acid groups with a pKa value of approximately 4.1 (the exact pKa value depends on temperature, ionic strength, and the microenvironment of the ionizable groups). Therefore, these side chains exhibit a negative charge at pH 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains are protonated and lose their charge.

[0025] Conversely, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values ​​of approximately 10 to 12. Therefore, these side chains exhibit a positive charge at pH 7.4. At higher pH values, these side chains are deprotonated and lose their charge.

[0026] Therefore, the overall (net) charge of a protein molecule is determined by the number of acidic and basic residues present in the protein (and the degree to which they are exposed to the surface) and the surrounding pH. Changing the surrounding pH changes the overall charge of the protein. Thus, for all proteins, there is a certain pH at which the number of positive and negative charges becomes equal and the protein no longer exhibits an overall net charge. This point is known as the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry that those skilled in the art will be familiar with.

[0027] Therefore, the isoelectric point (pI) is defined as the pH value at which a protein exhibits a net charge of zero. An increase in pI means that the protein requires a higher pH value to exhibit a net charge of zero. Thus, an increase in pI represents an increase in the net positive charge of the protein at a given pH. Conversely, a decrease in pI means that the protein requires a lower pH value to exhibit a net charge of zero. Thus, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH.

[0028] Methods for determining the pI of a protein are known in the art and are well known to those skilled in the art. For example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein. Alternatively, the pI of a protein can be experimentally determined using isoelectric focusing. In this method, proteins are separated by their pI using electrophoresis. Isoelectric focusing is generally performed using a gel with a fixed pH gradient. When an electric field is applied, the protein moves through the pH gradient to a pH where it has a net charge of zero, and this point becomes the pI of the protein.

[0029] The pI of a protein can be increased or decreased by altering the number of basic and / or acidic groups present on its surface. This can be achieved by modifying one or more amino acids in the protein. For example, an increase in pI can be achieved by reducing the number of acidic residues or increasing the number of basic residues. Such amino acid modifications will be discussed in more detail below.

[0030] The pI of natural (unmodified) Clostridium toxin is approximately 5 to 6. Therefore, at pH 7.4, natural botulinum toxin has a negative net charge. For example, the pI of BoNT / A is 6.4, and the BoNT / A molecule has a net charge of -8 at pH 7.4. These pI values ​​are calculated as described above.

[0031] [Table 1]

[0032] As described above, in one embodiment, the modified Clostridium toxin of the present invention comprises at least one amino acid modification, the at least one amino acid modification increasing the isoelectric point (pI) of the modified Clostridium toxin to a value at least 0.2 pI higher than that of the same Clostridium toxin except that lacks the at least one amino acid modification.

[0033] Therefore, in the present invention, a 0.2 unit increase in pI in the modified BoNT / A Clostridium toxin results in an increase in pI from 6.4 to 6.6.

[0034] As described above, in one embodiment, the modified Clostridium toxin of the present invention comprises at least one amino acid modification, the at least one amino acid modification increasing the isoelectric point (pI) of the modified Clostridium toxin to a value at least 1 pI higher than that of the same Clostridium toxin except that lacks the at least one amino acid modification.

[0035] Therefore, in the present invention, an increase of one unit of pI in the modified BoNT / A Clostridium toxin corresponds to an increase in pI from 6.4 to 7.4.

[0036] In one embodiment, the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin, making it at least 2 pI units higher than that of the same Clostridium toxin except that it lacks the at least one amino acid modification.

[0037] In one embodiment, the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin, making it 2 to 5 pI units higher than that of the same Clostridium toxin except that lacks the at least one amino acid modification.

[0038] In one embodiment, the modified Clostridium toxin has a pI of at least 6 (e.g., at least 6, at least 7, at least 8, or at least 9).

[0039] In one embodiment, the modified Clostridium toxin has a pI of at least 7.

[0040] In one embodiment, the modified Clostridium toxin has a pI of 6 to 10 (e.g., 7 to 9, or 8 to 9).

[0041] As described above, the modified Clostridium toxin of the present invention has increased tissue retention, which further increases potency and / or duration of action, making it possible to reduce the dose to be used (or increase the dose without adding any side effects) compared to known Clostridium toxin therapeutics. One way in which these advantageous properties (meaning an increase in the therapeutic index) can be defined is in terms of the safety margin of the modified Clostridium toxin. In this regard, the undesirable effects of Clostridium toxin (due to diffusion of the toxin from the administration site) can be experimentally evaluated by measuring the percentage of weight loss in relevant animal models (e.g., detecting weight loss within 7 days after administration in mice). Conversely, the effect of Clostridium toxin on the desired target can be experimentally evaluated by the Digital Abduction Score (DAS) assay, which is a measure of muscle paralysis. The DAS assay can be performed by injecting 20 μl of Clostridium toxin, formulated in gelatin phosphate buffer, into the mouse gastrocnemius / soleus muscle complex, and then evaluating the finger abduction score using Aoki's method (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response in which they extend their hind limbs and abduct their hind toes. After Clostridium toxin injection, the degree of finger abduction is scored on a 5-point scale (0 = normal to 4 = maximum decrease in finger abduction and limb extension).

[0042] The safety factor of Clostridium toxin can then be expressed as the ratio of the amount of toxin required to cause a 10% weight loss (measured at the peak effect within 7 days of administration to mice) to the amount of toxin required for a DAS score of 2. Therefore, a high safety factor score is desirable, indicating a toxin capable of effectively paralyzing the target muscle with little to no undesirable off-target effects. The modified toxin of the present invention has a higher safety factor than equivalent unmodified (natural) botulinum toxin.

[0043] Therefore, in one embodiment, the modified Clostridium toxin of the present 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), and the safety factor is such that the dose of toxin (pg / mouse) required for a -10% change in body weight is DAS ED 50 [ED]Calculated by dividing by (pg / mouse) 50 [=Dosage required to obtain a DAS score of 2]

[0044] In one embodiment, the modified Clostridium toxin of the present invention has a safety factor of at least 10. In another embodiment, the modified Clostridium toxin of the present invention has a safety factor of at least 15.

[0045] The modified Clostridium toxin of the present invention has at least one amino acid modification. The at least one amino acid modification increases the pI of the Clostridium toxin as described above. In the present invention, amino acid modification is a modification of the amino acid sequence of the Clostridium toxin. Such modifications can be achieved by replacing one amino acid in the sequence with another (i.e., substitution), inserting a new amino acid into the sequence, or deleting an amino acid from the sequence. Amino acids incorporated into the amino acid sequence of a protein are also called amino acid residues.

[0046] The 20 standard amino acids found in proteins are as follows:

[0047] [Table 2]

[0048] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0049] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acid and glutamic acid are called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0050] The following amino acids are considered non-charged amino acids (meaning they can participate in hydrogen bonding): asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0051] The following amino acids are considered uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0052] An increase in the pI of Clostridium toxin can be achieved by introducing one or more amino acid modifications into the Clostridium toxin that increase the ratio of positive charge to negative charge in the Clostridium toxin.

[0053] In one embodiment, at least one amino acid modification is selected from amino acid substitution, amino acid insertion, and amino acid deletion.

[0054] In amino acid substitution, an amino acid residue that forms part of the Clostridium toxin amino acid sequence is replaced with a different amino acid residue. The substituted amino acid residue may be one of the 20 standard amino acids mentioned above.

[0055] Alternatively, the substituted amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the 20 standard sets mentioned above). For example, the substituted amino acid may be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-forms 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 the nutrient-requiring host *Escherichia coli*.

[0056] Amino acid insertion involves incorporating an additional amino acid residue (which is not normally present) into the Clostridium toxin amino acid sequence, thereby increasing the total number of amino acid residues in the sequence. Amino acid deletion involves removing an amino acid residue from the Clostridium toxin amino acid sequence, thereby decreasing the total number of amino acid residues in the sequence.

[0057] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. For example, amino acid modification can be introduced by modifying the DNA sequence encoding Clostridium toxin. This can be achieved, for example, by site-directed mutagenesis using standard molecular cloning techniques, where a short chain (oligonucleotide) of DNA encoding a desired amino acid(s) is replaced with a polymerase enzyme, or by insertion / deletion of a portion of the gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized.

[0058] In one embodiment, at least one amino acid modification is selected from substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, substitution of an uncharged amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue, and deletion of an acidic amino acid residue.

[0059] In a preferred embodiment, at least one amino acid modification is a substitution that has the advantage of maintaining the same number of amino acid residues in Clostridium toxin. In one embodiment, the substitution is selected from substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, the basic amino acid residue is a lysine residue or an arginine residue. In one embodiment, the basic amino acid residue is a lysine residue. In one embodiment, the basic amino acid residue is an arginine residue. In one embodiment, if the substitution is substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine).

[0060] The modified Clostridium toxin of the present invention may contain one or more amino acid modifications. Therefore, in one embodiment, the modified Clostridium toxin (as described above) contains 1 to 80 amino acid modifications (e.g., 1 to 70, 1 to 60, 1 to 50, 4 to 40, 4 to 30, 5 to 40, 5 to 30, or 10 to 25 amino acid modifications). In one embodiment, the modified Clostridium toxin (as described above) contains 4 to 40 amino acid modifications. In one embodiment, the modified Clostridium toxin contains at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid modifications. In one embodiment, the modified Clostridium toxin contains at least 4 amino acid modifications (e.g., at least 4 amino acid substitutions). Each of the above amino acid modifications is one of the above-described amino acid modifications. Therefore, each of the above amino acid modifications contributes to an increase in the pI of the modified Clostridium toxin (compared to the pI of a Clostridium toxin identical except for the absence of the above amino acid modifications).

[0061] Any clostridial toxin amino acid (i.e., amino acid residue) can be modified as described above, provided that the result of the modification is an increase in the pI of the clostridial toxin (as described above). However, the inventors have identified a subset of clostridial toxin amino acids that are particularly suitable targets for modification.

[0062] Suitable target amino acids can have certain properties. As an example, suitable target amino acids can be (i) surface-exposed amino acids, (ii) those located outside the secondary structure of the clostridial toxin protein, (iii) those located within regions that are not essential for the protein function of the clostridial toxin protein, (iv) amino acids whose identity is not conserved among the types, subtypes, or serotypes of clostridial toxin, (iv) amino acids that do not form a predicted ubiquitination site upon their modification, or (v) any combination of the above.

[0063] As described above, clostridial toxin is formed from two polypeptide chains, namely a heavy chain (H chain) with a molecular mass of approximately 100 kDa and a light chain (L chain) with a molecular mass of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor-binding domain or H C domain) and an N-terminal translocation component (H N domain).

[0064] In one embodiment, at least one amino acid modification (as described above) is located in the receptor-binding domain (H C domain) of the clostridial toxin.

[0065] Examples of the reference sequences of the light chain include the following. Botulinum neurotoxin type A: Amino acid residues 1-448 Botulinum neurotoxin type B: Amino acid residues 1-440 Botulinum neurotoxin type C1: Amino acid residues 1-441 Botulinum neurotoxin type D: Amino acid residues 1-445 Botulinum neurotoxin type E: Amino acid residues 1-422 Botulinum toxin type F neurotoxin: amino acid residues 1-439 Botulinum toxin type G neurotoxin: Amino acid residues 1-441 Tetanus neurotoxin: Amino acid residues 1-457

[0066] The above reference sequences should be considered guidelines only, as slight variations may occur depending on the serotype. For example, US2007 / 0166332 (which, by citing its source, is considered part of this specification) lists a slightly different Clostridium sequence: Botulinum toxin type A neurotoxin: amino acid residues M1-K448 Botulinum toxin type B neurotoxin: amino acid residue M1-K441 Botulinum C1 neurotoxin: Amino acid residues M1-K449 Botulinum toxin type D neurotoxin: amino acid residues M1-R445 Botulinum toxin type E neurotoxin: amino acid residues M1-R422 Botulinum toxin type F neurotoxin: amino acid residues M1-K439 Botulinum toxin type G neurotoxin: amino acid residues M1-K446 Tetanus neurotoxin: Amino acid residue M1-A457

[0067] Clostridium toxin H C Examples of domain reference sequences include the following: 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

[0068] Clostridium toxin (BoNT, etc.) C The domain is H CC and H CNIt contains two distinct structural features called domains. The amino acid residues involved in receptor binding are H CC It is thought to be located within the domain.

[0069] In one embodiment, at least one amino acid modification (as described above) is used to modify the receptor-binding domain of Clostridium toxin (H C If located in the domain, the at least one amino acid modification is the H of Clostridium toxin. CN It is located in the domain (also called the translocation-promoting domain). In one embodiment, at least one amino acid modification (as described above) is located in the receptor-binding domain (H) of Clostridium toxin. C If located in the domain, the at least one amino acid modification is the H of Clostridium toxin. CC It is located within the domain.

[0070] Clostridium toxin H CN Examples of domain reference sequences include the following: Botulinum toxin type A neurotoxin: amino acid residues 872-1110 Botulinum toxin type B neurotoxin: amino acid residues 859-1097 Botulinum C1 neurotoxin: amino acid residues 867-1111 Botulinum toxin type D neurotoxin: amino acid residues 863-1098 Botulinum toxin type E neurotoxin: amino acid residues 846-1085 Botulinum toxin type F neurotoxin: amino acid residues 865-1105 Botulinum toxin type G neurotoxin: amino acid residues 864-1105 Tetanus neurotoxin: Amino acid residues 880-1127

[0071] The above sequence positions may vary slightly depending on the serotype / subtype, and the appropriate (reference) Clostridium toxin H CN Other examples of domains include: Botulinum toxin type A neurotoxin: amino acid residues 874-1110 Botulinum toxin type B neurotoxin: amino acid residues 861-1097 Botulinum C1 neurotoxin: amino acid residues 869-1111 Botulinum toxin type D neurotoxin: amino acid residues 865-1098 Botulinum toxin type E neurotoxin: amino acid residues 848-1085 Botulinum toxin type F neurotoxin: amino acid residues 867-1105 Botulinum toxin type G neurotoxin: amino acid residues 866-1105 Tetanus neurotoxin: Amino acid residues 882-1127

[0072] In one embodiment, at least one amino acid modification (as described above) is a modification of a surface-exposed amino acid residue. Surface-exposed amino acid residues are located on the outside of the folded protein and, in contrast to amino acid residues located on the inside of the folded protein, are accessible to the solvent. The degree of surface exposure of an amino acid residue, i.e., its exposure to the surrounding solvent, depends on its position within the folded protein and the conformation adopted by the protein. Therefore, modifications of amino acid residues with a high degree of surface exposure have a greater impact on the isoelectric point of the protein than modifications of amino acid residues with a low degree of surface exposure. Methods for determining the surface exposure of amino acid residues are known in the art. For example, the surface exposure of amino acid residues in a given protein can be calculated using the computer program AreaIMol (part of the CCP4 computer program suite). Surface-exposed amino acid residues can also be identified by visual inspection of the protein crystal structure (such as by X-ray crystallography). In one embodiment, surface-exposed amino acid residues have a total AreaIMol value of at least 40.

[0073] In one embodiment, at least one amino acid modification includes modification of an amino acid residue selected from aspartic acid residues, glutamic acid residues, histidine residues, serine residues, threonine residues, asparagine residues, glutamine residues, cysteine ​​residues, or tyrosine residues. The inventors have confirmed that amino acid residues from this group (charged residues and polar residues) represent particularly suitable targets for the modifications according to the present invention. While not wishing to be bound by any one theory, the inventors believe that amino acid residues within this group appear on the surface of Clostridium toxins at a higher frequency than undescribed hydrophobic residues.

[0074] In one embodiment, when the amino acid modification involves modification of an amino acid residue selected from (as described above) aspartic acid residue, glutamic acid residue, histidine residue, serine residue, threonine residue, asparagine residue, glutamine residue, cysteine ​​residue, or tyrosine residue, the amino acid residue is substituted with a lysine residue or an arginine residue. Therefore, in one embodiment, the charged residue or polar residue is substituted with a positively charged residue, which increases the ratio of positive charge to negative charge and thus increases the pI of Clostridium toxin.

[0075] In one embodiment, at least one amino acid modification (as described above) includes modification of an asparagine amino acid residue or a glutamine amino acid residue (both uncharged residues). In one embodiment, the asparagine or glutamine amino acid residue is substituted with a lysine residue or an arginine residue (both positively charged residues). In one embodiment, the asparagine or glutamine amino acid residue is substituted with a lysine residue. In one embodiment, the asparagine or glutamine amino acid residue is substituted with an arginine residue.

[0076] Asparagine and glutamine residues are polar, forming only weak dipole interactions with other residues, and they make up 14% of common Clostridium toxin molecules (such as BoNT / A), making them suitable for modification.

[0077] In one embodiment, the modified Clostridium toxin is BoNT / A. The reference BoNT / A sequence has UniProtKB accession number P10845.

[0078] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in BoNT / A Clostridium toxin.

[0079] In one embodiment, if the modified Clostridium toxin is BoNT / A, then the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU10 The modification includes at least one amino acid(s) selected from 83, ASP1086, and GLN1229 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 amino acids), wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0080] In one embodiment, if the modified Clostridium toxin is BoNT / A, then the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081 The modification includes at least one amino acid(s) selected from GLU1083, ASP1086, and GLN1229 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 amino acids), wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0081] In one embodiment, if the modified Clostridium toxin is BoNT / A, then the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS10 The modification includes at least one amino acid(s) selected from 64, ASN1080, GLU1081, GLU1083, ASP1086, and GLN1229 (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 amino acids), wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of the same BoNT / A except that which lacks the amino acid(s) modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0082] In one embodiment, if the modified Clostridium toxin is BoNT / A, then the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, The modification includes at least one amino acid(s) selected from HIS1064, ASN1080, GLU1081, GLU1083, and ASP1086 (for example, at least 1, 2, 3, 4, 5, 10, 15, or 20 amino acids), wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of the same BoNT / A except that which lacks the amino acid(s) modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0083] In one embodiment, if the modified Clostridium toxin is BoNT / A, then the modified BoNT / A is 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 The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30) amino acid(s) selected from ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0084] In one embodiment, if the modified Clostridium toxin is BoNT / A, then the modified BoNT / A is 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, G The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30) amino acid(s) selected from LY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0085] In one embodiment, if the modified Clostridium toxin is BoNT / A, then the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086 The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30) amino acid(s) selected from ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of the same BoNT / A except that which lacks the amino acid(s) modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0086] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, or GLN1229 (e.g., at least 1, 2, 3, 4, 5, or 6), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0087] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, or GLN1229 (e.g., at least 1, 2, 3, 4, 5, or 6), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0088] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) (e.g., at least 1, 2, 3, 4, 5, or 6) selected from ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0089] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0090] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0091] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0092] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one (e.g., at least 1, 2, 3, 4, 5, or 6) amino acids selected from ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid modification increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid modification. In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0093] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, or GLN1229 (e.g., at least 1, 2, 3, 4, 5, or 6), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0094] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) (e.g., at least 1, 2, 3, 4, 5, or 6) selected from ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0095] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by lysine residues or arginine residues. In one embodiment, the modifications include amino acid substitution by lysine residues. In one embodiment, the modifications include amino acid substitution by arginine residues.

[0096] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0097] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0098] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one (e.g., at least 1, 2, 3, 4, 5, or 6) amino acids selected from ASN930, SER955, GLN991, ASN1025, ASN1026, ASN1052, or GLN1229, wherein the amino acid modification increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / A except for the absence of the amino acid modification. In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0099] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN930, SER955, GLN991, ASN1025, ASN1026, ASN1052, or GLN1229 (e.g., at least 1, 2, 3, 4, 5, or 6), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0100] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) (e.g., at least 1, 2, 3, 4, 5, or 6) selected from ASN930, SER955, GLN991, ASN1025, ASN1026, ASN1052, or GLN1229, wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0101] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN930, SER955, GLN991, ASN1025, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0102] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN930, SER955, GLN991, ASN1025, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0103] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following seven amino acids: ASN930, SER955, GLN991, ASN1025, ASN1026, ASN1052, or GLN1229, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0104] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN1188, ASP1213, GLY1215, ASN1216, ASN1242, ASN1243, SER1274, THR1277 (e.g., at least 1, 2, 3, 4, 5, 6, or 7), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0105] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN1188, ASP1213, GLY1215, ASN1216, ASN1242, ASN1243, SER1274, THR1277 (e.g., at least 1, 2, 3, 4, 5, 6, or 7), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0106] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN1188, ASP1213, GLY1215, ASN1216, ASN1242, ASN1243, SER1274, and THR1277 (e.g., at least 1, 2, 3, 4, 5, 6, or 7), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0107] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following eight amino acids: ASN1188, ASP1213, GLY1215, ASN1216, ASN1242, ASN1243, SER1274, THR1277, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by lysine residues or arginine residues. In one embodiment, the modifications include amino acid substitution by lysine residues. In one embodiment, the modifications include amino acid substitution by arginine residues.

[0108] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following eight amino acids: ASN1188, ASP1213, GLY1215, ASN1216, ASN1242, ASN1243, SER1274, THR1277, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by lysine residues or arginine residues. In one embodiment, the modifications include amino acid substitution by lysine residues. In one embodiment, the modifications include amino acid substitution by arginine residues.

[0109] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modifications to the following eight amino acids: ASN1188, ASP1213, GLY1215, ASN1216, ASN1242, ASN1243, SER1274, and THR1277, wherein the amino acid modifications increase the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the lack of the amino acid modifications. In one embodiment, the modifications include amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modifications include amino acid substitution by a lysine residue. In one embodiment, the modifications include amino acid substitution by an arginine residue.

[0110] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN930, SER955, GLN991, ASN1026, ASN1052, HIS1064, and GLN1229 (e.g., at least 1, 2, 3, 4, 5, 6, or all 7), wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0111] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, HIS1064, ASN1080, ASN1147, and GLN1229 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11), wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0112] In one embodiment, if the modified Clostridium toxin is BoNT / A, the modified BoNT / A includes modification of at least one amino acid(s) selected from ASN886, ASN905, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, HIS1064, ASN1080, ASN1147, and GLN1229 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13), wherein the amino acid(s) modification(s) increases the isoelectric point (pI) of the modified BoNT / A to a value at least 1 pI higher than that of an identical BoNT / A except for the absence of the amino acid(s) modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In another embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0113] In one embodiment, the modified Clostridium toxin is BoNT / B. The reference BoNT / B sequence has UniProtKB accession number P10844.

[0114] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in BoNT / B Clostridium toxin.

[0115] In one embodiment, if the modified Clostridium toxin is BoNT / B, then the modified BoNT / B is ASN873, ASN874, GLU892, ASP895, ASN906, ASP940, ASN948, GLU949, ASN958, ASN959, ASN979, ASN990, GLU993, ASP994, GLU997, ASN1012, ASN1019, ASP1030, ASP1047, ASP1049, GLU1065, GLU1072, GLN1176, GL The modification includes at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) selected from U1189, GLU1252, and ASN1273, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / B to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / B except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0116] In one embodiment, if the modified Clostridium toxin is BoNT / B, then the modified BoNT / B is ASN873, ASN874, GLU892, ASP895, ASN906, ASP940, ASN948, GLU949, ASN958, ASN959, ASN979, ASN990, GLU993, ASP994, GLU997, ASN1012, ASN1019, ASP1030, ASP1047, ASP1049, GLU1065, GLU1072, GLN1 The modification includes at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) selected from 176, GLU1189, GLU1252, and ASN1273, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / B to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / B except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0117] In one embodiment, if the modified Clostridium toxin is BoNT / B, then the modified BoNT / B is ASN873, ASN874, GLU892, ASP895, ASN906, ASP940, ASN948, GLU949, ASN958, ASN959, ASN979, ASN990, GLU993, ASP994, GLU997, ASN1012, ASN1019, ASP1030, ASP1047, ASP1049, GL The modification includes at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) selected from U1065, GLU1072, GLN1176, GLU1189, GLU1252, and ASN1273, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / B to a value at least 1 pI higher than that of the same BoNT / B except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0118] In one embodiment, the modified Clostridium toxin is BoNT / C1. The reference BoNT / C1 sequence has UniProtKB accession number P18640.

[0119] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in BoNT / C1 Clostridium toxin.

[0120] Therefore, in one embodiment, if the modified Clostridium toxin is BoNT / C1, then the modified BoNT / C1 is ASN881, ASP898, GLU916, GLU927, ASN952, ASN964, ASN965, ASN984, GLU985, ASP986, ASP996, ASN1000, GLU1036, ASN1041, ASP1062, ASP1064, GLU1079, and ASP1081 or The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, or 15) amino acid modifications selected from the above, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / C1 to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / C1 except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0121] Therefore, in one embodiment, if the modified Clostridium toxin is BoNT / C1, then the modified BoNT / C1 is ASN881, ASP898, GLU916, GLU927, ASN952, ASN964, ASN965, ASN984, GLU985, ASP986, ASP996, ASN1000, GLU1036, ASN1041, ASP1062, ASP1064, GLU1079, and AS The modification includes at least one amino acid modification (e.g., at least 1, 2, 3, 4, 5, 10, or 15) selected from P1081, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / C1 to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / C1 except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0122] In one embodiment, if the modified Clostridium toxin is BoNT / C1, the modified BoNT / C1 includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, or 15) amino acid modification selected from ASN881, ASP898, GLU916, GLU927, ASN952, ASN964, ASN965, ASN984, GLU985, ASP986, ASP996, ASN1000, GLU1036, ASN1041, ASP1062, ASP1064, GLU1079, and ASP1081, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / C1 to a value at least 1 pI higher than that of an identical BoNT / C1 except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In another embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0123] In one embodiment, the modified Clostridium toxin is BoNT / D. The reference BoNT / D sequence has UniProtKB accession number P19321.

[0124] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in BoNT / D Clostridium toxin.

[0125] In one embodiment, if the modified Clostridium toxin is BoNT / D, then the modified BoNT / D is ASN877, ASP893, ASN894, ASN898, ASN920, ASN945, ASN948, GLU957, GLN958, ASN959, ASN968, ASN979, GLU1030, ASP1031, ASP1033, GLU1047, GLU1051, ASN1052, GLU1066, and GLN11 The modification includes at least one amino acid modification (e.g., at least 1, 2, 3, 4, 5, 10, 15, or 20) selected from 22, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / D to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of the identical BoNT / D except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0126] In one embodiment, if the modified Clostridium toxin is BoNT / D, then the modified BoNT / D is ASN877, ASP893, ASN894, ASN898, ASN920, ASN945, ASN948, GLU957, GLN958, ASN959, ASN968, ASN979, GLU1030, ASP1031, ASP1033, GLU1047, GLU1051, ASN1052, GLU1066, and The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid modification selected from GLN1122, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / D to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI higher than that of identical BoNT / D except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0127] In one embodiment, if the modified Clostridium toxin is BoNT / D, the modified BoNT / D includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid modification selected from ASN877, ASP893, ASN894, ASN898, ASN920, ASN945, ASN948, GLU957, GLN958, ASN959, ASN968, ASN979, GLU1030, ASP1031, ASP1033, GLU1047, GLU1051, ASN1052, GLU1066, and GLN1122, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / D to a value at least 1 pI higher than that of identical BoNT / D except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0128] In one embodiment, the modified Clostridium toxin is BoNT / E. The reference BoNT / E sequence has UniProtKB accession number Q00496.

[0129] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in BoNT / E Clostridium toxin.

[0130] In one embodiment, if the modified Clostridium toxin is BoNT / E, then the modified BoNT / E is ASN859, ASP860, ASN892, ASP893, ASP904, ASP909, ASN928, ASN932, ASN934, ASN935, GLU936, ASP945, ASN946, ASN947, ASN966, ASN976, ASN979, ASN981, ASP985, GLN1014, ASN1019, ASN1022, ASP1027, The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid modification selected from ASN1035 and ASN1140, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / E to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI higher than that of an identical BoNT / E except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0131] In one embodiment, if the modified Clostridium toxin is BoNT / E, then the modified BoNT / E is ASN859, ASP860, ASN892, ASP893, ASP904, ASP909, ASN928, ASN932, ASN934, ASN935, GLU936, ASP945, ASN946, ASN947, ASN966, ASN976, ASN979, ASN981, ASP985, GLN1014, ASN1019, ASN1022, AS The modification includes at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) selected from P1027, ASN1035, and ASN1140, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / E to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / E except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0132] In one embodiment, if the modified Clostridium toxin is BoNT / E, then the modified BoNT / E is ASN859, ASP860, ASN892, ASP893, ASP904, ASP909, ASN928, ASN932, ASN934, ASN935, GLU936, ASP945, ASN946, ASN947, ASN966, ASN976, ASN979, ASN981, ASP985, GLN1014, The modification includes at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) selected from ASN1019, ASN1022, ASP1027, ASN1035, and ASN1140, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / E to a value at least 1 pI higher than that of the same BoNT / E except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0133] In one embodiment, the modified Clostridium toxin is BoNT / F. The reference BoNT / F sequence has UniProtKB accession number YP_001390123.

[0134] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in BoNT / F Clostridium toxin.

[0135] In one embodiment, if the modified Clostridium toxin is BoNT / F, then the modified BoNT / F is ASN879, ASP896, ASN922, ASN923, ASN928, ASN947, ASN950, ASN952, ASN953, GLU954, ASN963, ASN964, ASN965, ASN987, GLN997, ASN1037, ASP1040, ASP1045, ASN1055, and ASP105 The modification includes at least one amino acid modification (e.g., at least 1, 2, 3, 4, 5, 10, 15, or 20) selected from 6, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / F to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / F except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0136] In one embodiment, if the modified Clostridium toxin is BoNT / F, then the modified BoNT / F is ASN879, ASP896, ASN922, ASN923, ASN928, ASN947, ASN950, ASN952, ASN953, GLU954, ASN963, ASN964, ASN965, ASN987, GLN997, ASN1037, ASP1040, ASP1045, ASN1055, and The modification includes at least one amino acid modification (e.g., at least 1, 2, 3, 4, 5, 10, 15, or 20) selected from ASP1056, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / F to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / F except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0137] In one embodiment, if the modified Clostridium toxin is BoNT / F, the modified BoNT / F includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid modification selected from ASN879, ASP896, ASN922, ASN923, ASN928, ASN947, ASN950, ASN952, ASN953, GLU954, ASN963, ASN964, ASN965, ASN987, GLN997, ASN1037, ASP1040, ASP1045, ASN1055, and ASP1056, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / F to a value at least 1 pI higher than that of identical BoNT / F except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0138] In one embodiment, the modified Clostridium toxin is BoNT / G. The reference BoNT / G sequence has UniProtKB accession number Q60393.

[0139] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in BoNT / G Clostridium toxin.

[0140] In one embodiment, if the modified Clostridium toxin is BoNT / G, then the modified BoNT / G is ASP900, ASN909, ASN910, GLU912, ASN913, ASN945, ASN947, GLU956, ASN965, ASP966, ASN986, ASN1001, ASN1038, ASP1040, ASN1046, ASP1057, GLU1073, ASN1075, and ASN1090 The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, or 15) amino acid modification selected from the above, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / G to a value at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / G except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0141] In one embodiment, if the modified Clostridium toxin is BoNT / G, then the modified BoNT / G is ASP900, ASN909, ASN910, GLU912, ASN913, ASN945, ASN947, GLU956, ASN965, ASP966, ASN986, ASN1001, ASN1038, ASP1040, ASN1046, ASP1057, GLU1073, ASN1075, and A The modification includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, or 15) amino acid modification selected from SN1090, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / G to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of an identical BoNT / G except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0142] In one embodiment, if the modified Clostridium toxin is BoNT / G, the modified BoNT / G includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, or 15) amino acid modification selected from ASP900, ASN909, ASN910, GLU912, ASN913, ASN945, ASN947, GLU956, ASN965, ASP966, ASN986, ASN1001, ASN1038, ASP1040, ASN1046, ASP1057, GLU1073, ASN1075, and ASN1090, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified BoNT / G, to a value at least 1 pI higher than that of an identical BoNT / G lacking the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0143] In one embodiment, the modified Clostridium toxin is TeNT. The reference TeNT sequence includes UniProtKB accession number P04958.

[0144] The inventors have identified specific amino acids that represent suitable targets for amino acid modification in TeNT Clostridium toxin.

[0145] In one embodiment, if the modified Clostridium toxin is TeNT, then the modified TeNT is selected from at least one of the following: ASN893, ASP894, ASP911, ASN919, ASN927, ASN928, GLU929, GLN968, ASN972, GLU973, GLU1010, ASP1018, ASN1079, ASN1080, ASN1081, and ASN1097. The modification also includes one (for example, at least 1, 2, 3, 4, 5, 10, or 15) amino acid modifications, the amino acid modification(s) raising the isoelectric point (pI) of the modified TeNT by at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units compared to the pI of the same TeNT except that lacking the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes amino acid substitution by an arginine residue.

[0146] In one embodiment, if the modified Clostridium toxin is TeNT, the modified TeNT includes at least one (e.g., at least 1, 2, 3, 4, 5, 10, or 15) amino acid modification selected from ASN893, ASP894, ASP911, ASN919, ASN927, ASN928, GLU929, GLN968, ASN972, GLU973, GLU1010, ASP1018, ASN1079, ASN1080, ASN1081, and ASN1097, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified TeNT to a value at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than that of identical TeNT except for the absence of the amino acid modification(s). In one embodiment, the modification includes the substitution of an amino acid by a lysine residue or an arginine residue. In one embodiment, the modification includes the substitution of an amino acid by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0147] In one embodiment, if the modified Clostridium toxin is TeNT, the modified TeNT includes modification of at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 10, or 15) selected from ASN893, ASP894, ASP911, ASN919, ASN927, ASN928, GLU929, GLN968, ASN972, GLU973, GLU1010, ASP1018, ASN1079, ASN1080, ASN1081, and ASN1097, wherein the amino acid modification(s) increases the isoelectric point (pI) of the modified TeNT to a value at least 1 pI higher than that of identical TeNT except for the absence of the amino acid modification(s). In one embodiment, the modification includes amino acid substitution by a lysine residue or an arginine residue. In one embodiment, the modification includes amino acid substitution by a lysine residue. In one embodiment, the modification includes the substitution of an amino acid by an arginine residue.

[0148] The present invention is suitable for application to a wide variety of Clostridium toxins. Accordingly, in the present invention, the term "Clostridium toxin" includes the toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, and G), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified Clostridium toxins or derivatives derived from any of the above. The term "Clostridium toxin" further includes botulinum neurotoxin serotype H.

[0149] Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex consisting of BoNT itself combined with numerous accessory proteins. Currently, there are eight different classes of botulinum neurotoxin, namely serotypes A, B, C1, D, E, F, G, and H, all of which share similar structures and mechanisms of action. Different BoNT serotypes can be distinguished by classification based on serotyping correlated with the percentage of sequence identity at the amino acid level, based on inactivation with specific neutralizing antisera. The BoNT protein of a given serotype is further divided into different subtypes based on the percentage of sequence identity at the amino acid level.

[0150] BoNT is absorbed in the gastrointestinal tract and enters the systemic circulation, where it binds to the presynaptic membrane of cholinergic nerve terminals, inhibiting the release of their 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 25kDa synaptosome-associated protein (SNAP-25), and BoNT / C1 cleaves syntaxin.

[0151] Tetanus toxin is produced by C. tetani in a single serotype. C. butyricum produces BoNT / E, and C. baratii produces BoNT / F.

[0152] The term "Clostridium toxin" further encompasses modified Clostridium toxin and its derivatives, and includes, but is not limited to, those described below. A modified Clostridium toxin or derivative may contain one or more modified amino acids compared to the natural (unmodified) form of Clostridium toxin, or may contain one or more inserted amino acids that are not present in the natural (unmodified) form of Clostridium toxin. For example, a modified Clostridium toxin may contain an amino acid sequence modified in one or more domains compared to the natural (unmodified) Clostridium toxin sequence. Such modifications may alter the functional aspects of the toxin, such as its biological activity or persistence. Therefore, in one embodiment, the modified Clostridium toxin of the present invention is a modified Clostridium toxin, or a modified Clostridium toxin derivative, or a modified Clostridium toxin derivative.

[0153] Modified Clostridium toxins have one or more modifications (modified H) in the amino acid sequence of the heavy chain. C The modified heavy chain may have domains, etc., and the modified heavy chain binds to target nerve cells with a higher or lower affinity than natural (unmodified) Clostridium toxin. C Modifications in the domain alter the binding of target neurons to ganglioside receptors and / or protein receptors. C This may include modifying residues within the ganglioside binding site or the protein (SV2 or synaptotagmin) binding site of the domain. Examples of such modified Clostridium toxins are described in WO2006 / 027207 and WO2006 / 114308, which together include their entire disclosures as part of this specification with proper attribution.

[0154] Modified Clostridium toxins may have one or more modifications in the amino acid sequence of the light chain, for example, modifications in the substrate-binding or catalytic domain that can alter or modify the SNARE protein specificity of the modified light chain. Examples of such modified Clostridium toxins are described in WO2010 / 120766 and US2011 / 0318385, which together include their entire disclosures as part of this specification with proper attribution.

[0155] Modified Clostridium toxins may contain one or more modifications that increase or decrease the biological activity and / or persistence of the modified Clostridium toxin. For example, a modified Clostridium toxin may contain a leucine-based or tyrosine-based motif that increases or decreases the biological activity and / or persistence of the modified Clostridium toxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, 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 Clostridium toxins containing leucine-based and tyrosine-based motifs are described in WO2002 / 08268, which, with proper attribution, constitutes part of this specification in its entirety.

[0156] The term "Clostridium toxin" includes hybrid and chimeric Clostridium toxins. A hybrid Clostridium toxin comprises at least a portion of a light chain derived from a Clostridium toxin or a subtype thereof, and at least a portion of a heavy chain derived from another Clostridium toxin or a Clostridium toxin subtype. In one embodiment, a hybrid Clostridium toxin may comprise the entire light chain of a Clostridium toxin subtype and a heavy chain derived from another Clostridium toxin subtype. In other embodiments, a chimeric Clostridium toxin may comprise a portion of a heavy chain (e.g., a binding domain) derived from a Clostridium toxin subtype together with the other portion of a heavy chain derived from another Clostridium toxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain portions derived from different groups of Clostridium toxins. Such hybrid or chimeric Clostridium toxins are useful as a means of delivering the therapeutic benefits of these Clostridium toxins to patients, for example, those who have immune resistance to a particular Clostridium toxin subtype, those who may have lower-than-average concentrations of receptors for the heavy chain binding domain of a particular Clostridium toxin, or those who may have protease-resistant variants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric Clostridium toxins are described in US8,071,110, the entire disclosure of which is included in this specification by citing the source. Accordingly, in one embodiment, the modified Clostridium toxin of the present invention is a modified hybrid Clostridium toxin or a modified chimeric Clostridium toxin.

[0157] The term "Clostridium toxin" encompasses retargeted Clostridium toxin. In retargeted Clostridium toxin, the Clostridium toxin is modified to include an exogenous ligand known as the Targeting Moiety (TM). The TM is selected to provide binding specificity to the desired target cells and, as part of the retargeting process, the native binding moiety of the Clostridium toxin (e.g., H) is modified. C Domain, or HCC The domains can be removed. Retargeting methods include, for example, EP-B-0689459, WO1994 / 021300, EP-B-0939818, US6,461,617, US7,192,596, WO1998 / 007864, EP-B-0826051, US5,989,545, US6,395,513, US6,962,703, WO1996 / 033273, EP-B-0996468, US7,052,702, WO1999 / 0 This information is described in 17806, EP-B-1107794, US6,632,440, WO2000 / 010598, WO2001 / 21213, WO2006 / 059093, WO2000 / 62814, WO2000 / 04926, WO1993 / 15766, WO2000 / 61192, and WO1999 / 58571, all of which, by clearly indicating their sources, constitute part of the present specification. Accordingly, in one embodiment, the modified Clostridium toxin of the present invention is a modified retargeted Clostridium toxin.

[0158] The present invention further includes Clostridium toxins having non-natural protease cleavage sites. In such Clostridium toxins, the natural protease cleavage site (also called the activation site, as described above) is modified or replaced by a protease cleavage site that is not natural to the Clostridium toxin (i.e., an exogenous cleavage site). These sites require an exogenous protease for cleavage that allows for enhanced control over the timing and location of the cleavage event. Non-natural protease cleavage sites that can be utilized in Clostridium toxins include: Enterokinase (DDDDK↓) Factor Xa (IEGR↓ / IDGR↓) TEV (Tobacco Sex Disease Virus) (ENLYFQ↓G) Thrombin (LVPR↓GS) PreScission (LEVLFQ↓GP).

[0159] Additional protease cleavage sites include recognition sequences cleaved by non-cytotoxic proteases, such as the light chains of Clostridium neurotoxins. These include SNARE (e.g., SNAP-25, syntaxin, VAMP) protein recognition sequences cleaved by non-cytotoxic proteases such as the light chains of Clostridium neurotoxins. Clostridium toxins containing non-natural protease cleavage sites are described in US7,132,259, EP1206554-B2, and US2007 / 0166332, all of which, with proper attribution, constitute part of this specification. The term protease cleavage site also includes inteins, which are self-cleaving sequences. Self-splicing reactions can be controlled, for example, by changing the concentration of the present reducing agent.

[0160] The present invention further includes Clostridium toxins containing “destructive cleavage sites.” In the Clostridium toxins, the non-natural protease cleavage sites are incorporated into the Clostridium toxin at a location selected such that cleavage at the site reduces the activity of the Clostridium toxin or inactivates it. Destructive protease cleavage sites can be made more readily cleaved by local proteases if the Clostridium toxin moves to a non-target site following administration. Suitable non-natural protease cleavage sites include those described above. Clostridium toxins containing destructive cleavage sites are described in WO2010 / 094905 and WO2002 / 044199, which together include their entire disclosure as part of this specification with due attribution.

[0161] The modified Clostridium toxin of the present invention, particularly its light chain component, may be PEGylated, which can be effective in increasing stability, for example, the duration of action of the light chain component. PEGylation is particularly preferred when the light chain contains a BoNT / A, B, or C1 protease. PEGylation preferably involves the addition of PEG to the N-terminus of the light chain component. As an example, the N-terminus of the light chain may be extended by one or more identical or different amino acid (e.g., cysteine) residues. One or more of these amino acid residues may have PEG molecules attached to them (e.g., covalently bonded). An example of this technique is described in WO2007 / 104567, which, with due attribution, incorporates its entire disclosure as part of this specification.

[0162] The modified Clostridium toxin of the present invention does not need to contain the complex-forming protein present in the naturally occurring Clostridium toxin complex.

[0163] The modified Clostridium toxin of the present invention may contain a limited number of non-standard amino acids. Therefore, in addition to the 20 standard amino acids, amino acid residues of the modified Clostridium toxin of the present invention may be substituted with non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine). Clostridium polypeptide amino acid residues may also be substituted with a limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids. The modified Clostridium toxin of the present invention may also contain unnaturally occurring amino acid residues.

[0164] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNA can be utilized. 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 a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, for example, 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 performed in African clawed frog oocytes by microinjection of mutant mRNA with chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in a state where the natural amino acid to be replaced (e.g., phenylalanine) is absent, but the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine) is present.Unnaturally occurring amino acids are incorporated into polypeptides in place of their corresponding native amino acids. See Koide et al., Biochem. 33:7470-6, 1994.

[0165] The modified Clostridium toxin of the present invention can be produced using recombinant nucleic acid technology. Therefore, in one embodiment, the modified Clostridium toxin (described above) is a recombinant modified Clostridium toxin.

[0166] In another embodiment, the present invention provides a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding the modified Clostridium toxin described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator.

[0167] In a preferred embodiment, the vector has a promoter selected from the following: [Table A]

[0168] In another preferred embodiment, the vector has a promoter selected from the following: [Table B]

[0169] The nucleic acid molecules of the present invention can be prepared using any suitable process known in the art. Therefore, the nucleic acid molecules can be prepared using chemical synthesis methods, or they can be prepared using molecular biological methods.

[0170] The DNA construct of the present invention is preferably designed in silico and then synthesized by conventional DNA synthesis methods.

[0171] The nucleic acid sequence information described above is arbitrarily modified to account for codon bias depending on the expression system of the final host cell (e.g., E. coli) being used.

[0172] In one embodiment, the nucleic acid sequence encoding the modified Clostridium toxin described above is a nucleic acid sequence having at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, or 99%) sequence identity with the nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, and 9.

[0173] In one embodiment, the present invention provides a nucleic acid sequence having at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, or 99%) sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, and 9. In one embodiment, the nucleic acid sequence has at least 90% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, and 9.

[0174] The present invention further provides polypeptides encoded by the nucleic acid sequences described above. Accordingly, in one embodiment, the present invention provides a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, or 99%) sequence identity with an amino acid sequence selected from SEQ ID NOs: 4, 6, 8, and 10. In one embodiment, the amino acid sequence has at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 4, 6, 8, and 10.

[0175] In one embodiment, the modified Clostridium toxin of the present invention is the modified BoNT / A described above, wherein the modified BoNT / A includes (or comprises) an amino acid sequence having at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) sequence identity with an amino acid sequence selected from SEQ ID NOs: 4, 6, 8, and 10.

[0176] In one embodiment, the modified Clostridium toxin of the present invention is the modified BoNT / A described above, wherein the modified BoNT / A comprises (or consists of) the amino acid sequence of SEQ ID NOs: 4, 6, 8, or 10.

[0177] In one embodiment, the present invention provides a polypeptide comprising (or consisting of) the amino acid sequence of SEQ ID NOs: 4, 6, 8, or 10.

[0178] In one embodiment, the present invention provides a nucleic acid encoding the modified Clostridium toxin described above, comprising a nucleic acid sequence having at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, and 9. In one embodiment, the nucleic acid comprises (or consists of) the nucleic acid sequence SEQ ID NOs: 3, 5, 7, or 9.

[0179] In one embodiment, the present invention provides a nucleic acid comprising (or consisting of) the nucleic acid sequence of SEQ ID NOs: 3, 5, 7, or 9.

[0180] The "sequence identity percentage" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions the sequences share. Therefore, the identity percentage can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying the result by 100. The calculation of the sequence identity percentage 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 the two or more sequences. Sequence comparison and determination of the identity percentage between two or more sequences can be performed using specific mathematical algorithms such as BLAST, which are well known to those skilled in the art.

[0181] In one embodiment, the present invention provides a method for producing a single-chain modified Clostridium toxin protein having a light chain and a heavy chain, comprising: expressing a nucleic acid (such as the nucleic acid described above) in a suitable host cell; lysing the host cell to provide a homogenate of the host cell containing the single-chain modified Clostridium toxin protein; and isolating the single-chain modified Clostridium toxin protein.

[0182] In another embodiment, the present invention provides a method for activating a modified Clostridium toxin, which includes providing a single-chain modified Clostridium toxin protein obtainable by the method for producing the single-chain modified Clostridium toxin protein described above, and a method comprising contacting a polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between a light chain and a heavy chain, thereby converting the polypeptide into a double-chain polypeptide in which the light chain and heavy chain are linked to each other by disulfide bonds.

[0183] The modified Clostridium toxins of the present invention may be used to prevent or treat certain medical or cosmetic diseases and conditions. Accordingly, in another embodiment, the present invention provides the above-mentioned modified Clostridium toxins for use in pharmaceuticals.

[0184] In a related embodiment, the present invention provides the above-described modified Clostridium toxin for use in the prevention or treatment of diseases or conditions selected from: strabismus, blepharospasm, squint, dystonia (e.g., spasmodic dystonia, mangooral dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic (beauty) applications where incapacitation of cells / muscles (by downregulation or inactivation of SNARE) is effective, neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, hypertropia, lateral rectus palsy, nystagmus, hypothyroid myopathy), writer's cramp, blepharospasm, bruxism. Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasms, muscle rigidity, tension headache, levator pelvis syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache), forehead wrinkles, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary neuropathy, chronic neurogenic inflammation, and smooth muscle disorders.

[0185] In use, the present invention utilizes a pharmaceutical composition comprising a modified Clostridium toxin together with at least one component selected from pharmaceutically acceptable carriers, excipients, adjuvants, sprays, and / or salts.

[0186] The modified Clostridium toxin of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation, or topical administration. Compositions suitable for injection may be in the form of solutions, suspensions, or emulsions, or as a dry powder to be dissolved or suspended in a suitable solvent before use.

[0187] In the case of modified Clostridium toxins delivered locally, the modified Clostridium toxin can be formulated as a cream (e.g., for topical application) or for subcutaneous injection.

[0188] Local delivery means may include aerosols or other sprayers (e.g., nebulizers). In this regard, aerosol formulations of modified Clostridium toxin enable delivery to the lungs and / or other nasal and / or bronchial or airway passages.

[0189] The modified Clostridium toxin of the present invention can be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal cord segments involved in the nerve supply of the affected organ.

[0190] The preferred route of administration is laparoscopic injection and / or local injection, particularly intramuscular injection.

[0191] The dosage range for the modified Clostridium toxin of the present invention is such that it produces the desired therapeutic effect. The required dosage range is determined according to the exact properties of the modified Clostridium toxin or composition, the route of administration, the properties of the dosage form, the patient's age, the nature, range, or severity of the patient's condition, any contraindications, and the judgment of the attending physician. These changes in dosage levels can be adjusted using standard empirical routines for optimization.

[0192] An appropriate daily dose (per kg of patient body weight) is in the range of 0.0001 to 1 ng / kg, preferably 0.0001 to 0.5 ng / kg, more preferably 0.002 to 0.5 ng / kg, and particularly preferably 0.004 to 0.5 ng / kg. The unit dose can be varied from less than 1 picogram to 30 ng, but is generally in the range of 0.01 to 1 ng per dose and can be administered daily, or preferably at an even lower frequency, for example, weekly or every six months.

[0193] A particularly preferred dosing regimen is one based on 0.05 ng of modified Clostridium toxin as a 1× dose. In this case, the preferred dose ranges from 1× to 100× (i.e., 0.05 to 5 ng).

[0194] Fluid administration forms are generally prepared using modified Clostridium toxin and a pyrogen-free sterile solvent. Modified Clostridium toxin can be dissolved or suspended in the solvent, depending on the solvent and the concentration used. In solution preparation, the modified Clostridium toxin can be dissolved in the solvent, and the solution is made isotonic by adding sodium chloride if necessary, filtered through a bacterial filter using aseptic techniques, and then filled into appropriate sterile vials or ampoules and sealed. Alternatively, if the solution is sufficiently stable, the solution in the sealed container can be sterilized by autoclaving. This has the advantage that additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers, and / or local anesthetics can be dissolved in the solvent.

[0195] The dry powder, which is dissolved or suspended in a suitable solvent before use, can be prepared by filling a sterile container with pre-sterilized material using aseptic techniques in a sterile area. Alternatively, the material may be dissolved in a suitable container using aseptic techniques in a sterile area. The product is then freeze-dried, and the container is sealed under sterile conditions.

[0196] Parenteral solutions suitable for intramuscular, subcutaneous, or intradermal injection are prepared by substantially the same method, except that sterile components are suspended in a sterile solvent instead of being dissolved, and sterilization cannot be performed by filtration. Components may be isolated under sterile conditions, or sterilized by gamma irradiation after isolation.

[0197] Including a suspending agent, such as polyvinylpyrrolidone, in the composition(s) has the advantage of promoting the uniform dispersion of the components.

[0198] The administration method according to the present invention may utilize various delivery technologies, including microparticle encapsulation, virus delivery systems, or high-pressure aerosol collision. [Brief explanation of the drawing]

[0199] [Figure 1] Figure 1 shows the isoelectric focusing (IEF) gel of a cationic construct. [Figure 2]Figure 2 shows an overview of the SNAP-25 cleavage percentage and pEC50 compared to nBoNT / A1 in rat embryonic spinal cord neurons (eSCNs) of Cat5v2(K1064H / N954K)(A), Cat5v2(K1064H / N886K)(B), and Cat5v2(K1064H / N1025K)(C). (A, B, C) Rat embryonic spinal cord neurons were cultured for 3 weeks, treated with Cat5v4 for 24 hours, and then Western blotted with SNAP-25 specific antibody. The data are the mean ± standard deviation from three independent experiments performed in triplicates. (D) Rat eSCN The figure shows the relative potency of Cat5v2(K1064H / N886K), Cat5v2(K1064H / N954K), and Cat5v2(K1064H / N1025K) relative to nBoNT / A1 (List Biological Laboratories) in the SNAP-25 cleavage potency assay. Each point corresponds to an individual batch and is the mean of three independent pEC50 judgments based on an 8-point concentration-response curve (CRC). Each concentration in the CRC is evaluated in triplicates. The potency comparison is performed against the mean of the pooled data (n=24) of the List batch. The data is the mean ± standard deviation of batches of n=3 per Cat5v4. [Figure 3] Figure 3 shows the efficacy (t50) of nBoNT / A1 and Cat5v4 in the mouse phrenic nerve hemi-diaphragm assay (mPNHD). Mouse phrenic nerve hemi-diaphragm tissue was incubated with either Cat5v4 or native BoNT / A1, and diaphragmatic contraction force was recorded until contraction was no longer detectable or after 140 minutes. Each point corresponds to an individual determination, and the t50 value is the time required to inhibit the contraction force of the mouse unilateral diaphragm by 50%.

[0200] [array]

[0201] Sequence ID 1. BoNT / A1 nucleic acid sequence

[0202] Sequence ID 2. BoNT / A1 amino acid sequence

[0203] Sequence ID 3. Modified BoNT / A1 "Cat-A" nucleic acid sequence

[0204] Sequence ID 4. Modified BoNT / A1 "Cat-A" amino acid sequence

[0205] Sequence ID 5. Modified BoNT / A1 "Cat-B" nucleic acid sequence

[0206] Sequence ID 6. Modified BoNT / A1 "Cat-B" amino acid sequence

[0207] Sequence ID 7. Modified BoNT / A1 "Cat-C" nucleic acid sequence

[0208] Sequence ID 8. Modified BoNT / A1 "Cat-C" amino acid sequence

[0209] Sequence ID 9. Modified BoNT / A1 "Cat-D" nucleic acid sequence

[0210] Sequence ID 10. Modified BoNT / A1 "Cat-D" amino acid sequence [Examples]

[0211] The following examples illustrate specific embodiments of the present invention and do not in any way limit the scope of the present invention as defined in the claims.

[0212] Example 1

[0213] Three examples of modified BoNT / A1 molecules according to the present invention were created.

[0214] The amino acids selected for modification (mutation sites) were chosen using a number of different criteria.

[0215] The criteria for residue substitution are as follows: 1. Types of residues, 2. Degree of surface exposure, 3. Position in relation to secondary / tertiary structure, 4. Location of BoNT in relation to known functional domains, 5. Degree of sequence conservation between BoNT / A or BoNT / E subtypes. 6. Probability of introducing additional ubiquitination sites.

[0216] In this example, asparagine (Asn, N) and glutamine (Gln, Q) were selected for mutation because they are polar, similar in size to Lys, form only weak dipole interactions with other residues, and these two residues constitute 14% of the molecule.

[0217] Asn and Gln residues visible on the molecular surface were identified from the crystal structure of BoNT / A1 (PDB ID: 3BTA). This ensures that all substituted residues are able to externally display their charge. By applying points 3 to 5 (iterative process) of the above selection criteria, it was possible to exclude residues unsuitable for substitution from this list.

[0218] Non-conserved residues within BoNT / A1 were identified by alignment with BoNT / A and other subtypes of functionally similar BoNT / E serotypes. Those appearing as basic residues in other sequences were selected as top-level replacement candidates.

[0219] After successively repeating the selection criteria described in the above section, a final list of candidate residues was identified. These were screened for their potential to generate additional ubiquitination consensus sequences (using the CKSAAP_UbSite server). Some of those identified were removed by replacing the lysine residue with arginine.

[0220] Examples of the final cationic constructs synthesized from the BoNT / A1 sequence are listed below and named Cat-A, Cat-B, and Cat-C. Each construct had a molecular weight of 149,637 daltons. Cat-A: N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N886K. Cat-B: N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N954K. Cat-C: N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N1025K.

[0221] Example 2

[0222] The amino acid sequences of BoNT / B, F, and E were evaluated for potential residues that can be substituted with Lys or Arg. This initial evaluation identified residues that, when substituted, can produce BoNT / B, E, or F proteins with increased pI.

[0223] The primary sequences of BoNT / B (Ac:P10844), BoNT / E (Ac:Q00496), and BoNT / F (Ac:P30996) were analyzed. A summary of the amino acid composition is shown in the table below.

[0224] [Table 3]

[0225] According to the table, a large number of polar Asn / Gln residues were present in the amino acid sequence, similar to what was observed for BoNT / A1. A relatively large number of acidic (Asp / Glu) residues were also present, which could be converted to their corresponding neutral (Asn / Gln) or basic (Lys or Arg) residues.

[0226] Example 3

[0227] Identification of Clostridium toxin amino acids suitable for modification.

[0228] Full-length structural data were available for BoNT / A, BoNT / B, and BoNT / E; however, theoretical models were generated for the remaining four serotypes using the LOOPP computer program based on their sequences and associated structural homologies.

[0229] Each structure was analyzed using AreaIMol (CCP4 suite), and exposed residues were identified as those with a total value exceeding 40. From this list, residues with polar side chains were selected, and from these, acidic residues (Asp and Glu) or residues with hydrogen bond acceptor side chains (Asn and Gln) were prioritized. The final calculation step included selecting residues between (multiple) α-helices and (multiple) β-strands based on analysis data from the Stride server. A visual inspection of the structure of each molecule was performed to identify and avoid residues located in interface regions.

[0230] For BoNT / A1, the list of suitable residues was supplemented with residues that are functionally nonconserved in at least 90% of the aligned sequence [large nonpolar side chains (Met, Pro, Phe, Trp) were considered equivalent, small nonpolar side chains (Gly, Ala, Val, Leu, Ile) were considered equivalent, acidic side chains (Asp, Glu) were considered equivalent, and basic side chains (Arg, Lys) were considered equivalent]. More specifically, these nonconserved residues that appear as basic residues in at least 10% of the sequence, along with nonconserved Asn, Gln, Asp, or Glu in the reference sequence, were selected as candidates.

[0231] For the remaining serotypes, multiple sequence alignments were performed between subtypes to identify functionally non-conserved residues that appear as basic residues in at least 10% of the sequences.

[0232] Clostridium toxin amino acids suitable for modification

[0233] BoNT / A: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN10 25, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086.

[0234] BoNT / B: ASN873, ASN874, GLU892, ASP895, ASN906, ASP940, ASN948, GLU949, ASN958, ASN959, ASN979, ASN990, GLU993, ASP99 4, GLU997, ASN1012, ASN1019, ASP1030, ASP1047, ASP1049, GLU1065, GLU1072, GLN1176, GLU1189, GLU1252, ASN1273.

[0235] BoNT / C1: ASN881, ASP898, GLU916, GLU927, ASN952, ASN964, ASN965, ASN984, GLU985, ASP986, ASP996, ASN1000, GLU1036, ASN1041, ASP1062, ASP1064, GLU1079, ASP1081.

[0236] BoNT / D: ASN877, ASP893, ASN894, ASN898, ASN920, ASN945, ASN948, GLU957, GLN958, ASN959, ASN968, ASN979, GLU1030, ASP1031, ASP1033, GLU1047, GLU1051, ASN1052, GLU1066, GLN1122.

[0237] BoNT / E: ASN859, ASP860, ASN892, ASP893, ASP904, ASP909, ASN928, ASN932, ASN934, ASN935, GLU936, ASP945, ASN946, ASN947, ASN966, ASN976, ASN979, ASN981, ASP985, GLN1014, ASN1019, ASN1022, ASP1027, ASN1035, and ASN1140.

[0238] BoNT / F: ASN879, ASP896, ASN922, ASN923, ASN928, ASN947, ASN950, ASN952, ASN953, GLU954, ASN963, ASN964, ASN965, ASN987, GLN997, ASN1037, ASP1040, ASP1045, ASN1055, ASP1056.

[0239] BoNT / G: ASP900, ASN909, ASN910, GLU912, ASN913, ASN945, ASN947, GLU956, ASN965, ASP966, ASN986, ASN1001, ASN1038, ASP1040, ASN1046, ASP1057, GLU1073, ASN1075, ASN1090.

[0240] TeNT: ASN893, ASP894, ASP911, ASN919, ASN927, ASN928, GLU929, GLN968, ASN972, GLU973, GLU1010, ASP1018, ASN1079, ASN1080, ASN1081, ASN1097.

[0241] The array used Accession number: BoNT / A: P10845 BoNT / B: P10844 BoNT / C1: P18640 BoNT / D: P19321 BoNT / E: Q00496 BoNT / F: YP_001390123 BoNT / G: Q60393 TeNT: P04958

[0242] Structural data source The crystal structures of BoNT / A(3BTA.pdb), BoNT / B(1EPW), and BoNT / E(3FFZ.pdb) were obtained from RCSB. Homology models for BoNT / C1, BoNT / D, BoNT / F, BoNT / G, and TeNT were performed using LOOPP and the following sequences: P18640, P19321, YP_001390123, Q60393, and P04958.

[0243] structural analysis Exposed residues were determined using AreaIMol from the CCP4 suite. The assignment of secondary structures was determined using Stride. Interfacial residues were determined by visual inspection using RasMol.

[0244] Sequence analysis The full-length BoNT sequence was obtained from NCBI. Alignment was performed using ClustalX.

[0245] Example 4

[0246] Cloning, Expression, and Purification

[0247] A DNA construct encoding the modified BoNT / A molecule described in Example 1 was synthesized, cloned into the pJ401 expression vector, and then transformed into Escherichia coli BL21(DE3). This enabled the soluble overexpression of recombinant Cat-A, Cat-B, and Cat-C proteins in Escherichia coli BL21(DE3).

[0248] The recombinant modified BoNT was purified from the Escherichia coli lysate using classical chromatography techniques. After using an initial purification step with a cation exchange resin, an intermediate purification step was performed using a hydrophobic interaction resin. Next, the single chain of the recombinant modified BoNT was cleaved by proteolysis to obtain the activated double-stranded modified BoNT. Thereafter, a final purification step was utilized to remove residual contaminants.

[0249] Example 5

[0250] Characterization of the Purified Modified BoNT

[0251] The modified BoNT described in Example 1 above was experimentally characterized as follows.

[0252] Measurement of the pI revealed that the isoelectric point of the modified BoNT was higher than that of unmodified (native) BoNT / A1. See Figure 1 and the table below.

[0253] [Table 4]

[0254] The ability of modified BoNT to enter neurons and cleave SNAP-25 (the target of BoNT / A1) was evaluated using rat embryonic spinal cord neurons (eSCNs). Figure 2 shows that the ability of modified BoNT to enter neurons and cleave SNAP-25 is similar to that of natural BoNT / A1.

[0255] The efficacy of the modified BoNT was further evaluated using the mouse unilateral phrenic nerve diaphragm assay (mPNHD). Figure 3 shows that the modified BoNT retains the same ability to inhibit the contractile capacity of the mouse unilateral diaphragm as natural BoNT / A1.

[0256] The relative efficacy and safety of both molecules were evaluated using an in vivo mouse finger abduction score (DAS) assay compared to natural BoNT / A1. Both molecules showed a higher safety margin and slightly higher efficacy compared to natural BoNT / A1. These data are described below (Table 5 (the second Table 4 in the original text)).

[0257] [Table 5]

[0258] The safety margin is a measure of the adverse effects (weight loss) of BoNT treatment on efficacy (maximum half-length finger abduction score (DAS)). -10% body weight (BW) and DAS ED 50 It is calculated as a ratio to ED, where -10%BW represents the amount of BoNT (pg / animal) required for a 10% reduction in body weight, and ED 50 This indicates the amount of BoNT (pg / animal) at which DAS becomes 2.

[0259] The DAS assay is performed by evaluating finger abduction as reported by Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001) after injecting 20 μl of modified Clostridium toxin formulated in gelatin phosphate buffer into the mouse gastrocnemius / soleus muscle complex.

[0260] Example 6

[0261] Other modified Clostridium toxins according to the present invention were designed using the criteria described in Example 1 above.

[0262] This cationic construct was also derived from BoNT / A1, had a calculated pI of 7.4, and a molecular weight of 149,859. This construct was named Cat-D. Constructs Cat-A, Cat-B, and Cat-C contained residues mutated to lysine, while Cat-D contained residues mutated to arginine. Cat-D: N1188R, D1213R, G1215R, N1216R, N1242R, N1243R, S1274R, T1277R.

[0263] Example 7

[0264] Treatment of patients with cervical dystonia

[0265] A 50-year-old woman with spasmodic torticollis who was in the hospital was previously treated with a therapeutically effective amount of a standard BoNT / A preparation into the neck muscles. However, the toxin spread to the oropharynx and caused dysphagia. This patient was treated by injection of the modified BoNT / A of the present invention at approximately 1.5 ng (or more) into the neck muscles. The patient's torticollis improved significantly 3 to 7 days later without causing dysphagia, and the patient was able to keep the head and shoulders in a normal position for at least 5 months. Since the modified BoNT / A molecule has enhanced tissue retention and reduced spread, physicians can inject more product without fear of side effects, and an increase in dosage leads to an extended duration of action.

[0266] Example 8

[0267] Treatment of patients with blepharospasm

[0268] A 47-year-old male is being treated for blepharospasm. This patient is treated with injections of 5 to 25 pg of the modified BoNT / A of the present invention into the orbicularis oculi muscle at the anterior lateral aspect of the tarsal plate of the upper eyelid and the orbicularis oculi muscle at the anterior lateral aspect of the tarsal plate of the lower eyelid. The patient's symptoms are relieved within about one week and continue for at least five months without causing ptosis. The increased safety of the polypeptide of the present invention allows physicians to increase the dosage, thereby extending the duration of the clinical effect.

[0269] Example 9

[0270] A 27-year-old male with cerebral palsy was receiving outpatient treatment for debilitating equinus deformity and difficulty walking. This patient had been treated with a therapeutically effective dose of BoNT / A, but the palliative improvement in walking was accompanied by muscle weakness and limb pain. The patient was treated by injecting approximately 20 pg / kg of the modified BoNT / A of the present invention into two sites: the medial and lateral heads of the gastrocnemius muscle in both affected lower limbs. The patient's walking improved within one week without the previously observed side effects, and the symptom relief continued for at least four months. The ability to administer larger doses of the formulation leads to a treatment with a longer duration of action.

[0271] [Item 1] A modified Clostridium toxin comprising at least one amino acid modification, wherein the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin to a value at least 0.2 pI higher than that of an identical Clostridium toxin except lacking the at least one amino acid modification. [Item 2] The modified Clostridium toxin according to item 1, wherein the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin to a value at least 0.5 pI higher than that of the identical Clostridium toxin except for lacking the at least one amino acid modification. [Item 3] The modified Clostridium toxin according to item 1, wherein the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin, making it at least 1 pI unit higher than that of the same Clostridium toxin except that lacking the at least one amino acid modification. [Item 4] The modified Clostridium toxin according to item 1, wherein the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin, making it at least 2 pI units higher than that of the same Clostridium toxin except that lacking the at least one amino acid modification. [Item 5] The modified Clostridium toxin according to item 1, wherein the at least one amino acid modification increases the isoelectric point (pI) of the modified Clostridium toxin, making it 2 to 5 pI units higher than that of the same Clostridium toxin except that lacking the at least one amino acid modification. [Item 6] The modified Clostridium toxin is a modified Clostridium toxin described in any of the preceding items, wherein the pI is at least 6. [Item 7] The modified Clostridium toxin is a modified Clostridium toxin described in any of the preceding items, wherein the pI is 6 to 10. [Item 8] The modified Clostridium toxin is the modified Clostridium toxin according to item 7, wherein the pI is 7 to 9, preferably 8 to 9. [Item 9] The modified Clostridium toxin described in any of the preceding items, wherein the at least one amino acid modification is selected from amino acid substitution, amino acid insertion, and amino acid deletion. [Item 10] The modified Clostridium toxin according to item 9, wherein the at least one amino acid substitution is selected from substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. [Item 11] The modified Clostridium toxin is a modified Clostridium toxin described in any of the preceding items, comprising 1 to 80 amino acid modifications. [Item 12] The modified Clostridium toxin is the modified Clostridium toxin described in item 11, comprising 4 to 40 amino acid modifications. [Item 13] The modified Clostridium toxin described in any of the preceding items, wherein the at least one amino acid modification is located in the receptor-binding domain (HC domain) of the Clostridium toxin. [Item 14] The aforementioned at least one amino acid modification is located in the HCN domain of the Clostridium toxin, as described in item 13. [Item 15] A modified Clostridium toxin as described in any of the preceding items, wherein the at least one amino acid modification is a modification of a surface-exposed amino acid residue. [Item 16] A modified Clostridium toxin as described in any of the preceding items, wherein the at least one amino acid modification comprises modification of an amino acid residue selected from aspartic acid residue, glutamic acid residue, histidine residue, serine residue, threonine residue, asparagine residue, glutamine residue, cysteine ​​residue, or tyrosine residue. [Item 17] The modified Clostridium toxin described in item 16, wherein the aforementioned amino acid residue is substituted with a lysine residue or an arginine residue. [Item 18] A nucleic acid comprising a nucleic acid sequence encoding a modified Clostridium toxin as described in any of items 1 through 17. [Item 19] A method for producing a single-chain modified Clostridium toxin protein having a light chain and a heavy chain, comprising: expressing a nucleic acid described in item 18 in a suitable host cell; lysing the host cell to provide a homogenate of the host cell containing the single-chain modified Clostridium toxin protein; and isolating the single-chain modified Clostridium toxin protein. [Item 20] A method for activating a modified Clostridium toxin, comprising: providing a single-chain modified Clostridium toxin protein obtainable by the method described in item 19; contacting a polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the light chain and the heavy chain; and converting the polypeptide into a double-chain polypeptide in which the light chain and the heavy chain are linked to each other by disulfide bonds. [Item 21] A modified Clostridium toxin as described in any of items 1 through 17, used in pharmaceuticals. [Item 22] Strabismus, blepharospasm, strabismus (squint), dystonia (e.g., spastic dystonia, maxillofacial dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic (beauty) applications where cell / muscle incapacitation (by downregulation or inactivation of SNARE) is effective, neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, hypertropia, lateral rectus palsy, nystagmus, hypothyroid myopathy), writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting from abnormal bladder control, animus, Modified Clostridium toxins as described in any of items 1 to 17, for use in the prevention or treatment of diseases or conditions selected from the following: back spasms, muscle rigidity, tension headaches, levator pelvis syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain due to muscle spasms), headaches (e.g., tension headaches), forehead wrinkles, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary neuropathy, chronic neurogenic inflammation, and smooth muscle disorders. [Item 23] Modified Clostridium toxins as substantially described herein with reference to the accompanying drawings.

Claims

1. Pharmaceuticals containing double-stranded modified botulinum toxin type A (BoNT / A) for the prevention or treatment of cosmetic conditions: Here, the dose of the double-stranded modified BoNT / A administered is 1 pg to 30 ng. The double-stranded modified BoNT / A includes a light chain and a heavy chain linked to each other by disulfide bonds. Modified double-stranded BoNT / A maintains its ability to bind to target cells, translocate, and cleave target SNARE proteins. Double-stranded modified BoNT / A contains 4 to 80 amino acid modifications. The heavy chain includes a Clostridium toxin receptor-binding domain (HC domain), and the HC domain is modified from the HC domain of BoNT / A, shown as Sequence ID No. 2, by at least four amino acid substitutions located within it. The at least four amino acid substitutions located in the HC domain increase the isoelectric point (pI) of the modified double-stranded BoNT / A, making it at least 0.4 pI higher than that of an identical double-stranded BoNT / A except for the lack of the at least four amino acid substitutions located in the HC domain. The at least four amino acid substitutions located in the HC domain include substitutions of four or more amino acids 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 by lysine or arginine residues.

2. The pharmaceutical product according to claim 1, wherein the aforementioned cosmetic condition is frown lines, hyperactive facial wrinkles, and / or nasolabial wrinkles.

3. Pharmaceuticals containing double-stranded modified botulinum toxin type A (BoNT / A) for the prevention or treatment of headaches: Here, the dose of the double-stranded modified BoNT / A administered is 1 pg to 30 ng. The double-stranded modified BoNT / A includes a light chain and a heavy chain linked to each other by disulfide bonds. Modified double-stranded BoNT / A maintains its ability to bind to target cells, translocate, and cleave target SNARE proteins. Double-stranded modified BoNT / A contains 4 to 80 amino acid modifications. The heavy chain includes a Clostridium toxin receptor-binding domain (HC domain), and the HC domain is modified from the HC domain of BoNT / A, shown as Sequence ID No. 2, by at least four amino acid substitutions located within it. The at least four amino acid substitutions located in the HC domain increase the isoelectric point (pI) of the modified double-stranded BoNT / A, making it at least 0.4 pI higher than that of an identical double-stranded BoNT / A except for the lack of the at least four amino acid substitutions located in the HC domain. The at least four amino acid substitutions located in the HC domain include substitutions of four or more amino acids 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 by lysine or arginine residues.

4. A pharmaceutical product containing double-stranded modified botulinum toxin type A (BoNT / A) for the prevention or treatment of blepharospasm or hemifacial spasm: Here, the dose of the double-stranded modified BoNT / A administered is 1 pg to 30 ng. The double-stranded modified BoNT / A includes a light chain and a heavy chain linked to each other by disulfide bonds. Modified double-stranded BoNT / A maintains its ability to bind to target cells, translocate, and cleave target SNARE proteins. Double-stranded modified BoNT / A contains 4 to 80 amino acid modifications. The heavy chain includes a Clostridium toxin receptor-binding domain (HC domain), and the HC domain is modified from the HC domain of BoNT / A, shown as Sequence ID No. 2, by at least four amino acid substitutions located within it. The at least four amino acid substitutions located in the HC domain increase the isoelectric point (pI) of the modified double-stranded BoNT / A, making it at least 0.4 pI higher than that of an identical double-stranded BoNT / A except for the lack of the at least four amino acid substitutions located in the HC domain. The at least four amino acid substitutions located in the HC domain include substitutions of four or more amino acids 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 by lysine or arginine residues.

5. Pharmaceuticals containing double-stranded modified botulinum toxin type A (BoNT / A) for the prevention or treatment of limb spasticity: Here, the dose of the double-stranded modified BoNT / A administered is 1 pg to 30 ng. The double-stranded modified BoNT / A includes a light chain and a heavy chain linked to each other by disulfide bonds. Modified double-stranded BoNT / A maintains its ability to bind to target cells, translocate, and cleave target SNARE proteins. Double-stranded modified BoNT / A contains 4 to 80 amino acid modifications. The heavy chain includes a Clostridium toxin receptor-binding domain (HC domain), and the HC domain is modified from the HC domain of BoNT / A, shown as Sequence ID No. 2, by at least four amino acid substitutions located within it. The at least four amino acid substitutions located in the HC domain increase the isoelectric point (pI) of the modified double-stranded BoNT / A, making it at least 0.4 pI higher than that of an identical double-stranded BoNT / A except for the lack of the at least four amino acid substitutions located in the HC domain. The at least four amino acid substitutions located in the HC domain include substitutions of four or more amino acids 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 by lysine or arginine residues.

6. The pharmaceutical product according to any one of claims 1 to 5, wherein the dose of the double-stranded modified BoNT / A administered is 5 ng or less.

7. The pharmaceutical product according to any one of claims 1 to 6, wherein the dose of the double-stranded modified BoNT / A administered is 0.05 ng to 5 ng.

8. A pharmaceutical product containing double-stranded modified botulinum toxin type A (BoNT / A) for the prevention or treatment of spasmodic torticollis: Here, the dose of the double-stranded modified BoNT / A administered is 1.5 ng or more. The double-stranded modified BoNT / A includes a light chain and a heavy chain linked to each other by disulfide bonds. Modified double-stranded BoNT / A maintains its ability to bind to target cells, translocate, and cleave target SNARE proteins. Double-stranded modified BoNT / A contains 4 to 80 amino acid modifications. The heavy chain includes a Clostridium toxin receptor-binding domain (HC domain), and the HC domain is modified from the HC domain of BoNT / A, shown as Sequence ID No. 2, by at least four amino acid substitutions located within it. The at least four amino acid substitutions located in the HC domain increase the isoelectric point (pI) of the modified double-stranded BoNT / A, making it at least 0.4 pI higher than that of an identical double-stranded BoNT / A except for the lack of the at least four amino acid substitutions located in the HC domain. The at least four amino acid substitutions located in the HC domain include substitutions of four or more amino acids 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 by lysine or arginine residues.

9. H C The pharmaceutical product according to any one of claims 1 to 8, wherein the at least four amino acid substitutions located in the domain consist of substitutions by lysine or arginine residues of the following seven amino acids: ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, or GLN1229.

10. H C The pharmaceutical product according to any one of claims 1 to 8, wherein the at least four amino acid substitutions located in the domain consist of substitutions by lysine or arginine residues of the following seven amino acids: ASN930, ASN954, SER955, GLN991, ASN1026, ASN1052, or GLN1229.

11. H C The pharmaceutical product according to any one of claims 1 to 8, wherein the at least four amino acid substitutions located in the domain consist of substitutions by lysine or arginine residues of the following seven amino acids: ASN930, SER955, GLN991, ASN1025, ASN1026, ASN1052, or GLN1229.

12. H C The pharmaceutical product according to any one of claims 1 to 8, wherein the at least four amino acid substitutions located in the domain consist of substitutions by lysine or arginine residues of the following eight amino acids: ASN1188, ASP1213, GLY1215, ASN1216, ASN1242, ASN1243, SER1274, or THR1277.

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